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Regulation of Mitochondrial Biogenesis
in Human Embryonic Stem Cells
Li-Pin Kao
A thesis submitted for the degree of Doctor of Philosophy at
The University of Queensland in 2015
Australian Institute for Bioengineering & Nanotechnology
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Abstract
Human embryonic stem cells (hESCs) are established from the inner cell mass of
the pre-implantation blastocyst and are both pluripotent (being able to be differentiated into
all cell types of the body) and immortal (being able to proliferate indefinitely). When grown
in culture, they provide a unique in vitro model system that allows us to study the earliest
steps of human embryogenesis, a developmental process that is otherwise inaccessible to
experimentation.
Mitochondria are not only the essential organelles for generating energy in cells but
also generate reactive oxygen species (ROS) as side-products of aerobic energy
production. The presence of ROS leads to the progressive accumulation of mutations
within mitochondrial DNA (mtDNA), resulting in dysfunctional mitochondria. To counteract
this, it is thought that a selective amplification of healthy mitochondria occurs during
pre-implantation development, aiming at maintaining the mitochondrial fitness in the
germline (the ‗bottleneck‘ theory). Embryonic stem cells harvested from the
pre-implantation blastocyst can be cultured indefinitely in vitro and contain
metabolically-active mitochondria. Therefore, in this study, we wished to investigate how
the mtDNA copy number is regulated in cultured pluripotent stem cells.
Initially, the mitochondrial-encoded as well as the mitochondrial biogenesis-related
gene expression were analysed in spontaneously differentiating hESCs and during different
lineage-specific differentiation of hESCs in a time-dependent manner. The
spontaneously-differentiated hESCs possess more copies of mitochondrial genome in
mitochondria and early-differentiated hESCs display more mature mitochondrial
morphology and a higher mitochondrial membrane potential as compared to
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undifferentiated hESCs. After 7 days of spontaneous differentiation of hESCs, the transcript
levels of D-loop and PGC1α, the master regulatory gene of the mitochondrial biogenesis,
were upregulated. We also demonstrated that the early ectoderm and cardiomyocyte
differentiation is accompanied by an upregulation of PGC1α. While this shows that
mitochondrial gene expression and biogenesis changes upon differentiation it remained to
be determined what role mitochondria play in pluripotent stem cells and their differentiated
derivatives.
To address this question, we investigated the effect of interference with
mitochondrial DNA replication (using EtBr) or mitochondrial protein synthesis (using
chloramphenicol) in fibroblasts. Compared with untreated primary human foreskin
fibroblasts, the mitochondrial impaired rho-minus fibroblasts showed a lower rate of
proliferation and a decreased level of mitochondrial gene expression, as well as lower
mitochondrial membrane potential and greater rate of lactate production. Moreover, the
mitochondrial-encoded and mitochondrial biogenesis-related genes were upregulated after
the treatments had been removed. While fibroblasts were able to tolerate mitochondrial
depletion or inhibition of mitochondrial protein synthesis repeated attempts to generate
mitochondria depleted, let alone mitochondria null, hESC failed, suggesting that despite the
notion that hESCs are mainly glycolytic mitochondria are required for pluripotent stem cell
proliferation and survival.
These observations next led us to investigate whether the mtDNA copy number
affects the hESC‘s survival. To this end, we chose to perturb the expression of
mitochondrial transcription factor A (TFAM), a member of the high-mobility group (HMG)
family of proteins, which is known to have a role in controlling the mtDNA copy number in
other cell types. Overexpression of TFAM in hESCs led to an increase in the mtDNA copy
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number but did not affect hESC morphology, whereas knockdown of TFAM expression
resulted in dramatically cell death in H9 and Mel 1 hESCs. The data indicate that in contrast
to other cell types TFAM does not appear to play a central role in mitochondrial biogenesis
in hESC and that PGC1α may be more important in this respect.
To investigate the mechanism of mitochondrial turnover in hESC, we investigated
mitophagy in pluripotent and differentiating hESC. Following a short-term treatment of
hESCs, with rapamycin (mTOR inhibition), CCCP (depolarisation of the mitochondrial
membrane), or ethidium bromide (mtDNA damage). Autophagosome numbers were
increased. Furthermore, after longer-term, EtBr triggered mitophagy events in every single
cell in a hESC culture. Interestingly we found that during early hESC differentiation, the
mitophagic activity transiently increased, perhaps indicating that mitochondrial turnover
may be involved in mitochondrial quality control during very early differentiation of hESCs.
While there remain many questions to be answered regarding the role and regulation of
mitochondria in hESC and hESC differentiation this thesis provides several novel insights
into these processes.
hESCs represent a unique system to study the molecular mechanisms that control
mitochondrial quality and quantity and investigating these processes in the most primitive
human stem cells is highly relevant to understanding the effects of environmental toxins on
early human development and regenerative medicine approaches for in particular
mitochondrial diseases.
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Declaration by Author
This thesis is composed of my original work and contains no material previously
published or written by another person except where due reference has been made in the
text. I have clearly stated the contribution by others to jointly-authored works that I have
included in my thesis.
I have clearly stated the contribution of others to my thesis as a whole, including
statistical assistance, survey design, data analysis, significant technical procedures,
professional editorial advice and any other original research work used or reported in my
thesis. The content of my thesis is the result of work I have carried out since the
commencement of my research higher degree candidature and does not include a
substantial part of work that has been submitted to qualify for the award of any other degree
or diploma in any university or other tertiary institution. I have clearly stated which parts of
my thesis, if any, have been submitted to qualify for another award.
I acknowledge that an electronic copy of my thesis must be lodged with the University
Library and, subjected to the General Award Rules of The University of Queensland,
immediately made available for research and study in accordance with the Copyright Act
1968.
I acknowledge that the copyright of all material contained in my thesis resides with the
copyright holder(s) of that material. Where appropriate I have obtained copyright
permission from the copyright holder to reproduce material in this thesis.
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Publications during candidature
1. Kao LP., Ovchinnikov DA., and Wolvetang EJ. Regulations of Mitochondrial
Biogenesis in Chloramphenicol- and Ethidium Bromide-Treated Primary Human
Fibroblasts. Toxicol Appl Pharmacol. 2012 May 15:261(1):42-49.
2. Chious SS., Wang SS., Wu DC., Lin YC., Kao LP., Kuo KK., Wu CC., Chai CY., Lin CL.,
Lee CY., Liao YM., Wuputra K., Yang YH., Wang SW., Ku CC., Nakamura Y., Saito S.,
Hasegawa H., Yamaguchi N., Miyoshi H., Lin CS., Eckner R., Yokoyama KK (2013)
Control of oxidative stress and generation of induced pluripotent stem cell-like cells by
Jun Dimerization protein 2. Cancer 5(3):959-984.
3. Wu JM., Chen CT., Coumar MS., Lin WH., Chen ZJ., Hsu JT., Peng YH., Shiao HY., Lin
WH., Chu CY., Wu JS., Lin CT., Chen CP., Hsueh CC., Chang KY., Kao LP., Huang
CY., Chao YS., Wu SY., Hsieh HP., Chi YH. (2013) Auora kinase inhibitors reveal
mechanisms of HURP in nucleation of centrosomal and kinetochore microtubules. Proc.
Natl. Acad. Sci. USA. 110 (19): E1779-1787.
4. Chen BZ., Yu SL., Singh S., Kao LP., Tsai ZY., Yang PC., Chen BH., Li SS. (2011)
Identification of microRNAs expressed highly in pancreatic islet-like cell clusters
differentiation from human embryonic stem cells. Cell Biol Int 35(1):29-37.
5. Wang KH., Kao AP., Singh S., Yu SL., Kao LP., Tsai ZY., Lin SD., Li SS (2010)
Comparative expression profiles of mRNAs and microRNAs among human
mesenchymal stem cells derived from breast, face, and abdominal adipose tissues.
Kaohsiung J Med Sci 26(3):113-122.
6. Tsai ZY., Singh S., Yu SL., Kao LP., Chen BZ., Ho BC., Yang PC., Li SS
(2010)Identification of microRNAs regulated by activin A in human embryonic stem cells.
J Cell Biochem 109(1):93-102.
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7. Li SS., Yu SL, Kao LP, Tsai ZY., Singh S., Chen BZ., Ho BC., Liu YH., Yang PC. (2009)
Target identification of microRNAs expressed highly in human embryonic stem cells.
Journal of Cellular Biochemistry. 106(6): 1020-1030.
8. Kao LP, Yu SL, Singh S, Wang KH, Kao AP, Li SS (2008) Comparative profiling of
mRNA and microRNA expression in human mesenchymal stem cells derived from adult
adipose and lipoma tissues. The Open Stem Cell Journal. 1:1-9.
Publications included in this thesis
1) Kao LP., Ovchinnikov DA., and Wolvetang EJ. Regulations of Mitochondrial Biogenesis
in Chloramphenicol- and Ethidium Bromide-Treated Primary Human Fibroblasts.
Toxicol Appl Pharmacol. 2012 May 15:261(1):42-49. PMID: 22712077 –incorporated in
Objective 2- Kao was responsible for 50% of experiments design, analysis and
interpretation of data, drafting and writing; Ovchinnikov was responsible for 5% of
editing and interpretation of data; Wolvetang was responsible for 45% of conception,
experimental design, analysis and interpretation of data, drafting and writing.
2) Tra T., Gong L., Kao LP, Li XL., Grandela C., Devenish RJ., Wolvetang E., Prescott M.
(2011) Autophagy in human embryonic stem cells. PLoS One. 6(11):e27485. -Kao was
responsible for 5% of experimental performances. Wolvetang was responsible for 70%
of drafting and writing
3) Briggs JA., Sun J., Shepherd J., Ovchinnikov DA., Chung TL., Nayler SP., Kao LP.,
Morrow CA., Thakar NY., Soo SY., Peura T., Grimmond S., Wolvetang EJ (2013)
Integration-free induced pluripotent stem cells model genetic and neural development
features of down syndrome etiology. Stem Cells.31 (3):467-478-Kao was responsible
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for 5% of drafting and writing. Wolvetang was responsible for 70% of drafting and
writing.
Oral and Poster presentations based on this thesis
1. Li-Pin Kao, D.A. Ovchinnikov, M. Prescott, T. Tra, J.P. Turner., J.J. Cooper-White., E.J.
Wolvetang (2013). Regulation of mitochondrial biogenesis in human embryonic stem
cells. The fifth international system biology and bioinformatics (SSBC), Russia. Oral
presentation.-Second best oral presenter and poster presentation-received paper
submission invitation.
2. Li-Pin Kao, M. Prescott, T. Tra, J.P. Turner., J.J. Cooper-White., E.J. Wolvetang (2012).
Mitochondria turnover in human embryonic stem cells and can be modulated by
environmental factors. The annual international conference on stem cell research (SCR),
Malaysia. Oral presentation and poster presentation -received paper submission
invitation.
3. Li-Pin Kao, Mark Prescott, Thien Tra, Rachel Horne and Ernst Wolvetang (2009)
Mitophagy in human embryonic stem cells. The seventh international society for stem
cell research (ASSCR), Australia. Poster presentation.
4. Li-Pin Kao, Mark Prescott, Thien Tra, Rachel Horne and Ernst Wolvetang (2009)
Mitophagy in human embryonic stem cells. The seventh international society for stem
cell research (ISSCR) USA. Poster presentation.
5. Li-Pin Kao, Mark Prescott, Thien Tra, Rachel Horne and Ernst Wolvetang (2009)
Mitophagy in human embryonic stem cells. 2009 MMRI Stem Cell and Regenerative
Medicine Symposium, Australia. Poster presentation.
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Contributions by others to the thesis
1) A/Prof. Ernst Wolvetang and Dr. Dmitry Ovchinnikov contributed significantly to the
concept and design of projects, as well as data analysis and interpretation, revision of
work, editing of manuscripts and the thesis as a whole.
2) Briggs JA., Sun J., Shepherd J., Ovchinnikov DA., Chung TL., Nayler SP., Kao LP.,
Morrow CA., Thakar NY., Soo SY., Peura T., Grimmond S., Wolvetang EJ (2013)
Integration-free induced pluripotent stem cells model genetic and neural development
features of down syndrome etiology. Stem Cells.31 (3):467-478. - Drs. Briggs and Sun
contributed to the hiPSCs and protocol and cDNAs of ectoderm (neural) differentiation
to this thesis.
3) Hudson J, Titmarsh D, Hidalgo A, Wolvetang E, Cooper-White J. Primitive cardiac cells
from human embryonic stem cells (2012) Stem Cells
Dev.:0;21(9):1513-23.(PMID:21933026)- Dr. Hudson contributed to the protocol and
cDNAs of mesendoderm and cardiomyocyte differentiation to this thesis.
4) Dr. Samah Alharbi contributed to the protocol, cDNAs generation and
immunocytochemistry of definitive endoderm differentiation to this thesis.
5) Dr. Jennifer Turner assisted with other part of mitochondrial metabolism analysis
laboratory work on human embryonic stem cells including optimisation and validation of
lactate, glucose and oxygen measurement methods.
Statement of parts of the thesis submitted to qualify for the
award of another degree
None
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Acknowledgements
I would like to express my gratitude to Assoc. Prof. Ernst Wolvetang, my primary
supervisor for his guidance and support during this period. Ernst, thank you so much for
waiting for my manuscript and delays patiently, and it's so great of you to act as a good
supervisor with positive as well as encouraging attitude towards my work. Thanks for
accompanying me to get through the tough time and to achieve this project, and to my two
associate supervisors, Dr. Dmitry Ovchinnikov and Dr Tung-Liang Chung for being willing
to be my co-supervisor during my PhD and conducting follow-up of this thesis. If either I
hadn't met you during the period of my studying or you hadn't been involved in my advisory
committee, I couldn't have made it through. Of course, there would have been a different
result if we had never met each other. I couldn't express how much I appreciate your
continuous help, support and encouragement, with which I could defeat the difficulties I
encountered while I was developing this thesis. Again, thanks for your dedication to the
stem cell research.
I also want to express my gratitude to the two members of my thesis committee, both
of them are listed in my PhD program: Prof. Justin Cooper-White, thanks for spending your
time and supporting my proposals in each milestone review meeting; and Assoc. Prof.
Christine Wells, thank you for always giving me advice. I also want to show my appreciation
to the members in the entire Cooper-White and Wells groups, past and present, for their
continuous support, encouragement and helping me with my project ideas.
Furthermore, my sincere expressions of thanks are extended to Ms. Victoria Turner
and Mrs. Michelle Brush from the Brisbane core facility of the Australian Stem Cell Centre
for their continuous provision of human embryonic stem cell lines. I would also like to
express my appreciation to Dr. Geoff Osborne and Mr. John Wilson from the FACS facility
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of the Queensland Brain Institute and the Centre for Microscopy and Microanalysis, Dr.
Elena Taran from the Australian National Fabrication Facility, and members of the Centre
for Microscopy and Microanalysis for their technical support and friendship. Without their
wholehearted devotion and support, this thesis would not have been completed.
This study was made possible because of the support from a scholarship and topup
provided by the Australian Postgraduate Award and the Australian Institute Bioengineering
and Nanotechnology. My appreciation also extends to the staff and postgraduate students
of the AIBN, University of Queensland, for their help and friendship. To all my past and
present supervisors (Prof. Mao SJ, Li SS, Yokoyama KK, and Prof. Chi YH) presence in my
career in the U.S.A, Taiwan and Japan, I am deeply appreciated your support, discussion
and review of my entire thesis when I encountered a bottleneck during my PhD study.
Special thanks should also go to my friends in Australia who gave me the benefit of
their tremendous suggestions. I particularly thank them for being with me and listening to
my research problems even though for most of them, my research topic is not even close to
theirs. With their understanding and assistance, the journey of getting the PhD was no
longer tedious. Each of them deserves credit for the quality and style of this paper. In
addition, I owe a debt of gratitude to members in the Wilson family and their two lovely dogs,
Gucci and Thor. They are the best companions to talk to, to be my ears, and to look in my
eyes as if they truly feel what I have been through during my pursuing PhD in Australia. I
have spent so much quality time running and walking around St Lucia campus with them. I
hope I can run and walk with them again in Australia. Furthermore, I am very grateful to
family members (LKK, Diana YHH, and Leo LYK) for their presence in comfort me. Finally,
to my Marley, thanks for all the joy and happiness you brought into my life and I wish you
have a good time in heaven.
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Keywords
human embryonic stem cells, human foreskin fibroblast cells, mitochondrial biogenesis,
mitochondrial rho minus cells, mitophagy, transcription factors
Australian and New Zealand Standard Research Classifications
(ANZSRC)
ANZSRC code: 060103, Cell development, Proliferation and Death 50%
ANZSRC code: 060199, Biochemistry and Cell Biology not elsewhere classified, 50%
Fields of Research (FoR) Classification
FoR code:0601, Biochemistry and Cell Biology, 50%
FoR code:1004, Medical Biotechnology 50%
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Table of Contents
Page
Abstract 2
Declaration by Author 5
Publications 6
Acknolwedgments 10
Keywords 12
Research Classifications 12
List of Tables 14
List of Figures 15
List of Videos 17
Chapter 1. Introduction 18
Chapter 2. Review of Literature 23
Chapter 3. Methods and Procedures 87
Chapter 4. Results 113
Chapter 5: Discussion 135
Chapter 6 Final Conclusion 158
References 160
Tables 189
Figures and Videos 203
Appendix Tables 235
Supplementary Figures 255
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List of Tables
Page
Table 2.1 Diseases that are associated with perturbations to the mitochondrial fusion/fission machinery
40
Table 2.2 Methods for analysis of mtDNA 46
Table 2.3 Mouse gene knockouts of nuclear-encoded regulators of transcription implicated in mitochondrial biogenesis
54
Table 4.1 Comparison of TRA-1-60 and mitochondrial staining in undifferentiated and differentiated hESCs
189
Table 4.2 Changes in gene expression associated with ectoderm-specific differentiation of hESCs
190
Table 4.3 Changes in gene expression associated with endoderm-specific differentiation of hESCs
192
Table 4.4 Changes in gene expression associated with mesendoderm-specific differentiation of hESCs
193
Table 4.5 Changes in gene expression associated with cardiomyocyte-specific differentiation of hESCs
194
Table 4.6 Gene expression changes of mitochondria-encoded genes, transcription factors, and mtDNA copy number in control, ethidium bromide- or chloramphenicol-treated cells after treatment for 6 weeks and release from treatment for 2 weeks
195
Table 4.7 Relative amounts of glucose, lactate, GSH, and GSSG, and intracellular ATP, ADP concentrations in control, ethidium bromide– or chloramphenicol-treated cells after treatment for 6 weeks and release from treatment for 2 weeks
196
Table 4.8 Comparison of the mitochondrial membrane potential in control, ethidium bromide– or chloramphenicol-treated cells after treatment for 6 weeks and release from treatment for 2 weeks
198
Table 4.9 Target sequence of shRNAs against human TFAM 199
Table 4.10 Levels of TFAM expression in hESC lines (gain-of-function) 200
Table 4.11 Levels of TFAM expression in hESC lines (loss-of-function) 201
Table 4.12 Quantification of autophagosomes and mitophagy events per hES cells
202
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List of Figures
Page
Figure 2.1 hESC isolation and culture procedures 25
Figure 2.2 ES cells can give rise to cell types from the three primitive germ
layers
32
Figure 2.3 Structure of mitochondrion 33
Figure 2.4 Schematic representation of the human mitochondrial genome 35
Figure 2.5 Stylized representation of the electron transport chain (ETC), which
is the final stage of cellular respiration where oxidative
phosphorylation (OXPHOS) takes place
37
Figure 2.6 Schematic of general mitochondrial replication 48
Figure 2.7 Transcriptional activation from the D-loop promoter region of mtDNA 53
Figure 2.8 TFAM alignment with different species 55
Figure 2.9 Changes in mtDNA copy number per cell during implantation stages 65
Figure 2.10 The possible origin of pathogenic mtDNA deletions 66
Figure 2.11 Stages of autophagosome formation in selective and non-selective
pathways
75
Figure 2.12 The mTOR signaling pathway 79
Figure 4.1 Schematic of lineage-specific differentiation in hESCs 203
Figure 4.2 Comparison of TRA-1-60 and mitochondrial staining in
undifferentiated and differentiated hESCs
204
Figure 4.3 Characterization of mitochondria in undifferentiated and
differentiated hESCs
206
Figure 4.4 Mitochondrial biogenesis profiles during ectoderm differentiation 208
Figure 4.5 Mitochondrial biogenesis profiles during endoderm differentiation 209
Figure 4.6 Mitochondrial biogenesis profiles during mesendoderm differentiation 210
Figure 4.7 Mitochondrial biogenesis profiles during cardiomyocytes
differentiation
211
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Figure 4.8 Morphology and karyotypes of rho minus cells 212
Figure 4.9 Relative expression of mitochondrial-encoded, and mitochondrial
biogenesis-related genes after treatment for 6 weeks (6w) and
release from treatment for 2 weeks (6+2w)
213
Figure 4.10 Metabolic effect of ethidium bromide and chloramphenicol treatment
on human fibroblasts after treatment for 6 weeks and release from
treatment for 2 weeks
215
Figure 4.11 Mitochondrial membrane potential in ethidium bromide- or
chloramphenicol-treated cells after treatment for 6 weeks and
release from treatment for 2 weeks
217
Figure 4.12 Assessment of mitochondrial ultrastructure by transmission electron
microscopy after treatment for 6 weeks and release from treatment
for 2 weeks
219
Figure 4.13 Levels of TFAM expression in hESC lines (gain-of-function) 220
Figure 4.14 Constitutive knockdown of TFAM levels in hESCs 222
Figure 4.15 Levels of TFAM expression in hESC lines (loss-of function) 223
Figure 4.16 Morphological phenotypes associated with TFAM loss- and gain-of
function in hESC lines
225
Figure 4.17 Characterization of the hESC-LC3-GFP reporter cell line 226
Figure 4.18 Mitochondrial toxin and differentiation-induced mitophagy in
hES3-LC3-GFP cells
227
Figure 4.19 Quantification of mitophagy in hES3-LC3-GFP cells using
live-imaging flow-cytometry
229
Figure 4.20 Quantification of autophagosomes and mitophagosomes in hESCs 232
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List of Videos
Page
Video 4.1 Live-imaging of mitophagy in hES3-LC3-GFP 233
Video 4.2 Live-imaging of mitophagy in hES3-LC3-GFP with 200nM rapamycin 233
Video 4.3 Live-imaging of mitophagy in hES3-LC3-GFP with 50µM CCCP 233
Video 4.4 Live-imaging of mitophagy in hES3-LC3-GFP with 100ng/ml EtBr 233
Video 4.5 Live-imaging of mitophagy in spontaneously differentiated hES3-LC3-GFP without basic fibroblast growth factor for 7 days
234
Video 4.6 Live-imaging of mitophagy in hES3-LC3-GFP with 100ng/ml EtBr for 16 hours
234
Video 4.7 Live-imaging of mitophagy in hES3-LC3-GFP with 100ng/ml EtBr for one week
234
Video 4.8 Live-imaging of mitophagy in hES3-LC3-GFP for one week 234
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Chapter 1: Introduction
Embryonic stem cells (ESCs) are derived from the inner-cell-mass (ICM) of
blastocysts. ESC and induced pluripotent stem cell (iPSC) lines are immortal, meaning that
they have unlimited proliferation potential. They are also pluripotent; that is, they can
differentiate into lineages derived from all 3 major germ layers of the embryo. Consequently,
these cells have been widely investigated in the development of regenerative medicine
therapies. Human ESCs have been regarded as important research tools for the
investigation into early development of the human embryo. Mitochondria are the
powerhouses capable of providing the majority of energy within the cell and performing
important metabolic functions such as the Krebs cycle. The well-known endosymbiotic
theory (Martin, Hoffmeister et al. 2001) has suggested that the mitochondrion was originally
derived from a prokaryotic cell that invaded a larger, nucleated host cell. Mitochondria
indeed contain their own mitochondrial DNA (mtDNA) in a circular form, similar to the
bacterial genome. Mitochondrial genomes encode several essential genes of the
eukaryotic respiratory machinery. However, most of the components of the respiratory
machinery and factors controlling mitochondrial biogenesis are encoded in the nucleus.
The cooperation and communication between mitochondria and nuclei are conducted by
retrograde signals, such as energy supply and redox signaling and this currently
poorly-understood communication is essential for balancing energy production and
demand in the cell.
Mutations or deletions in the mitochondrial genome or nuclear-encoded
mitochondrial biogenesis-related genes usually result in a spectrum of mitochondrial
dysfunctions. This can affect a wide variety of tissues, including the brain, heart, liver,
skeletal muscles, kidney and the endocrine and respiratory systems. Differences in the
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organs affected may lead to different symptoms, such as myopathies, developmental
delays and respiratory complications. Human cells lacking mtDNA known as rho zero (ρ˚)
cells, provide an opportunity to examine cellular processes in the absence of functional
mitochondria; that is to mimic the cells from patients with mitochondrial depletion syndrome
(MDS).
Mitochondria are entirely derived from the oocyte and very little mitochondrial
biogenesis is thought to occur until the implantation of blastocyst into uterus. Implantation
results in dramatic environmental alterations, such as increased oxygen and nutrient supply.
Interestingly, this is also the stage at which the differentiation of the inner cell mass into the
various tissues and cell types of the body starts to occur. The development of an organism
demands energy. Mitochondrial dysfunction (stemming from low mtDNA content, for
example, or from mutations within the mtDNA) within an oocyte may contribute to the failure
of in vitro fertilization and may affect hESC maintenance and differentiation (Reynier,
May-Panloup et al. 2001). The role of mitochondria in human embryonic stem cells (hESCs)
and the mechanism, by which mitochondrial health is maintained in these immortal cells,
remain unclear.
Mitochondrial turnover eliminates defective mitochondria to maintain cellular
homeostasis (Diaz and Moraes 2008). Mitochondrial turnover occurs through a specific
form of autophagy, an evolutionarily-conserved process in eukaryotic cells that results in
the breakdown of some of cytoplasmic proteins and organelles within the lysosome in
response to stress conditions. A key signaling pathway in the regulation of autophagy is the
mammalian Target of Rapamycin (mTOR) kinase, which senses signals causes by the
nutrient levels. mTOR regulates protein synthesis in the cell, the status of energy and
oxygen supply, cell proliferation and cell growth. This mTOR signaling is essential for
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survival, differentiation, development and homeostasis in all organisms. Autophagy is
essential during degradation and development of maternal RNAs, proteins and organelles
in order to activate zygotic transcription and translation at different developmental stages.
Upon differentiation of hESCs, the mtDNA copy number increases to meet the energy
requirements of different cell types generated. Both ‗healthy‘ and ‗mutated‘ mitochondria
could be amplified upon differentiation. There exists a selective mechanism, called
mitophagy, by which autophagosomes uptake mitochondria. Mitophagic events are a
highly-selective process controlled by oxidative stress and mTOR and is accompanied by
loss of membrane potential and ensuing mitochondrial degradation.
The purpose of the current thesis aimed to examine the role of mitochondria in
hESCs during maintenance and differentiation. Four initial objectives were formulated to
guide the investigation:
Objective 1: To review the gene expression patterns of mitochondria-encoded and
mitochondrial biogenesis-related genes during spontaneous and directed
lineage-specific differentiation.
The gene expression patterns of mitochondria-encoded and mitochondrial
biogenesis-related genes during the differentiation of human pluripotent stem cells are not
known. Mitochondria are the major source of energy for eukaryotic cells. To meet rising
energy demands, cells often expand the pool of mitochondria, a process termed
mitochondrial biogenesis, which involves the replication of mitochondrial DNA (mtDNA) and
the synthesis, import, and assembly of essential mitochondrial proteins (Hock and Kralli
2009). Levels of mtDNA can affect oocyte maturation and subsequent developmental
processes (Van Blerkom 2004). During cellular differentiation, mitochondria also
differentiate in a process that involves structural and functional changes. For example,
21
mitochondria within spontaneously differentiating cells increase their membrane potential
(St John, Ramalho-Santos et al. 2005). This section describes the treatment conditions
used to promote differentiation and documents the expression of mitochondrial-encoded
and mitochondrial biogenesis-related genes during spontaneous, lineage-specific-, or
tissue-specific-differentiation of hESCs.
Objective 2: To investigate the effects of mitochondrial depletion on human
fibroblasts and hESCs.
Environmental toxins might cause mitochondrial dysfunction, which generates
retrograde signaling from the mitochondria to the nucleus. While this signaling pathway has
been well described in yeast, the manner in which mammalian cells respond to
mitochondrial DNA (mtDNA) depletion or the inhibition of mitochondrial protein synthesis
remains largely unclear. To investigate this response, mitochondria-depleted (rho-minus)
human fibroblasts were generated with agents that damage mitochondria: ethidium
bromide, which interferes with mitochondrial DNA replication, or chloramphenicol, which
interferes with mitochondrial protein synthesis. Gene expression, mitochondrial membrane
potential, morphology, and metabolic parameters were assessed during and after
treatment.
Objective 3: To characterize the effects of TFAM overexpression and knockdown on
the control of mitochondrial biogenesis during the proliferation of hESCs.
TFAM regulates the stability (Fisher and Clayton 1988) and copy number (Scarpulla
2008) of mtDNA, and mice lacking Tfam do not survive past embryonic day (E) 8.5
(Larsson, Wang et al. 1998). We hypothesized that TFAM also controls the mtDNA copy
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number in hESCs. Therefore, we investigated whether manipulation of TFAM expression
affected the proliferation or survival of hESCs. First approach was to assess how
constitutive overexpress or knockdown of TFAM affected the survival of stem cells. The
second approach was to switch from a constitutive expression system to an inducible
expression system in order to obtain a better understanding of the relationship between
TFAM expression and stem cell survival.
Objective 4: To compare mitochondrial turnover (mitophagy) following exposure to
mitochondrial toxins and during the differentiation of hESCs.
Mitochondrial turnover is essential for maintaining optimal cellular function within the
human body. Several diseases have been identified that result from the improper turnover
of mitochondria. LC3 initially forms a complex that resembles a pre-autophagosomal
structure, serving as a nucleation site on damaged mitochondria that sends an ―eat-me‖
signal. The pre-autophagosomal structure eventually forms a cup-shaped isolation
membrane that sequesters and delivers damaged mitochondria to autophagosomes (i.e.
mitophagosomes) and then to autolysosomes. In this study, we investigated the mitophagic
process during differentiation and after exposure to agents that interfere with mitochondrial
function. Firstly, we established the GFP-LC3 system as a means to quantitatively measure
mitophagy. Next, the system was used to understand the dynamics of mitophagy during
differentiation of hESCs or when cells were challenged with toxins that affect mitochondrial
function. Furthermore, various mitochondrial toxins were used to study mitophagy during
stem cell differentiation.
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Chapter 2: Reivew of Literature
2.1 Human Pluripotent Stem Cells
Stem cells are endowed with a unique capacity for self-renewal (Weissman 2000). In
mammalian development, fertilized oocytes are able to differentiate into all types of cells and
are thus classified as totipotent. ‗Totipotent‘ is derived from the Latin totus, which means
‗entire‘. A totipotent stem cell has the ability to generate all cell types found in the embryo
and in extra-embryonic tissues. Fertilized oocytes divide and progress into 8-cell embryos
and then blastocysts. Blastocysts are composed of an outer layer of cells, called
trophoblasts, and inner cells, which form the inner cell mass (ICM). The ICM can develop
into all cell types of the adult body and is therefore called ‗pluripotent‘, a term derived from
the Latin plures, meaning ‗several‘ or ‗many‘. Pluripotent stem cells, such as mouse
embryonic stem cells (mESCs), human embryonic stem cells (hESCs), and induced
pluripotent stem cells (iPSCs), can differentiate into ectoderm, mesoderm, endoderm, and
germ cells (Lemoli, Bertolini et al. 2005).
Once blastocyst-derived cells have fully differentiated into tissues or organs, stem
cells that reside in various tissues and organs remain in order to generate new tissue or
repair damaged tissue. These stem cells, known as multipotent stem cells or adult stem
cells (ASCs), include mesenchymal stem (or stromal) cells (MSCs) and hematopoietic stem
cells (HSCs). Their differentiation ability is limited compared to that of totipotent and
pluripotent cells. For example, hematopoietic stem cells, which are found in bone marrow,
can differentiate into erythrocytes and white blood cells (including macrophages). These
types of cells are important for homeostasis because they enable the steady self-renewal of
tissue.
24
There are different types of pluripotent stem cells: embryonic stem cells (ESCs),
induced pluripotent stem cells (iPSCs), embryonic germ cells (EGCs), and embryonic
carcinoma cells (ECCs). ESCs are isolated from the ICM of the blastocyst (Figure 2.1)
(Thomson, Itskovitz-Eldor 1998), whereas iPSCs are artificially generated by
reprogramming somatic cells using a defined set of transcription factors. iPSCs are similar
to hESCs in morphology, gene expression, and differentiation ability (Chen, Gulbranson et
al. 2011). Human embryonic germ cells (hEGCs) are derived from the primordial germ cells
(PGCs) and are pluripotent stem cells (Thomson and Odorico 2000). Embryonic carcinoma
cells (ECCs) are stem cells derived from teratocarcinomas, which are tumors that arise from
embryonic tissues such as those from the testis or ovary, or from cultures of explanted cells
(Przyborski, Christie et al. 2004). ECCs are considered the malignant counterparts of
hESCs.
2.2 Embryonic Stem Cells (ESCs) and induced pluripotent stem cells (iPSCs)
Evans and Kaufman (1981) were the first to derive ESCs from an early mouse
embryo (Evans and Kaufman 1981). In 1998, Thomson and colleagues reported the first
successful derivation of hESC lines (Norman, Fischer et al. 2010); these lines are still
widely used. They are capable of proliferating extensively in their undifferentiated state in
vitro and have the ability to differentiate into all three germ layers (Shamblott, Axelman et al.
1998, Thomson and Odorico 2000). The embryo-derived hESCs were established from
blastocysts discarded during in vitro fertilization (IVF) procedures.
iPSCs are an artificially generated type of pluripotent stem cell. iPSCs are
reprogrammed from adult somatic cells to acquire stem cell-like properties through the
forced expression of a combination of transcription factors, such as Oct4, Sox2, Nanog,
25
c-myc, KLF4, and Lin28. Takahashi and Yamanaka introduced four pluripotent genes-Oct4,
Sox2, c-myc, and Kruppel-like family transcription factor 4 (Klf4)—that could reprogram
mouse fibroblasts into mouse-induced pluripotent stem cells (miESCs) or human fibroblasts
into human-induced pluripotent stem cells (hiPSCs) (Takahashi and Yamanaka 2006).
Thomson and colleagues used Oct4 and Sox2 in combination with Nanog and Lin-28
homolog (Lin28), instead of c-myc and Klf4, to reprogram human fibroblasts into hiPSCs
(Yu, Vodyanik et al. 2007). These iPSCs express stem cell markers and can differentiate
into three germ layers in a teratoma in vivo.
iPSCs have the advantage that they do not require the destruction of an embryo.
Moreover, given their origin, they provide a perfect match to the cell donor (are fully
isogenic) and thus would likely avoid rejection by the donor‘s immune system.
Figure 2.1 hESC isolation and culture procedures. hESCs are derived from the inner cell
mass of the blastocyst (left). For long-term culture, hESCs are grown on inactivated mouse
embryonic fibroblasts used as a feeder layer (Hoffman and Carpenter 2005).
26
2.3 Characterization of Undifferentiated hESCs
Undifferentiated hESCs express high levels of cell surface antigens that can be used
as stem cell-specific pluripotency markers. These antigens include: (1) glycolipids, such as
the stage-specific embryonic antigens SSEA-3 and SSEA-4 (Kannagi, Cochran et al. 1983);
(2) glycoproteins, such as TRA-1-60 and TRA-1-81 (Badcock, Pigott et al. 1999); and (3)
alkaline phosphatase (Thomson, Itskovitz-Eldor et al. 1998, Reubinoff, Pera et al. 2000,
Henderson, Draper et al. 2002). Pluripotency markers include the transcription factors
Octamer-4, POU domain, class 5, transcription factor 1 (OCT4 or POU5F1) (Goto, Adjaye
et al. 1999, Hansis, grifo et al. 2000, Mitsui, Tokuzawa et al. 2003, Sperger, Chen et al.
2003, Hart, hartley et al. 2004, Zangrossi, Marabese et al. 2007);sex-determining region
Y-box 2 (SOX2) (Avilion, Nicolis et al. 2003); and Nanog homeobox (NANOG) (Ivanova,
Dimos et al. 2002, Pan and Thomson 2007).These molecular markers provide a means of
identifying pluripotent stem cells, and a decrease in their expression can be used to monitor
the onset of differentiation. The molecular mechanisms underlying the self-renewal of
hESCs have not been fully elucidated.
2.4 Gene Expression Analysis of hESCs
Investigating gene expression in various stem cell lines could give important insights
into how stem cells control pluripotency and differentiation. A number of studies have
measured hESC gene expression to investigate related molecular mechanisms and have
reported differential gene expression in different hESC lines. Rao and Stic (2004) reported
a 75% similarity in the microarray profiles of two lines; in a another study, 48% of the
expressed genes were restricted to one or two lines (Abeyta, Clark et al. 2004). However,
variations in gene expression have been observed in hESC lines derived within the same
laboratory (Ginis, Luo et al. 2004) and even in the same hESC lines (38%) after three
27
passages in different media (Rao, Calhoun et al. 2004). Gene expression also changes
during spontaneous differentiation (Aghajanova, Skottman et al. 2006). For example, the
expression of leukemia inhibitory factor (LIF) and its receptors is low in undifferentiated
hESCs, but increases during differentiation. Differential DNA methylation of
pluripotency-associated promoters such as NANOG and OCT4/POU5F1 has been
observed in pluripotent and differentiated cells. Understanding gene expression in hESCs
will help shed light on the molecular basis of normal differentiation and the abnormal
processes that underlie human developmental disorders.
Several external signals that maintain stem cell pluripotency have been
characterized. External signals are also thought to play important roles in the regulation of
ESC self-renewal and differentiation. For example, the pluripotency of hESCs is maintained
by several signaling pathways (Ohtsuka and Dalton 2008):
The Fibroblast Growth Factor (FGF) Signaling Pathway
Exogenous bFGF is an essential factor in a defined hESC culture medium used for
the maintenance of undifferentiated hESCs and hiPSCs in vitro. Withdrawal of bFGF
induces the downregulation of pluripotency markers and the differentiation of hESCs.
This suggests that FGF signaling plays an important role in self-renewal and
pluripotency regulation in human ES cells and iPSCs (Xu, Peck et al. 2005, Eisenberg,
Knauer et al. 2009).
The Transforming Growth Factor-β (TGF-β)/Activin/Nodal-SMAD2/3 Signaling Pathway
The TGF-β/Activin/Nodal branch is highly active in undifferentiated hESCs. The
pathway supports the self-renewal of undifferentiated hESCs by activating SMAD2/3
and inducing the expression of the pluripotency markers Oct4 and Nanog (James,
28
Levine et al. 2005, Vallier, Alexander et al. 2005). The TGF-β/Activin/Nodal-SMAD2/3
signaling pathway is important in maintaining the self-renewal and pluripotency of
hESCs (Valdimarsdottir and mummery 2005).
The Phosphoinositide-3-Kinase (PI3K) Signaling Pathway
The PI3K protein is highly expressed in undifferentiated cells and is downregulated
in differentiated ESCs (Di Cristofano, Pesce et al. 1998, Takahashi, Murakami et al. 2005,
Armstrong, Hughes et al. 2006). Blocking the PI3K signaling pathway with the PI3K
inhibitor LY294002 results in the loss of pluripotency markers and initiates cellular
differentiation (Paling, Wheadon et al. 2004). In addition, activation of the PI3K signaling
pathway induces the PI3K-dependent phosphorylation of PKB/Akt and GSK-3α/β proteins.
2.5 Propagation of Undifferentiated hESCs
Initially, hESCs were grown on irradiated mouse feeders or human foreskin
fibroblasts (Inzunza, Gertow et al. 2005). However, exposure to animal-derived culture
constituents is a drawback of the feeder-dependent systems (Sidhu, Walke et al. 2008).
Given that hESCs and hiPSCs are attractive candidates for future human cell
transplantation, it is important to optimize good manufacturing practice (GMP)-compliant
systems for the derivation, scale-up, and banking of cells and their corresponding quality
assurance controls (Unger, Skottman et al. 2008, Ausubel, Lopez et al. 2011). Therefore
feeder-free systems are increasingly used in combination with xeno-free defined culture
medium and GMP-compliant coating substrates specially designed for hESC growth (Yoon,
Chang et al. 2010), including laminin and fibronectin.
29
2.6 Pluripotent Stem Cell Differentiation
In vitro and in vivo, hES and iPS cells can differentiate into cell types from the three
primitive germ layers: ectoderm, mesoderm, and endoderm (Thomson, Itskovitz-Eldor et al.
1998, Reubinoff, Pera et al. 2000, Thomson and Odorico 2000) (Figure 2.2). The
differentiation capacity of these cells is typically tested by assessing their spontaneous
differentiation in cell culture (i.e. in vitro formation of embryonic bodies or EBs). Upon the
removal of growth factor (e.g. bFGF) or feeder layers and/or transfer to suspension
conditions, hESCs undergo spontaneous unguided differentiation into various cells
representative of the different germ layers. In two-dimensional (2D) spontaneously
differentiated hESCs, the different morphologies appeared to be epithelial cells, neural cells
with axons and dendrites, and cells with mesenchymal characteristics (Odorico, Kaufman
et al. 2001). In suspension, the hESCs form multicellular aggregates of differentiated and
undifferentiated cells called embryoid bodies (EBs), which resemble early post-implantation
embryos and frequently progress through a series of differentiation stages (Itskovitz-Eldor,
Schuldiner et al. 2000). Typically, EBs are allowed to grow for several days or weeks, with
samples taken at intervals for analysis via flow cytometry or immunocytochemical staining.
In vitro and in vivo assessments of differentiation involve determining whether the derived
cells have acquired a variety of ectoderm-, mesoderm-, and endoderm-like properties (and
loss of markers for pluripotency). In vivo assessment of pluripotency is performed by
xenografting hESCs and iPSCs into severe combined immune-deficient (SCID) mice and
observing the formation of teratomas with derivatives of all three germ layers, which
indicates that the injected stem cells have the ability to differentiation along three lineages
(Thomson, Itskovitz-Eldor et al. 1998, Reubinoff, Pera et al. 2000).
30
2.7 Directed Differentiation
To direct the differentiation of ESCs towards a particular cell type, such as a neuronal
cell type or a cardiomyocyte cell type, hESCs in monolayer culture are exposed to certain
growth factors or stimuli and extracellular matrix components, either directly or indirectly
through feeder cells (Schuldiner, Yanuka et al. 2000).
Retinoic acid (RA) induces human embryonic stem cells to differentiate into the
ectodermic lineage (Guan, Chang et al. 2001). RA and its receptors play important roles in
the development of the central nervous system by initiating the cellular differentiation of
neuronal precursors (Niederreither and Dolle 2008). Several papers have reported that RA
induces neuronal differentiation in neuroblastoma cell lines (SH-SY5Y human
dopaminergic neuroblastoma cells) (Constantinescu, Constantinescu et al. 2007, Lopes,
Schroder et al. 2010) and human promyelocytic leukemia HL-60 cells (Racanicchi,
Montanucci et al. 2008). Further, RA also induces embryonic stem cells to differentiate into
neuronal cells (Guan, Chang et al. 2001, Parsons, Teng et al. 2011, Liu, Liu et al. 2012),
including neurons and glial cells. In order to improve the efficiency and reproducibility of
neuronal differentiation, several studies have attempted to add small molecules. For
example, Idelson M et al. have demonstrated that nicotinamide promotes the differentiation
of hESCs into neural cells and subsequently into retinal pigment epithelium (RPE) cells
(Idelson, Alper et al. 2009). Moreover, Lu SJ et al. have described a robust system that
efficiently generates large numbers of hemangioblasts from multiple hESC lines and
produces functional homogeneous RBCs with oxygen-carrying capacity on a large scale
(Lu, Feng et al. 2010). The markers of early ectodermdifferentiation are paired box gene 6
(PAX6), SRY (sex determining region Y)-box 1 (SOX1), nesting, and glial fibrillary acidic
31
protein (GFAP). Briggs JA et al. have demonstrated successful neuronal differentiation via
a sophisticated protocol (Briggs, Sun et al. 2013).
Mesoderm differentiation has been extensively studied, particularly the families of
protein growth factors that control the early stages of mesoderm formation in
cardiomyocytes (Mummery, Zhang et al. 2012). Hudson J et al. have used small molecules
to target the wingless/INT (Wnt) signaling pathway in order to induce the differentiation of
hESCs into beating cardiomyocytes (Hudson, Titmarsh et al. 2012). Biomarkers for early
mesoderm-differentiation include T-box factor Brachyury (BRY or T), the homeodomain
protein MIXL1, and myosin (Hudson, Titmarsh et al. 2012).
Endoderm differentiation forms several tissues, including the liver, lung, thyroid, and
foregut endoderm. The families of protein growth factors that control the early stage of
endoderm differentiation into the anterior-ventral domain of the foregut endoderm are
targeted by signaling through Nodal, a member of the transforming growth factor-B (TGF-B)
superfamily and the SMAD signaling pathway (Kim, Yoon et al. 2011). The biomarkers of
early endoderm differentiation are insulin-like growth factor 2 (IGF2) and gata binding factor
(GATA4); SRY (sex determining region Y)-box 17 (SOX17) is also an indicator of the
definitive foregut endoderm (Kanai-Azuma, Kanai et al. 2002, Nakanishi, Kurisaki et al.
2009). Little is known about the expression of mitochondrial biogenesis-related genes
during hESC lineage-specific differentiation.
32
Figure 2.2 Undifferentiated hES cells (A) can give rise to cell types from the three primitive
germ layers: (B) neuronal cells in ectoderm-lineage differentiation, (C) pancreatic cells in
endoderm-lineage differentiation, and (D) cardiomyocytes in mesoderm-lineage
differentiation. This figure is adapted from Hoffman and Carpenter 2005, Wobus and
Boheler 2005).
33
2.8 Mitochondrial Biology: Introduction
The mitochondrion is a dual-membrane organelle which is found in all eukaryotic
cells except erythrocytes and lense tissue. The mitochondrion is composed of the outer
mitochondrial membrane, the intermembrane space (the space between the outer and
inner membranes), the inner mitochondrial membrane, the cristae space (formed by
in-folding of the inner membrane), and the matrix (the space within the inner membrane)
(Figure 2.3).
Figure 2.3 Structure of a mitochondrion. This figure is modified from (Frey and Mannella
2000).
The human mitochondrial genome consists of a 16.5-kb double-stranded circular
DNA molecule that contains both non-coding and coding regions. The largest non-coding
sequence in mammalian mtDNA is the displacement-loop (D-loop), which contains
promoters and origins of replication. The coding regions contain 37 genes, including 2
ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and 13 genes that encode
34
subunits of the respiratory complexes (I, III, IV, and V) (Figure 2.4) (Chan 2006). Alkaline
gradient centrifugation separates mtDNA into a heavy strand (H-strand) and a light strand
(L-strand) based on differential contents of guanosine (G) and cytosine (C). The H-strand
encodes 2 rRNAs, 12 polypeptides, and 14 tRNAs, while the L-strand encodes one
polypeptide (ND6) and 8 tRNAs. The genetic code of mitochondria is similar to the
universal genetic code, with three exceptions: 1) the TGA codon codes for tryptophan in
mtDNA instead of stop in nuclear DNA, 2) AGA and AGG code for stop in mtDNA instead of
arginine in nuclear DNA, and 3) ATA codes for methionine in mtDNA instead of isoleucine
in nuclear DNA (Anderson, Bankier et al. 1981). It is thought that mitochondria originally
derived from endosymbiotic prokaryotes because they share many features. For example,
the circular mitochondrial genome is very similar to the genomes of bacteria (Martin,
Hoffmeister et al. 2001).
35
Figure 2.4 Schematic representation of the human mitochondrial genome. mtDNA is a
double-stranded circular molecule of approximately 16.5 kb, consisting of a heavy (H)
strand and a light (L) strand. mtDNA consists of non-coding and coding regions. The
non-coding D-loop (displacement loop) region contains the origin of heavy strand
replication (OH), and the transcription promoters for both strands (HSP and LSP). The
origin of light strand replication (OL) is found approximately two-thirds of the way around the
genome (at 16024–16569 and 1–576). The coding region encodes 13 protein subunits of
the electron transport chain (ETC), 12 on the heavy strand and 1 on the light strand (ND6),
along with 22 tRNAs and 2 rRNAs that are necessary for mtDNA transcription and protein
synthesis. Proteins encoded by mtDNA, indicated in yellow, include ND1, 2, 3, 4, 4L, 5, and
6 (NADH dehydrogenase; Complex I); CYTB (cytochrome b; Complex III); COX1, 2, and 3
(cytochrome c oxidase; Complex IV); and ATP6 and 8 (ATP synthase; Complex V).
36
Mitochondrial respiratory activity occurs via the electron transport chain (ETC). The
ETC is composed of five complexes: Complex I (NADH dehydrogenase, also called ND),
Complex II (succinate dehydrogenase), Complex III (cytochrome reductase), Complex IV
(cytochrome C oxidase, also called COX), and Complex V (ATP synthase). Energy
obtained from the transfer of electrons through these complexes is used to pump protons
from the mitochondrial matrix into the intermembrane space. The sum of the pH gradient
and the membrane potential (Δψ), which arises from the net movement of positive charge
across the inner membrane, is known as the proton motive force (PMF; ΔP= ΔµH+ + Δψ),
which accumulates across the mitochondrial inner membrane. The proton motive force
allows ATP synthase (Complex V) to transfer protons back into the matrix to generate ATP
from ADP and inorganic phosphate (Pi). As electrons flow through the complexes, they
eventually reach Complex IV, where they are used to generate water from hydrogen ions
and molecular oxygen (Figure 2.5) (Wallace and Fan 2010).
37
Figure 2.5 Stylized representation of the electron transport chain (ETC), the final stage of
cellular respiration, where oxidative phosphorylation (OXPHOS) takes place. The ETC
consists of five protein complexes that contain polypeptides encoded by the nuclear and
mitochondrial genomes (except for Complex II). The flow of electrons through the first four
complexes releases protons (H+) from the mitochondrion and establishes the mitochondrial
membrane potential. The transfer of these protons back into the mitochondrion through
Complex V generates ATP. This figure is modified from (Zeviani and Di Donato 2004).
Abbreviations: CoQ, coenzyme Q; Cyt C, cytochrome C; ANT, adenosine nucleoside
transporter; ADP, adenosine diphosphate; ATP, adenosine triphosphate.
38
2.9 Mitochondria Are Dynamic Organelles
A mitochondrion is not a discrete, autonomous organelle; rather, it is part of a
dynamic network akin to the endoplasmic reticulum. Mitochondria are highly dynamic
organelles that are constantly engaged in fusion and fission in the cell, as demonstrated, for
instance, in mouse embryonic fibroblasts (Chen, Detmer et al. 2003). In dividing cells, the
mass of this mitochondrial network steadily increases throughout the cell cycle; it normally
divides more or less equally between daughter cells. During fusion and fission, the
synthesis of the vast majority of mitochondrial components is required, including lipids and
proteins that are imported into pre-existing organelles, thereby enlarging the structure.
Mitochondria have maintained a fission system in order to split the enlarged organelle mass.
Once a sufficient volume of organelle has been generated and the cell is ready to divide,
the mitochondria segregate between the cells of the next generation. The relative rates of
fusion and fission determine the morphology of the mitochondrial network at any given
time.
Multiple proteins are involved in mitochondrial fusion and fission. Several important
players are involved in the mitochondrial fusion mechanism in mammalian cells, such as
mitofusin 1 (MFN1), mitofusin 2 (MFN2), and optic atrophy 1 (OPA1) (Westermann 2010).
These three proteins are large GTPases that localize to different sites in mitochondria
(Detmer and Chan 2007, Liesa, Palacin et al. 2009). MFN1 and MFN2 insert into the outer
membrane and form homo- and hetero-oligomers; OPA1 is found at the intermembrane
space partially anchored to the inner membrane (liesa, Palacin et al. 2009, Westermann
2010). The presence of mitofusins on adjacent mitochondria is required during fusion
because they form complexes in-trans that tether the mitochondria together (Meeusen,
McCaffery et al. 2004). Inhibition of mitofusins results in mitochondrial fragmentation and
39
poor mitochondrial function (Chen, Detmer et al. 2003, Rakovic, Grunewald et al. 2011).
Inhibition of OPA1 leads to mitochondrial fragmentation and severe aberrations in the
structure of the cristae owing to a loss of mitochondrial fusion (Olichon, Baricault et al. 2003,
Griparic, van der Wel et al. 2004, Chen and Chan 2005). However, the specific functions of
these proteins in membrane fusion remain to be determined (Youle and Karbowski 2005).
In vitro, mitochondrial fusion of both the outer and inner membranes requires GTP
hydrolysis; the mitochondrial membrane potential is also required for inner membrane
fusion (Meeusen, McCaffery et al. 2004).
In mammalian cells, mitochondrial fission is regulated by fission 1 (mitochondrial
outer membrane) homologue (FIS1) and dynamin-related protein 1 (DRP1). FIS1 is
localized uniformly on the mitochondrial outer membrane. During mitochondrial fission,
DRP1 is recruited from the cytosol to the outer membrane. The majority of DRP1 is located
in the cytosol, but a subpool is localized to punctate spots on microtubules. A subset of
these puncta mark future sites of fission (Smirnova, Griparic et al. 2001, Youle and
Karbowski 2005), and DRP1 plays a role in membrane constriction during fission. Inhibition
of FIS1 and DRP1 inhibits fission, resulting in the elongation of mitochondrial tubules
(Zeviani and Di Donato 2004). However, inhibition of FIS1 does not disrupt the mitochondrial
localization of DRP1 (Zeviani and Di Donato 2004). DRP1 plays a role in membrane
constriction during mitochondrial fission. However, the precise mechanism of fission
remains to be determined.
Mitochondrial fission and fusion events are important processes not only in the
normal functioning of the cell but also in pathophysiological situations in different cells and
tissues (Table 2.1). For example, mitochondrial fission mediated by DRP1 is a necessary
step for cell death in apoptosis (Frank, Gaume et al. 2001).
40
Table 2.1 Diseases associated with perturbations in the mitochondrial fusion/fission
machinery. This table is modified from (Detmer and Chan 2007). Abbreviations: CMT2A,
Charcot-Marie-Tooth type 2A; MFN2, mitofusin-2; OPA1, optic atropohy-1; DRP1,
dynamin-related protein-1.
Diseases Mitochondrial
function Gene Description
CMT2A Fusion MFN2 Autosomal dominant peripheral neuropathy
ADOA Fusion OPA1 Autosomal dominant optic atrophy (ADOA)
Unnamed Fission DRP1 Neonatal lethality
2.10 Mitochondrial Defects
Mitochondrial diseases often result from mtDNA mutations and represent common
inherited conditions. One of every 7,634 newborns is affected by mitochondrial dysfunction
(Debray, Lambert et al. 2007). Mitochondrial diseases are a heterogeneous group of
disorders in which mitochondrial dysfunction can affect tissues with varying severity. The
effects are often more prominent in neurons of the brain and in skeletal muscles, which
require more energy than other tissues.
Within mtDNA, several types of mutations have been identified, including 1) point
mutations, 2) large-scale rearrangements, and 3) depletion of mtDNA resulting from a
reduction in the mtDNA copy number (Zeviani and Antozzi 1997, Zeviani and Di Donato
2004). Depletion of mtDNA is an autosomal recessive trait that can cause severe illnesses,
such as neurodegenerative disorders (Zeviani and Antozzi 1997, Zeviani and Di Donato
41
2004, St John and Lovell-Badge 2007). Profound reductions in mtDNA are responsible for
a series of syndromes that are collectively referred to as mtDNA-depletion syndromes
(Deodato F 2009). Patients with these syndromes present with myopathy,
encephalomyopathy, and mitochondrial neurogastrointestinal encephalomyopathy
syndromes (Deodato F 2009). Pathologies associated with these syndromes vary widely
and can manifest in utero or later in life. To study the consequences of mtDNA dysfunction,
animal and cellular models with mitochondrial mutations, deletions, or depletion have been
generated.
2.11 Mitochondrial Dysfunction in Animal and Cellular Models
The symptoms of mitochondrial dysfunction caused by mutations or deletions in the
mitochondrial genome or by mitochondrial depletion can be observed in whole animals and
cellular models. Several studies have described mice with abnormal mitochondria resulting
from mutations or deletions in the mitochondrial genome. These mutations cause
conditions that phenocopy Pearson and Kearns-Sayre syndromes (Wallace 1999, Inoue,
Nakada et al. 2000). The mice exhibit numerous symptoms indicative of mitochondrial
dysfunction, with clinical phenotypes associated with human diseases (Inoue, Nakada et al.
2000, Sligh, Levy et al. 2000), including: (1) abnormalities of the optic nerve in the retina, (2)
decreases in cytochrome c-oxidase (COX) activity, (3) ragged-red fibers in muscle, (4) a
high lactic acid concentration, and (5) death from kidney failure (acidosis). Furthermore, the
progeny of the mice exhibit several features: (1) inherited mtDNA mutations, homoplasmic
or heteroplasmic, (2) a severely affected survival rate, with many dying in utero or in the
first few days after birth, (3) growth retardation, (4) abnormal mitochondrial morphology in
skeletal and cardiac muscle, and (5) progressive myopathy, myofibril disruption and loss,
42
and dilated cardiomyopathy. Tissues derived from normal hESCs or tissues repaired using
a gene therapy approach carry normal mtDNA. Inducing hESCs with normal mtDNA to
differentiate into specific cell types might help in the treatment of mitochondrial dysfunction
generated by mutations, deletions, and depletion of the mitochondrial genome. Therefore,
hESCs could be a useful model for understanding the etiology of mitochondrial-related
diseases.
To demonstrate the importance of mitochondrial morphology and function in
cell-specific functions, mitochondria-depleted cells called rho-zero cells (ρ˚), which are
depleted of mtDNA, were generated in vitro through the application of different drugs, such
as ethidium bromide, antibiotics, or the nucleoside analogue reverse transcriptase inhibitor
(NARTI, an anti-HIV drug). ρ˚ cells exhibit several common features: (1) they become
autotrophic, relying on pyrimidine (uridine) and pyruvate supplementation for cell growth
(Armand, Channon et al. 2004, Miceli and Jazwinski 2005); (2) they have a low mtDNA
copy number and low expression of mitochondrial-encoded genes, but not of
nuclear-encoded genes; (3) they have low mitochondrial respiratory chain complex
activities, with the exception of complex II; (4) they have low ATP concentrations,
respiration rates (oxygen consumption), and mitochondrial membrane potential; (5) they
shift from aerobic to anaerobic metabolism if given supplemental pyruvate; and (6) they
have an immature mitochondrial structure with reduced numbers of cristae membranes,
circular morphology, and loss of tubular structure.
Upon a reduction in oxygen consumption, several studies have found that
antioxidants can reverse the increased ROS production in ρ˚ cells. In addition, ρ˚ cells have
decreased levels of cell proliferation, mitotic cyclin gene expression, cyclin-dependent
kinase inhibitors, retinoblastoma 1 phosphorylation, and telomerase activity (Park, Choi et
43
al. 2004, Schroeder, Gremmel et al. 2008). In ρ˚ cells, upregulation of mitochondrial
biogenesis-related genes, relative to expression in control cells, has been observed
(Miranda, Foncea et al. 1999, Joseph, Rungi et al. 2004).
Interestingly, it has been suggested that ρ˚ cells have increased resistance to
apoptosis. However, they exhibit a normal distribution of cytochrome c within mitochondria
during staurosporine-induced apoptosis (in spite of low mtDNA levels and respiratory
function deficiencies). Consistently, caspase 3 activation and DNA fragmentation are not
affected in ρ˚ cells. However, the localization of NF-κB is altered (i.e. more NF-κB in the
nucleus than in the cytoplasm), which might be related to the observed resistance to
apoptosis. Moreover, in ρ˚ cells, a greater amount of mass is associated with lysosome and
peroxidation production (King and Attardi 1989, Appleby, Porteous et al. 1999, Miranda,
Foncea et al. 1999). Remarkably, the differentiation of SH-SY5Y neuroblastoma cells into
neuron-like cells is not affected by defective mitochondria, as indicated by the presence of
long neurites and secretory granules, which are typical of differentiating neuroblastoma
cells (Miller, Trimmer et al. 1996).
2.12 Assessing mtDNA Integrity, Copy Number, and Mitochondrial Biogenesis
Mitochondria have their own protein-coding DNA but also import a large number of
nuclear-encoded proteins. Therefore, mitochondrial biogenesis requires precise
coordination between the nuclear and mitochondrial genomes. Because of the complex
and dynamic nature of mitochondria, the analysis of mitochondrial biogenesis within a
population of cells is difficult and requires measurements of multiple parameters, such as:
(1) the volume and number of mitochondria via microscopy, (2) mtDNA copy number, (3)
44
levels of mtDNA-encoded transcripts, (4) levels of mitochondrial biogenesis-related
transcription factors (e.g. TFAM, NRF1, POLG, and PGC1α) via qPCR, (5) levels of
biogenesis molecular markers via western blotting, and 6) mitochondrial rates of translation
by in vivo labeling (Medeiros 2008). Details of these techniques are provided below.
First, mitochondrial morphology can be visualized and measured by fixing,
dehydrating, sectioning, and staining cells and tissue sections for analysis with
transmission electron microscopy (TEM). The method is time-consuming, but generates
reliable, quantitative data. TEM analysis also requires expensive equipment and the
assistance of an experienced TEM operator.
Second, mitochondrial mass can be visualized and measured using fluorescent dyes.
Analysis of stained samples using microscopy or flow cytometry yields quantitative results.
Several fluorescent dyes are available for measuring the mitochondrial mass or Δψ
(mitochondrial potential). Mitochondrial mass is typically measured using MitoTracker
(Molecular Probes, Invitrogen) or 10-n-nonyl-acridine orange. The latter binds cardiolipin,
which is predominantly found in the inner mitochondrial membrane.
Third, common PCR and qPCR techniques can be used to assess the levels of
mtDNA, mtDNA-encoded transcripts, and mitogenesis-associated transcription factors (e.g.
TFAM, NRF1, and PGC1α). Because mitochondria have their own genomes, the amount of
mtDNA is roughly proportional to the number of mitochondria, although each mitochondrion
has multiple copies of mtDNA. mtDNA has traditionally been quantified using Southern blot
analysis (Tang, Halberg et al. 2012), but the use of qPCR techniques is becoming more
common. qPCR methods typically compare levels of mtDNA and nuclear DNA to determine
the mtDNA copy number (i.e. the number of mitochondrial genomes per cell) (Hoschele,
Wiertz et al. 2008). The measurements typically use a variety of primer sets, such as those
45
for NADH dehydrogenase subunit I (ND1), along with primers specific for nuclear-encoded
housekeeping genes, which are used for normalization purposes. In addition, the D-loop is
critical for the replication and transcription of mtDNA (Takamatsu, Umeda et al. 2002).
Expression of D-loop elements can be used to determine the mtDNA copy number.
However, when quantifying levels of mtDNA, it is important to use target sequences that
are not commonly deleted in disease or by aging.
The quality of mtDNA is typically determined via DNA sequencing or microarray
analysis (Maitra, Cohen et al. 2004) (Table 2.2). These techniques are expensive and must
be repeated multiple times because of the heteroplasmy of mtDNA. mtDNA sequencing
can be used to detect polymorphic sites and to determine maternal relationships between
individuals. Molecular techniques such as these have led to the creation of the Human
Mitochondrial Genome Database (MtDB), which contains many human mtDNA sequences,
a list of known mutation hotspots, and a catalogue of polymorphic sites (Ingman and
Gyllensten 2006).
Fourth, antibodies directed against proteins encoded by the mitochondrial genome
or proteins critical for mitogenesis (e.g. TFAM, NRF1, POLG, and PPARGC1A) can be
used (Williams, Scholte et al. 2001). Many of these antibodies are commercially available
(Invitrogen, Sigma-Aldrich, MitoSciences, and Santa Cruz Biotechnology). Mitochondria
can be measured by assessing complex (I, II, IV, and V) activities, fission/fusion rates, and
oxygen consumption. GFP, targeted by a leading signaling peptide to mitochondria, is used
to measure fission/fusion rates. During oxidative phosphorylation, the transfer of electrons
to oxygen and the oxidation of substrates is used to generate ATP. Thus, oxygen
consumption can be one of the most informative and direct measures of mitochondrial
function.
46
Table 2.2 Methods for the analysis of mtDNA mutations or mtDNA copy number
Method for the analysis of mtDNA
mutations Sample type Reference
Oligonucleotide array Total DNA (Maitra, Cohen et al. 2004,
Maitra, Arking et al. 2005)
Sequence analysis of PCR-amplified
products Total DNA
(Polyak, Li et al. 1998, Fliss,
Usadel et al. 2000, Maitra,
Cohen et al. 2004)
qPCR (mitochondrial-encoded gene,
ND1, COX2) cDNA
(Cunningham, Rodgers et al.
2007)
qPCR (mitochondrial-encoded genes and
non-coding region, D-loop) Total DNA
(Kanki, Ohgaki et al. 2004, St
John, Amaral et al. 2006,
Amaral, Ramalhoo-Santos et
al. 2007, Armstrong, Tilgner
et al. 2010)
PCR (ND4, CYTB, and COXI) and
western blot (COXI and COXII)
cDNA and
proteins (Jeng, Yeh et al. 2008)
Southern and northern blot
(mitochondrial-encoded genes)
Total DNA
and total RNA
(Larsson, Oldfors et al. 1994,
Garstka, Schmitt et al. 2003,
Ekstrand, Falkenberg et al.
2004, Maniura-Weber,
Goffart et al. 2004,
Pohjoismaki, Wanrooij et al.
2006).
Southern blot (Alpha-32P-dCTP labeled
random primed probes generated against
a 16-kb mtDNA template)
Total DNA (Rebelo, Williams et al. 2009
47
2.13 Mitochondrial Biogenesis: Introduction
Mitochondrial biogenesis to generate new mitochondria involves all cellular
processes. There are several transcription factor regulators of mitochondrial biogenesis.
During adaptation to changing environmental situations, inputs from all cellular processes
promote mitochondrial biogenesis, including the synthesis, import, and assembly of
essential mitochondrial components, as well as the replication of mtDNA (Hock and Kralli
2009).
2.14 Mitochondrial DNA Replication
Mitochondrial DNA is replicated and transcribed within mitochondria. In these
semi-autonomous organelles, mtDNA replication takes place independently from the cell
cycle and from nuclear DNA replication. Transcription factors involved in mtDNA biogenesis,
encoded by nuclear DNA, regulate mtDNA replication and mtDNA transcription (Moraes
2001).
The process of DNA replication differs in mitochondria and the nucleus. Replication
of mtDNA depends on nuclear DNA-encoded regulatory proteins, particularly
mtDNA-specific polymerase gamma (POLG). POLG consists of two subunits: the catalytic
subunit, responsible for elongation of the daughter DNA strands, and the accessory subunit,
responsible for primer recognition and proofreading activity (Gray and Wong 1992).
Synthesis starts at one of the multiple origins of replication of the heavy strand (OH) in the
D-loop region using a short RNA primer, which is complementary to the light strand DNA
(Xu and Clayton 1995). The leader sequence is then removed from the remainder of the
light strand transcript by the mitochondrial RNA processing endoribonuclease (RNase MRP)
(Figure 2.6) (Shadel and Clayton 1997), allowing it to form a triple-stranded RNA-DNA
48
hybrid. mtDNA synthesis continues until it reaches the origin of replication of the light strand
(OL), which is situated approximately 10 kbp downstream of the OH. At this time, synthesis
of the light strand starts (Garesse and Vallejo 2001). mtDNA replication is carried out by
POLG, which has 3′–5‘ exonuclease and 5′-deoxyribose phosphate lyase activities,
assisted by the helicase TWINKLE, which unwinds the DNA duplex, and by single-strand
binding proteins (mtSSB), which maintain the DNA in a single strand (Moraes 2001,
Falkenberg, Larsson et al. 2007).
Figure 2.6. Schedmatic of mitochondrial replication. The H-strand and L-strand are
depicted with bold and thin black lines, respectively. Firstly, TFAM (yellow box), TFBM
(green box), and mtRNA polymerase (light blue box) bind to the L-strand promoter to
produce an RNA transcript (red arrow). Secondly, the RNA transcript extends across the
conserved sequence blocks (CSBs) and forms an RNA/DNA hybrid (dashed lines) in a
stable loop configuration. This loop configuration subsequently generates RNA primers
(green arrow). The RNase MRP (dark blue circle) then cleaves the RNA primers from the
loop structure. Lastly, the RNA primer (thin line attached to the circle) and DNA polymerase
49
initiate heavy-strand (OH) DNA replication (heavy arrows with attached circle). This figure
was modified from (Shadel and Clayton 1997).
Two types of mtDNA replication models have been proposed: the asynchronous
strand displacement model (Clayton 1982) and the strand-coupled bidirectional replication
model (Holt, Lorimer et al. 2000). In the asynchronous strand displacement
model,replication of mtDNA is unidirectional and asynchronous, with initiation of replication
at two replication origins (OH and OL) (Clayton 1982, Brown, Cecconi et al. 2005).
Transcription starts from a light-strand promoter (LSP), which forms an R-loop, a triplex
structure containing the transcript RNA. The RNA serves as a primer for the initiation of
H-strand synthesis from the replication origin of H-strand, OH, in the D-loop region. The
D-loop is a DNA triplex structure composed of a displacement loop and a stretch of DNA.
Many of the DNA synthesis events prematurely terminate at about 700 bases, resulting in
the formation of a D-loop structure. This 700-base nascent H-strand is termed 7S DNA or
the D-loop strand. If H-strand synthesis does not terminate prematurely, DNA synthesis
undergoes a complete replication cycle. Displacement of the parental H-strand as
single-stranded DNA continues until synthesis reaches the replication origin of L-strand, OL,
located approximately two-thirds of the genome downstream from OH. Once exposed on a
single-stranded H-strand, the OL region assumes a special stem-loop structure, which
initiates the synthesis of an RNA primer for L-strand DNA synthesis (Clayton 1992, Schmitt
and Clayton 1993, Shadel and Clayton 1997, Kang and Hamasaki 2006, Krishnan, Reeve
et al. 2008). The two new DNA molecules are ligated to form the circular double-stranded
mtDNA. Because the lagging L-strand synthesis is delayed, replication proceeds
asymmetrically. This unique property of mtDNA replication has not been observed with
either mammalian nuclear DNA or bacterial DNA.
50
The second model, proposed by Holt and Jacobs, is a bidirectional replication model
of mtDNA replication in which replication initiates at one replication zone and proceeds
symmetrically in both directions. The model is based on the observation of replication
intermediates in 2D gels (Holt, Lorimer et al. 2000, Yang, bowmaker et al. 2002, Bowmaker,
Yang et al. 2003, Krishnan, Reeve et al. 2008). Both mtDNA strands replicate at the same
time from the same replication origin in the D-loop; this might be an alternative replication
mechanism, with the choice of mechanism determined by variations in transcription factor
binding or in the number of dNTPs present within mitochondria in different cell types. In the
studies describing strand-coupled bidirectional replication, no replication intermediates
harboring long single-stranded DNA, indicative of asymmetric replication, were found. In
contrast, Y-fork intermediates, typical of nuclear DNA replication, were observed. On the
basis of these observations, it was concluded that lagging strand synthesis must occur
simultaneously and symmetrically with eading strand synthesis, as is generally seen in
DNA replication. Because mitochondria contain a large amount of RNA, Holt and Jacobs
proposed that the long single-stranded ‗intermediates‘ observed by Clayton and Shadel
(Shadel and Clayton 1997) were due to degradation of mitochondrial RNA during mtDNA
preparation (Yang, Bowmaker et al. 2002). In addition, they proposed that H-strand
synthesis initiated from a broad 5-kb region located downstream of the D-loop region and
not from OH (Bowmaker, Yang et al. 2003, Yasukawa, Yang et al. 2005, Kang and
Hamasaki 2006, Falkenberg, Larsson et al. 2007).
The exact contributions of the two mechanisms of mtDNA replication remain to be
determined. The balance between the two replication mechanisms is thought to influence
the mtDNA copy number under several physiological and developmental conditions (Holt,
Lorimer et al. 2000). The asynchronous mechanism of mtDNA replication occurs in growing
51
cells with a lower mtDNA synthesis rate that maintain a stable mtDNA copy number for the
daughter cells. In cells in which the mtDNA copy number is increasing, the bidirectional
replication mechanism is predominantly observed. The bidirectional replication mechanism
is also more common in cells transiently depleted of mtDNA (Holt, Lorimer et al. 2000,
Moraes 2001). These findings suggest that the first model is responsible for the
maintenance of mtDNA and the second model is used primarily when mtDNA amplification
is required (Holt, Lorimer et al. 2000).
2.15 Mitochondrial Transcription
The transcription of mtDNA depends upon factors encoded within the nucleus.
These include mitochondrial transcription factor A and B (TFAM, TFB1M and TFB2M),
nuclear respiratory factors 1 and 2 (NRF1 and NRF2), and members of the peroxisome
proliferator-activated receptor gamma coactivator 1 family of regulated coactivators
(including PGC1α and PGC1β) (Scarpulla 2008).
A number of factors regulate mitochondrial biogenesis. For example, DNA
polymerase gamma (POLG) is the catalytic subunit of the mtDNA polymerase and is
responsible for mtDNA replication (Graziewicz, Longley et al. 2006). Mitochondrial
transcription factors (e.g. TFAM, TFB1M, and TFB2M) localize to mitochondria, bind
mtDNA, and stimulate the transcription of mtDNA (Kelly and Scarpulla 2004, Gleyzer,
Vercauteren et al. 2005). TFAM, TFB1M, and TFB2M are induced in response to signals
that promote mitochondrial biogenesis (Figure 2.7) (Scarpulla 2008, Hock and Kralli 2009).
TFAM is a key activator of mtDNA transcription during mitochondrial genome replication.
TFAM binds the two major promoters in the mitochondrial genome: the light-strand and
52
heavy-strand promoters (Larsson, Wang et al. 1998, garstka, Schmitt et al. 2003). There
are other regulators of nuclear-encoded transcription factors, such as nuclear respiratory
factors (NRF1 and NRF2). NRF1 and NRF2 (also known as GA-binding protein, GABP) are
transcription factors that act within the nucleus to regulate genes that encode components
of the mitochondrial respiratory system and many genes involved in the replication of
mtDNA (Evans and Scarpulla 1990, Virbasius, Virbasius et al. 1993). PGC1α also affects
mitochondrial biogenesis by regulating the expression of numerous transcription factors
(e.g. the NRFs) that are involved in the transcription and replication of mtDNA (Puigserver,
Rhee et al. 2003).
By examining the regulatory network that controls mitochondrial function and
biogenesis, many relevant transcription factors, coactivators, and signaling pathways have
been identified. As one would expect, perturbation of the mitochondrial biogenesis
machinery dramatically affects cellular physiology and development. Studies have provided
important insights into the dynamic and coordinated control of nuclear and mitochondrial
genes during development (Table 2.3) (Scarpulla 2008). For example, knockout of NRF1 in
mice results in mtDNA depletion, loss of Δψ, and blastocyst growth defects, resulting in
embryonic lethality between E3.5 and E6.5 (Huo and Scarpulla 2001). Knockout of the
catalytic subunit of POLG in mice results in mtDNA depletion, cytochrome oxidase
deficiency, and embryonic lethality between E7.5 and E8.5 (Hance, Ekstrand et al. 2005).
Knockout of TFAM in mice results in mtDNA depletion and severe respiratory chain
deficiency, with embryonic lethality between E8.5 and E11.5 (Larsson, Wang et al. 1998).
A variety of therapeutic interventions (e.g. dietary supplements, exercise therapy,
and chemicals) have been used to induce mitochondrial biogenesis. For example,
resveratrol induces mitochondrial biogenesis and improves aerobic capacity in mice
53
through histone modifications and activation of PGC1α (Lagouge, Argmann et al. 2006).
Moreover, upregulation of the expression of PGC1 family members stimulates
mitochondrial oxidative phosphorylation (OXPHOS). Caffeine and
5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) enhance PGC1α
expression in the skeletal muscles of rodents in vivo (Irrcher, Ljubicic et al. 2009, McConell,
Ng et al. 2010). Benzafibrate is also a known activator of the PPAR/PGC1α pathway. When
added to cell lines with defective mitochondria, it improves several indicators of
mitochondrial function (e.g. the activity of some mitochondrial enzymes and the levels of
mitochondrial proteins) (Bastin, Aubey et al. 2008).
Figure 2.7 Transcriptional activation from the D-loop promoter region of mtDNA. The
transcription complex is composed of TFAM, TFB (comprising TFB1M and TFB2M), and
mitochondrial RNA polymerase (POLRMT). TFAM unwinds the DNA and forms a complex
with TFB and POLRMT. Together, they initiate transcription from the bidirectional
promoters present within the D-loop (Scarpulla 2008).
54
Table 2.3 Mouse gene knockouts of nuclear-encoded regulators of transcription that have
been implicated in mitochondrial biogenesis (Scarpulla 2008).
Gene
knockout Viability Mitochondrial phenotype Reference
TFAM
(germ line)
Embryonic lethal
between E8.5 and
E11.5
mtDNA depletion, severe
respiratory chain deficiency
(Larsson,
Wang et al.
1998).
Catalytic
subunit of
POLG
Embryonic lethal
between E7.5 and E8.5
mtDNA depletion, cytochrome
oxidase deficiency
(Hance,
Ekstrand et al.
2005)
NRF-1 Embryonic lethal
between E3.5 and E6.5
mtDNA depletion, loss of Δψ,
blastocyst growth defect
(Huo and
Scarpulla
2001)
NRF-2
(GABP) Pre-implantation lethal Not determined
(Ristevski,
O'Leary et al.
2004)
2.16 Mitochondrial Transcription Factor A (TFAM)
Several transcription factors play important roles in mitochondrial biogenesis. One of
them is TFAM, which controls the mtDNA copy number (Ekstrand, Falkenberg et al. 2004)
and stimulates mtDNA transcription in vitro and in vivo (Maniura-Weber, Goffart et al. 2004).
TFAM (also known as mitochondrial transcription factor 1, mtTFA, mtTF1, TCF6, and
TCF6L2) was initially characterized as a monomeric 25-kDa protein comprising a signal
peptide (mitochondrial leading peptide, MLP) and two high-mobility group (HMG) motifs,
which share strong similarities across species (Figure 2.8) (Fisher and Clayton 1988,
Diffley and Stillman 1991, Parisi, Xu et al. 1993, Dairaghi, Shadel et al. 1995, Ohgaki, Kanki
et al. 2007). Abf2p, the yeast homologue, shares similar features but lacks the
55
carboxy-terminal tail, which is essential for transcription activity. If added to the Abf2p
protein, the tail is sufficient to convert the protein into a transcriptional activator (Dairaghi,
Shadel et al. 1995). The Abf2p protein does not have a role in transcription but packages
mtDNA. This has led many to speculate that TFAM might also have DNA-packaging
activities. While there is 81% similarity between the mouse and human TFAM peptide
sequences, human TFAM is a poor activator of mouse mitochondrial transcription in vitro,
despite its strong capacity for non-specific DNA binding (Ekstrand, Falkenberg et al. 2004).
Conversely, mouse TFAM cannot substitute for human TFAM.
Figure 2.8 TFAM alignment across a wide range of species. The image shows the
N-terminal region, HMG boxes 1 and 2, linker region, and the C-terminal tail of TFAM in
human, mouse, rat, and yeast. The amino acid residues conserved in all 4 species are
indicated in red. The amino acid residues conserved in 3 species are indicated in blue. The
dashes indicate sequence gaps due to different amino acid compositions. Abbreviations:
Human, Homo sapiens; Mouse, Mus musculus; Rat, Rattus norvegicus; and Yeast,
Saccharomyces cerevisiae.
Levels of TFAM were altered in a patient suffering from mtDNA depletion and are
greatly decreased in tissue culture cell lines lacking mtDNA (Larsson, Oldfors et al. 1994).
56
The HMG motifs in TFAM recognise specific mtDNA regulatory sequences and bind
upstream of the light- and heavy-strand promoters (OL and OH) of the mitochondrial
genome in a non-specific fashion (Fisher, Lisowsky et al. 1992, Garstka, Schmitt et al.
2003), while facilitating the synthesis of RNA primers that are necessary to initiate mtDNA
replication and transcription (Xu and Clayton 1995).
TFAM has a higher affinity for DNA near the light strand promoter (LSP) than for
DNA near the heavy strand promoter (HSP) and stimulates transcription to a much greater
extent from the LSP than from the HSP in vitro (Fisher and Clayton 1985, Falkenberg,
Gaspari et al. 2002).
Early in vitro transcription studies dissecting the human mitochondrial transcription
machinery showed that, in addition to mitochondrial RNA polymerase, a number of
transcription factors were necessary for specific transcription initiation from mitochondrial
promoters (Fisher and Clayton 1988). Low levels of TFAM are sufficient to recruit other
replication factors (TFBMs and POLRMT) for mtDNA replication in order to increase the
mtDNA copy number (Seidel-Rogol and Shadel 2002, Alam, Kanki et al. 2003). TFAM is
absolutely necessary for mtDNA maintenance during development (Larsson, Wang et al.
1998). Moreover, in addition to its transcription factor activity, TFAM might function to
protect and package mtDNA (Kanki, Ohgaki et al. 2004, Kang, Kim et al. 2007). Its
non-specific DNA binding activity shows that TFAM is involved in the transcriptional
regulation of mtDNA. Studies have shown that the estimated molar ratio between TFAM
and mtDNA in mammals varies from 1 TFAM molecule per 15-20 bp of mitochondrial
genome (Alam, Kanki et al. 2003) to 1 TFAM molecule per 35-50 bp of mitochondrial
genome (Maniura-Weber, Goffart et al. 2004, Cotney, Wang et al. 2007). Additionally,
several studies have shown that mtDNA is organized in a chromatin-like higher-order
57
structure, of which TFAM might be an integral part (Spelbrink, Li et al. 2001, Alam, Kanki et
al. 2003, Garrido, griparic et al. 2003). The studies indicate that TFAM could be a stabilizing
factor in mitochondrial chromatin that plays a role in maintaining chromatin integrity.
2.17 Manipulation of TFAM Expression Levels in Cellular Models
Overexpression of TFAM in a human epithelial carcinoma cell line (HeLa) results in
an increase in mtDNA copy number, whereas knockdown of TFAM results in a gradual
decrease in mtDNA, which corresponds to the gradual decrease in TFAM protein (Kanki,
Ohgaki et al. 2004). Thus, TFAM plays an important role in the replication and transcription
of the mitochondrial genome. Moreover, PGC1α, an upstream regulator of TFAM,
enhances the expression of TFAM and increases levels of mtDNA (Wu, Puigserver et al.
1999). Interestingly, while TFAM is absolutely required for in vitro mitochondrial
transcription, physiologically high levels of TFAM negatively impact mitochondrial
transcription (Parisi, Xu et al. 1993, Falkenberg, Gaspari et al. 2002, Ylikallio, Tyynismaa et
al. 2010).
The amount of TFAM localized to the mitochondria decreases during
spermatogenesis in humans (Larsson, barsh et al. 1997) and mice (Larsson, Garman et al.
1996). This has been proposed as a functional mechanism for specifically reducing mtDNA
copy number in maturing sperm and thereby reducing paternal mtDNA transmission to
future generations. Interestingly, the reduction in mtDNA copy number resulting from
reduced TFAM levels is not mirrored by an increase in mtDNA copy number following
overexpression of TFAM in cultured HEK cells (Maniura-Weber, Goffart et al. 2004). The
majority of TFAM protein is found in the chromatin fraction of PC3 and SKR1 cells.
SiRNA-mediated knockdown of TFAM expression downregulates 596 genes, including
58
baculoviral IAP repeat-containing 5 (BIRC5). Conversely, BIRC5 is upregulated when
TFAM is overexpressed. In contrast, cyclin-dependent kinase inhibitor p21 (CDKN1A) is
upregulated in TFAM-knockdown cells. Chromatin immunoprecipitation was used to
demonstrate that TFAM binds the BIRC5 promoter in the nucleus. Among
TFAM-overexpressing cells,a greater proportion of cells are in the G2/M phase of the cell
cycle; in contrast, among TFAM-knockdown cells, more are in G1 and fewer are in G2/M.
This suggests that TFAM localizes to the nucleus and regulates nuclear genes and cellular
growth in a manner dependent on the level of TFAM expression. The studies demonstrate
that TFAM overexpression has different effects depending on the cell type. Overall, these
results suggest that TFAM is involved in mitochondrial biogenesis.
TFAM-deficient human fibroblasts have low mitochondrial activity (as assessed by
MitoTracker staining), more hydrogen peroxide, more aerobic glycolysis, and greater
L-lactate secretion than control cells (Balliet, Capparelli et al. 2011). Furthermore,
TFAM-deficient fibroblasts promote breast cancer tumor growth in vivo without increasing
tumor angiogenesis. TFAM-deficient fibroblasts increase the mitochondrial activity of
adjacent cancerous epithelial cells. This might mean that the downregulation of TFAM
enhances processes that support tumorigenesis in fibroblasts, including glycolytic
metabolism. In conclusion, the transcription factor TFAM is indispensable for the
maintenance of active mammalian mitochondria. TFAM recognizes sequences upstream of
mitochondrial promoters, and it is absolutely essential for mitochondrial transcription
initiation.
59
2.18 Animal Models of TFAM Knockdown
The necessity of TFAM for the maintenance of mtDNA during embryonic
development has been well documented (Larsson, Wang et al. 1998). Several studies have
described mouse models of TFAM depletion and TFAM knockout. Larsson et al. first
established a TFAM knockout mouse model (Larsson, Wang et al. 1998). The
heterozygous knockout mice exhibited reduced mtDNA copy number in all tissues and
cardiac respiratory chain deficiency, while the homozygous knockout mice did not develop
beyond E10.5 because of severe mtDNA depletion and the absence of electron transport
chain (ETC) function. Homozygous knockout mouse embryos exhibited numerous enlarged
mitochondria with abnormal cristae and severe mtDNA depletion accompanied by the
absence of oxidative phosphorylation. At day E8.5, TFAM-deficient embryos were smaller
than littermate controls; the fomer had delayed neural development, no optic discs, no
recognizable cardiac structures, and indistinct somites (Larsson, Wang et al. 1998). These
data indicate that TFAM is critical for controlling the number of mtDNA copies during
development.
To clarify the role of TFAM during embryonic development, conditional knockout
strategies have been used to eliminate TFAM from specific tissues. When TFAM
expression was specifically disrupted in the heart and muscle of mice, all conditional
tissue-specific TFAM knockouts presented with mitochondrial disease in the affected tissue,
resulting from a loss of mtDNA, loss of mitochondrial transcripts, and severe respiratory
chain deficiency (Wang, Wilhelmsson et al. 1999, Li, Wang et al. 2000, Sorensen, Ekstrand
et al. 2001, Wredenberg, Wibom et al. 2002, hansson, Hance et al. 2004, Silva, Aires et al.
2008). As a result, respiratory chain activity decreased dramatically in heart tissue, which
also exhibited dilated cardiomyopathy and atrioventricular heart conduction blocks (Wang,
60
Silva et al. 2001). Symptoms of heart failure were also identified, such as an increase in
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression and a decrease in
calcium ATPase 2. However, there was no indication of fibrosis, necrosis, or infiltration of
inflammatory cells. Apoptosis was detected in hearts with TFAM knockout: levels of TUNEL
staining (DNA fragmentation), active caspases 3/7, Bax, and Bcl-xL were all elevated.
Increased levels of GPX and SOD2 activated the oxidative defense mechanism. These
studies demonstrate that the loss of TFAM results in increased levels of apoptosis (li, Wang
et al. 2000). When TFAM was eliminated from pancreatic beta cells, the mice developed
diabetes approximately 5 weeks later (Silva, Kohler et al. 2000). The characteristics of the
pancreatic beta cells included severe mtDNA depletion, deficient oxidative phosphorylation,
morphologically abnormal mitochondria, and the inability to release insulin when stimulated
by glucose. Another study showed that TFAM bound to damaged DNA and that binding
increased in the presence of p53, suggesting that TFAM has a direct role in the regulation
of apoptosis (Yoshida, Izumi et al. 2002). TFAM has been shown to bind with high affinity to
cisplatin-damaged DNA, suggesting that TFAM acts as a recruitment factor for the repair
mechanism in mitochondria and the nucleus (Yoshida, Izumi et al. 2002).
Mice with tissue-specific knockout of TFAM in forebrain neurons (MILON mice)
survived for a surprisingly long time (Sorensen, Ekstrand et al. 2001). When the mice were
4 months of age, many neurons had severe respiratory chain deficiencies and displayed
increased sensitivity to excitotoxic stress, but the mice still appeared normal. MILON mice
died from massive synchronized neurodegenerative events in the neocortex and
hippocampus at 5-6 months of age without showing any major upregulation of oxidative
defenses. In cortical neurons that lacked TFAM, apoptosis, DNA fragmentation, and the
61
expression of caspase 3 and 7 increased. In addition, the neurons were more vulnerable to
excitotoxic stress.
TFAM has also been specifically knocked out in dopaminergic (DA) neurons,
generating a transgenic mouse termed ―MitoPark‖ (Galter, Pernold et al. 2010). Two other
studies generated mice in which the TFAM gene was specifically inactivated in DA neurons
(Ekstrand, Terzioglu et al. 2007, Ekstrand and Galter 2009). In early adulthood, MitoPark
mice exhibited a progressive Parkinson‘s disease (PD) phenotype, which slowly impaired
motor function. They developed progressive symptoms that replicated many of the clinical
features of PD, such as bradykinesia, rigidity, and abnormal gait. The behavioral
disturbances correlated with a slow degeneration of the nigro-striatal DA pathway. Primarily,
DA neurons in the substantia nigra pars compacta were lost; at later stages, the ventral
tegmental area neurons also succumbed. Many surviving DA neurons displayed abnormal
morphology and contained alpha-synuclein and ubiquitin-immunoreactive inclusions,
similar to Lewy bodies observed in PD. This phenotype was accompanied by reduced
mtDNA expression and respiratory chain defects within midbrain neurons. Intraneuronal
inclusions also developed, and the cells eventually died (Ekstrand, Terzioglu et al. 2007,
Galter, Pernold et al. 2010). These findings suggest that mitochondrial dysfunction plays a
role in PD. Collectively, these studies demonstrate that TFAM is important for mtDNA
maintenance. Novel strategies to treat mitochondrial disorders and neurological diseases
will likely involve the manipulation of TFAM expression (Keeney, Quigley et al. 2009).
These studies also support the experimental rationale for the manipulation of TFAM
expression in this thesis.
Mice that lack TFAM in Schwann cells (TFAM-SCKO mice) are viable, but with age
they develop sensory and motor deficits, resulting from a progressive peripheral
62
neuropathy (Viader, Golden et al. 2011). The mice exhibit early loss of small, unmyelinated
fibers, followed by demyelination and degeneration of large-calibre axons. Within Schwann
cells of TFAM-SCKO mice, the mtDNA content was severely depleted, and respiratory
chain abnormalities were prevalent. However, the proliferation and survival of Schwann
cells were not affected. Moreover, TFAM has been selectively eliminated from neurons and
glia of the enteric nervous system (ENS), leading to an 80% reduction in total mtDNA
content within the ENS, which induced severe respiratory chain deficiencies. Many
enlarged mitochondria with distorted cristae were found specifically within enteric neurons
and glia. Mitochondrial dysfunction in these cells resulted in high levels of apoptosis and
disruption of gastrointestinal motility (Viader, Wright-Jin et al. 2011). The mice (designated
TFAM-ENSKO) were initially viable, but by 4 weeks of age they were significantly smaller
than littermate controls. At 6–8 weeks of age, TFAM-ENSKO mice developed abdominal
distension with mild phenotypic abnormalities. At 10-12 weeks of age, enteric
neurodegeneration in the proximal small intestine (SI) was profound. Massive dilation of the
proximal SI, accompanied by contraction of the distal SI, was also observed. By 12 weeks
of age, the majority of TFAM-ENSKO animals had died. Interestingly, the ENS is not
uniformly affected in TFAM-ENSKO mice: both regional and neuronal subtype-specific
effects are observed. This regional and subtype-specific variability in susceptibility to
mitochondrial defects results in an imbalance of inhibitory and excitatory neurons that likely
accounts for phenotypes associated with TFAM-ENSKO mice. At 12 weeks of age, the
relative abundance of inhibitory input to the proximal SI results in distension, while the
relative loss of inhibitory input to the distal SI leads to constriction. Small molecules (such
as rotenone and antimycin) were used to inhibit the mitochondrial electron transport chain.
Disrupting mitochondrial respiration induced neurite degeneration within 24 h and
63
subsequently induced enteric neuron apoptosis. Together, these results suggest that
mitochondrial defects stimulate axonal degeneration, which precedes the loss of enteric
neurons. Overall, the study suggests that mitochondria are essential for the survival of
axonal neurons and for normal peripheral nerve function; that mitochondria play a role in
Schwann cells, glial cells in the enteric nervous system (ENS); and that mitochondrial
dysfunction within neurons contributes to human peripheral neuropathies.
The multiple links between TFAM expression or activity and mitochondrial
dysfunction suggest that TFAM is an important driving force in neurodegenerative disorders,
including Parkinson‘s disease, as well as in diabetes and mtDNA depletion disorders, such
as infantile mitochondrial myopathy, fatal childhood myopathy, skeletal muscle and
mitochondrial encephalomyopathy, ocular myopathy, exercise intolerance, and muscle
wasting (Larsson, Oldfors et al. 1994, Poulton, Morten et al. 1994, Siciliano, Mancuso et al.
2000, Tessa, Manca et al. 2000, Choi, Kim et al. 2001, Belin, Bjork et al. 2007).
2.19 Role of the Mitochondrion during Pre-implantation Stages: Introduction
The pre-implantation stages of development include the fertilized zygote; the two-,
four-, eight-, and 16-cell morula (pre-embryo); and the blastocyst. The amount of mtDNA
present during these stages depends on the number of mtDNA copies in the oocyte at
fertilization, because mtDNA replication is initiated only post-implantation. As such, the
mtDNA copy number at fertilization is an important determinant of subsequent
developmental success (Spikings, Alderson et al. 2007), and a certain minimum mtDNA
copy number is required for an oocyte to reach implantation (Spikings, Alderson et al. 2007).
In addition, embryonic mtDNA copy numbers decrease between the 2- and 8-cell stages,
suggesting an active mtDNA degradation process. In mature murine and human oocytes,
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the critical threshold is estimated at 100,000 mtDNA copies (Piko and Taylor 1987, Reynier,
May-Panloup et al. 2001, Santos, El Shourbagy et al. 2006). It has also been shown that
low levels of mtDNA (or mtDNA mutations) in the ovary are associated with ovarian
dysfunction (May-Panloup, Chretien et al. 2005).
2.20 Mitochondrial Bottleneck during Pre-implantation Stages
During the earliest stages of development (between fertilization and implantation),
the mtDNA inherited by an organism appears to pass through a genetic bottleneck. This
implies that there is selective elimination of mitochondrial genomes, which ensures that
only a small number of mtDNA molecules are passed to the next generation.
Very little mitochondrial replication occurs during the pre-implantation stages of
development (Piko and Matsumoto 1976, Ebert, Liem et al. 1988) (Figure 2.9). The mtDNA
copy number within a single primordial germ cell (PGC) is ~1350–3600 at 7.5-13.5 days
post-coitum (Cao, Shitara et al. 2007). To establish the number of mtDNA molecules within
PGCs, Wai et al. (2008) used OCT4-GFP to identify these cells. They measured a low
mtDNA copy number in early PGCs, suggesting that a mitochondrial bottleneck occurs in
the early PGC. Furthermore, the number of mtDNA copies in the female germ line is lower
than in male germ cells, suggesting that the mitochondrial genetic bottleneck is less
important in the male germ line than in the female one. However, mtDNA mutations can
alter sperm motility (Spiropoulous, Tumbull et al. 2002, Cree, Samuels et al. 2008) but do
not affect fertility (Wai, Ao et al. 2010). Moreover, partial knockdown of TFAM in blastocysts
does not affect fertilization or pre-implantation development in mice, but it does alter
post-implantation embryonic viability. In contrast, male fertility is not affected by TFAM
knockdown (Wai, Ao et al. 2010).
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Figure 2.9 Changes in mtDNA copy number per cell during implantation stages. This figure
is modified from (Wai, Ao et al. 2010).
In theory, all mitochondrial genomes within an organism should be identical.
However, because mutations to mtDNA are inherited from the maternal line, wild-type and
mutant mtDNA can coexist (heteroplasmy) (Figure 2.10). Examination of heteroplasmic
mice and humans has revealed that mtDNA genotypes shift extremely rapidly in offspring
and can even return to homoplasmy within a few generations (at least in some progeny)
(Kaneda, Hayashi et al. 1995, Marchington, Hartshorne et al. 1997, Inoue, Nakada et al.
2000, Fan, Waymire et al. 2008). The number of heteroplasmic mutations varies widely
among offspring, suggesting that mutant mtDNA molecules are eliminated during
development.
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Figure 2.10 The possible origin of pathogenic mtDNA deletions. This figure is modified from
(Chinnery, DiMauro et al. 2004).
Overall, these studies indicate that a drastic reduction in mtDNA copy number does
not occur in PGCs. Rather, the genetic bottleneck associated with mtDNA seems to result
from the selective replication of a subpopulation of mitochondrial genomes. This is
accompanied by strong selection against mutations within the germ line. This has been
demonstrated in studies of an ND6 frame-shift mutation (Fan, Waymire et al. 2008) and a
large deletion (Sato, Nakada et al. 2007, Cao, Shitara et al. 2009).
It has been proposed that primordial female germ cells contain a small number of
mtDNA molecules to permit rapid genetic drift toward populations of pure mutant or pure
wild-type mtDNA. A process for selecting mitochondrial mutations has been proposed for
mice and humans (Kaneda, Hayashii et al. 1995, Marchington, Hartshorne et al. 1997, Fan,
Waymire et al. 2008, Inoue, Noda et al. 2010). Although the mechanism by which
deleterious mtDNA mutations are recognized and eliminated remains unclear, selection
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might be based on mitochondrial ‗fitness‘. For example, mitochondria that produce the most
ROS undergo apoptosis or mitochondrial turnover. Understanding the genetic bottleneck
associated with mitochondria will facilitate the development of therapeutic strategies that
block the transmission of mitochondrial disease from mother to progeny and ensure the
safety of hESC-derived therapeutic agents.
2.21 Analysis of Mitochondrial Behavior in Stem Cells
Mitochondrial properties have been used to assess oocyte competence during
oocyte development in various species, including Xenopus laevis, Artemia franciscana,
Equus caballus, Sus scrofa, Mus musculus, and Rattus norvegicus (Vallejo, Lopez et al.
1996, El Shourbagy, Spikings et al. 2006, Facucho-Oliveira, Alderson et al. 2007,
Kameyama, Filion et al. 2007, Mohammadi-Sangcheshmeh, Held et al. 2011). Changes in
mitochondrial morphology from the late morula to the early post-implantation stages of
embryogenesis have been closely examined in monkeys and rats (Shepard, Muffley et al.
2000). During this time, the number of cristae increases, but the cristae appear tubular or
vesicular. New cristae begin to form from blebs of the inner mitochondrial membrane,
resulting in a lamellar appearance. Mitochondrial properties have also been investigated in
a variety of species, using adult monkey stem cells, porcine oocytes, mESCs, hESCs, and
hiPSCs (St John, Ramalho-Santos et al. 2005, Cho, Kwon et al. 2006, St John, Amaral et al.
2006, Spikings, Alderson et al. 2007, Armstrong, Tilgner et al. 2010).
The extent to which the analysis of mitochondria in human pluripotent stem cells
reflects the inner cell mass of the blastocyst remains to be determined, given that hESCs
and iPSCs are usually cultured for long periods of time in vitro before analysis. It is likely
that mitochondrial properties shift in culture as mitochondria adapt to the culture conditions.
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Indeed, hESCs grown in hMSC-CM (conditioned medium derived from human
mesenchymal stem cells) or mTeSR exhibited lower mitochondrial membrane potential (Δψ)
and lower expression of mtDNA-encoded genes and mitochondrial biogenesis-related
genes then cells grown in hFF-CM (conditioned medium derived from human foreskin
fibroblasts) (Ramos-Mejia, Bueno et al. 2012). Moreover, mtDNA and the bioenergetics
profiles of hESCs and hiPSCs can be altered to provide a growth advantage for culture
passage (Maitra, Arking et al. 2005, Prigione, Lichtner et al. 2011).
Prigione and co-workers (2011) have shown that hiPSC lines exhibit varying levels
of homoplasmic and heteroplasmic mtDNA mutations and display a number of point
mutations, but not large-scale rearrangements (e.g. long insertions or deletions) (Prigione,
Lichtner et al. 2011). These studies suggest that the accumulation of mitochondrial
mutations correlates with the level of mitochondrial damage and could affect the
differentiation ability of neural stem cells and hematopoietic stem cells (Norddahl, Pronk et
al. 2011, Wang, Esbensen et al. 2011).
Overall, undifferentiated hESCs and hiPSCs share similar features (Oh, Kim et al.
2005, St John, Ramalho-Santos et al. 2005, Cho, Kwon et al. 2006, Prigione, Lichtner et al.
2011, Panopoulos, Yanes et al. 2012). These features include: (1) high expression of
pluripotency markers, (2) a low mtDNA copy number and a low level of
mitochondrial-encoded genes, (3) a low level of mitochondrial complex activities and
mitochondrial biogenesis, (4) a spherical and immature mitochondrial structure (i.e. poorly
developed cristae) located around the nucleus, (5) restricted oxidative capacity, including a
lower mitochondrial membrane potential and low levels of oxygen consumption, ROS
production, and intracellular ATP generation, (6) a greater reliance on glycolysis (anerobic
metabolism) than on oxidative phosphorylation (aerobic metabolism) for energy generation,
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(7) low levels of intracellular peroxide and mitochondrial superoxide, and (8) defensive
systems, including glutathione S transferase (GSTA3), glutathione peroxidise 2 (GPX2),
and heat shock protein 1 (HSPA1B and HSPB)1 (Armstrong, Tilgner et al. 2010).
hESCs and hiPSCs have a similar metabolic status. They are more reliant on
anaerobic metabolism, which breaks down glucose to form two molecules of pyruvic acid
and generates ATP without oxygen in a manner that is less efficient than ATP production
through oxidative phosphorylation. Studies have shown that a stimulator
(D-fructose-6-phosphate, F6P) of anaerobic metabolism improves the reprogramming from
somatic cells (aerobic metabolism) by shifting to hiPSCs, which have an hESC-like
morphology (anaerobic metabolism), when compared to the effects of an anaerobic
metabolism inhibitor (2-deoxy-D-glucose, 2-DG) (Panopoulos, Yanes et al. 2012). However,
other studies have shown that inhibition of mitochondrial complexes (i.e. using a Complex
III inhibitor such as antimycin) in hESCs with low mitochondrial activities does not alter the
expression of pluripotency markers and that mitochondrial morphology is immature (Varum,
Momcilovic et al. 2009). Moreover, inhibition of oxidative phosphorylation in mitochondria
(i.e. with protonophore carbonyl cyanide m-chlorophenylhydrazone, CCCP, which
depolarizes the mitochondrial membrane potential) does not affect pluripotent gene
expression. The cells still use similar energy sources from anaerobic metabolism (i.e. low in
oxygen consumption and ATP production and more glycolytic flux), have a similar
methylation pattern, and are able to differentiate into three different lineages in mESCs and
hESCs (Mandal, Lindgren et al. 2011). Therefore, glycolysis is required to support the
proliferation of self-renewing ESCs.
hESCs and hiPSCs have a similar metabolic status, although small differences exist.
hESCs have more unsaturated fatty acid metabolites (i.e. ω-6 and ω-3 fatty acids,
70
arachidonic acid, linoleic acid, docosapentaenoic acid, and adrenic acid) than do hiPSCs,
which generate energy by breaking down fatty acids in the beta-oxidation pathway to
acetyl-coA, which then enters the Citric Acid Cycle (TCA) in mitochondria (Panopoulos,
Yanes et al. 2012). This suggests that inhibition of oxidative pathways is important for
maintaining pluripotency in pluripotent stem cells. Early in vivo and in vitro studies
confirmed that the metabolic pathway from lipids is significantly upregulated during oocyte
maturation. There is a higher concentration of unsaturated lipids, such as linoleic acids and
palmitic acid, and a lower concentration of total saturates in human preimplanation
embryos. The concentrations increase upon oocyte differentiation in the course of embryo
development, indicating that energy generation plays an essential role in the maturation of
oocytes (Waterman and Wall 1988). Unsaturated fatty acids induce the expression of
uncoupled proteins (UCPs). UCP2 has been found in the inner mitochondrial membrane; it
is able to uncouple the oxidation of substrates in the electron transport chain from ATP
synthesis, thus dissipating energy as heat and potentially affecting metabolic efficiency
(Pecqueur, Bui et al. 2008). Pecqueur and Bui‘s study demonstrated that undifferentiated
hESCs express more UCP2 than do differentiated hESCs and that the level of UCP2
expression does perturb the metabolic transition or impair hESC differentiation.
Gain-of-function (GOF) UCP2 has several effects: (1) it promotes mitochondrial fatty
acid oxidation but does not cause a major metabolic shift (i.e. there is no significant change
in oxygen, glucose consumption, ATP, or lactate production), (2) it does not affect
three-lineage differentiation, but does alter the quantity and quality of embryonic bodies
(EBs), (3) it reduces cell proliferation and viability, (4) it increases ribose generation from
glucose in undifferentiated hESCs, relative to that in differentiated hESCs, and (5) it
enhances resistance to glucose starvation by inducing autophagy. These findings indicate
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that UCP2 has differential effects on proliferation and survival in undifferentiated and
differentiated hESCs because of differences in the cells‘ metabolism. Expression of GOF
UCP2 might help shunt pyruvate away from oxidation in mitochondria and towards the
pentose phosphate pathway (PPP) by upregulating glycolytic flux. Loss-of-function (LOF)
UCP2 also has several effects: (1) it maintains the expression of pluripotency markers, (2) it
improves cell proliferation, and (3) it facilitates the metabolic transition from fatty acid
oxidation to oxidative metabolism (i.e. reduced lactate and ATP production). This suggests
that loss of UCP2 reduces the metabolic activity of hESCs but does not affect their
pluripotency status. Overall, these findings demonstrate that UCP2 acts as a metabolic
regulator of hESC energy metabolism by preventing mitochondrial glucose oxidation and
facilitating glycolysis via a substrate shunting mechanism in order to maintain the
pluripotency of undifferentiated hESCs or facilitate early differentiation. This suggests that
mitochondria are directly involved in controlling the pluripotency of hESCs.
The mitochondrial membrane potential (Δψ) has been used as a measure of
mitochondrial competence during development. One study observed an increase in
mitochondrial membrane potential, evaluated using JC-1, during the preimplantation stages
(Acton, Jurisicova et al. 2004). This suggests that the mitochondrial membrane potential
progressively changes during the stages of development, reflecting constant fusion and
fission and demands for different amounts of energy. Schieke and co-workers (2008)
demonstrated a correlation between the mitochondrial membrane potential and
differentiation ability in mESCs (Schieke, Ma et al. 2008). mESCs colonies have different
levels of mitochondrial metabolism; those with a higher ΔΨ have a higher overall metabolic
rate. Stem cells with a high Δψ also exhibit high levels of tumor formation, lactate
production, oxygen consumption, and ATP production. In contrast, stem cells with a low Δψ
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are more likely to differentiate into mesodermal lineage cells (Schieke, McCoy et al. 2008).
Under serum-starvation conditions, mESCs with a low Δψ are more likely to be in early G1
phase, whereas those with a higher Δψ are more likely to be in late G1 phase (Schieke,
McCoy et al. 2008). This suggests that changes in the mitochondrial membrane potential
correlate with pluripotency and/or the efficiency of differentiation.
2.22 Mitochondrial Property Changes upon Differentiation
Different tissues have different energy demands, and mitochondrial morphology can
therefore vary widely. The number and structure of mitochondria in skeletal muscle, heart,
and liver, can vary widely; developing heart cells contain larger mitochondria than other
cells, such as skin or neural tubes. However, the protein compositions of mitochondria in
different tissues are similar (Forner, Foster et al. 2006). Mitochondrial morphology in
cardiac myocytes and neurons can change during the aging process, possibly because
oxidative damage leads to mitochondrial damage and a reduction in the mitochondrial
membrane potential (Terman, Dalen et al. 2004).
Several studies have investigated the relationship between mitochondrial properties
and cell-type specific differentiation, such as neurogenesis, osteogenesis, adipogenesis,
and cardiogenesis (Cordeau-Lossouam, Vayssiere et al. 1991, Komarova, Ataullakhanov
et al. 2000, St John, Ramalho-Santos et al. 2005, Chung, Dzeja et al. 2007, Ducluzeau,
Priou et al. 2011). Several methods can be used to stimulate hESC differentiation, such as
the formation of embryoid bodies (St John, Ramalho-Santos et al. 2005, Cho, Kwon et al.
2006). Differentiating hESCs have a greater mitochondrial mass and a higher mtDNA copy
number when compared with undifferentiated hESCs (Cho, Kwon et al. 2006, Chung, Dzeja
73
et al. 2007, Saretzki, Walter et al. 2008). Common changes in mitochondrial properties
include: (1) upregulation of mitochondrial-encoded and mitochondrial biogenesis-related
genes (TFAM and POLG) (Facucho-Oliveira, Alderson et al. 2007, Facucho-Oliveira and St
John 2009, Armstrong, Tilgner et al. 2010), (2) more mature mitochondrial morphology (i.e.
distinct cristae and a dense matrix), (3) upregulated mitochondrial respiratory activity and
increased ATP production (Cho, Kwon et al. 2006, Ramalho-Santos, Varum et al. 2009),
and (4) a metabolic shift from glycolysis (anaerobic metabolism) to oxidative
phosphorylation (aerobic metabolism) in spontaneous or induced-differentiation cells. The
same metabolic shift has been observed during cardiomyogenesis from hESCs (Chung,
Dzeja et al. 2007). During cardiomyogenesis, the mitochondrial morphology changes from
spherical, cristae-poor, and peri-nuclear to elongated with abundant and well-organized
cristae and a cytosolic pattern in cardiomyocytes. The metabolic shift from anaerobic to
aerobic metabolism during cardio-differentiation indicates that the maturation of
mitochondrial properties is essential during cardiomyogenesis.
2.23 Autophagy: Introduction
Autophagy is derived from the Greek words auto, which means self, and phagy,
which means to eat. The autophagic process breaks down cellular components and plays a
particularly important role during periods of starvation and when organelles are damaged.
Autophagy allows cells to maintain a critical balance between the synthesis, degradation,
and recycling of cellular structures. This balance is important during development,
homeostasis, and cell proliferation. Three types of autophagy have been described:
chaperone-mediated, microautophagy, and macroautophagy (Cuervo 2004).
74
Chaperone-mediated autophagy involves the direct translocation of proteins with specific
protein motifs across lysosomal membranes; chaperone proteins that interact with
lysosomal membrane proteins facilitate the translocation. Microautophagy is the direct
intake of cytoplasm by invagination of lysosomal membranes. Macroautophagy, often
referred to simply as autophagy, involves the formation of double membrane vesicles
around organelles and other cytoplasmic components that then fuse with lysosomes
(Levine and Klionsky 2004).
Autophagosomes are double-membrane cytosolic vesicles that encapsulate and
break down cellular components to supply nutrients to the cell (Tolkovsky, Xue et al. 2002).
Autophagy is a major mechanism by which starving cells reallocate nutrients from
non-essential to essential processes. However, insufficient or excessive regulation of
autophagy can cause cell injury (Levine and Yuan 2005). Therefore, appropriate regulation
of autophagy is essential.
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Figure 2.11 Stages of autophagosome formation in selective and non-selective pathways.
Autophagosome formation can be stimulated by the environment or drugs. Following an
inductive signal, autophagosome formation, mitochondrial engulfment, mitochondrial
breakdown, and export of metabolic components occur in both pathways. This figure is
modified from (Yen and Klionsky 2008).
The process of autophagy involves five discrete steps: (1) induction at the
pre-autophagosomal structure (PAS), (2) growth and elongation of the isolation membrane
of autophagosomes, (3) sequestration of cargo within the autophagosome, (4) fusion with
the lysosome/vacuole to form the autolysosome, and (5) degradation of the cellular
components by lysosomal proteases and release of the metabolic products into the
cytoplasm (Kundu and Thompson 2008) (Figure 2.11). The autophagosome membrane
was initially thought to derive from ribosome-free regions of the rough endoplasmic
reticulum (RER). However, other studies have presented evidence supporting that
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autophagosome membranes derive from the RER (Suzuki, Kirisako et al. 2001, McEwan
and Dikic 2010, Rambold and Lippincott-Schwartz 2011). Thus the origin of phagosome
membranes remains to be determined.
The majority of the molecular mechanisms and genes that control autophagy (ATG
genes) were discovered in a yeast model (Ohsumi and Mizushima 2004), but the
mechanism of mitochondrial autophagy (mitophagy) remains to be investigated. Atg
proteins are categorized by function. For example, Uth1p, which resides in the outer
mitochondrial membrane, is involved in autophagic induction (Kissova, Deffieu et al. 2004).
Atg9 functions in membrane protein recycling. The Atg12-Atg8 complex, comprising a pair
of ubiquitin-like proteins, promotes vesicle extension and completion (Yorimitsu and
Klionsky 2004). Studies have shown that Atg3, 5, and 7 participate in quality control and
cell remodelling during T-lymphocyte and adipocyte differentiation (Lafontan 2008, Pua,
guo et al. 2009, Jia and He 2011). During the oocyte to embryo transition, autophagy is
activated within four hours of fertilization. The degradation of maternal proteins and the
regeneration of amino acids and proteins is important for the subsequent differentiation of
the fertilized oocyte (Mizushima and Levine 2010). During the recycling of cytosolic material,
autophagy might provide building blocks for cells to generate ATP. Autophagy also plays a
crucial role in programmed cell death during mammalian morphogenesis (Qu, Zou et al.
2007). Beclin1 and Atg5 might be essential for cavitation, the process by which the
proamniotic cavity is formed during embryonic development. Collectively, these data
suggest that autophagy provides cells with ATP for essential processes and contributes to
tissue sculpting during embryonic development.
The Atg12-Atg5-mediated pathway for the sequestration and formation of
autophagosomes in yeast involves the binding of Atg16, which translocates to the isolation
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membrane and functions as a linker in the formation and elongation of autophagosomes
(Ohsumi and Mizushima 2004, matsushita, Suzuki et al. 2007). Studies suggest that the
yeast protein Atg32 is a transmembrane receptor that directs autophagosome formation to
mitochondria for mitophagy. Atg32 localizes on mitochondria and binds to the adaptor
protein Atg11 during selective types of autophagy; it is then recruited to and imported into
the vacuole along with mitochondria (Kanki and Klionsky 2009, Tolkovsky 2009, Okamoto
and Kondo-Okamoto 2012). This suggests that mitochondria are specifically targeted for
degradation. In some instances, mitochondria have been shown to be ubiquitin-tagged for
autophagy. This is the case for sperm mitochondria that are eliminated after oocyte
fertilization (Sutovsky, Moreno et al. 2000). Although it has been suggested that
mitochondria are selected for autophagy depending on the level of oxidative damage to
their membrane, this hypothesis has not been investigated (Terman and Brunk 2004).
LC3 is a mammalian autophagosomal orthologue of the yeast protein Atg8. In
mammalian cells, Atg4B cleaves newly synthesized pro-LC3 into its cytosolic form, LC3-I,
thus exposing the C-terminal glycine 120. LC3-I is then activated by Atg7p, an E1-like
enzyme, and conjugated to phosphatidylethanolamine (PE) by Atg3p, an E2-like enzyme
(Kabeya, Mizushima et al. 2000, Tanida, Ueno et al. 2004). LC3-II (16 kDa) has slightly
higher mobility than LC3-I (18 kDa) when run on SDS-PAGE gels. LC3-II is selectively
incorporated into forming and newly formed autophagosomes. After the autophagosome
fuses with the lysosome, LC3-II is trapped on the inner surface of the double membrane
autophagosome and degraded when deconjugated from PE by Atg4 (Mizushima,
Yamamoto et al. 2004).
Autophagy has been demonstrated during neuronal, adipocyte, and T-lymphocyte
differentiation. A reduction in autophagic activity abolishes differentiation or downregulates
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differentiation in adipocyte cells, which exhibit decreased expression of differentiation
markers, lipid accumulation, and low levels white adipose, leading to a lean body mass
(Zeng and Zhou 2008, Singh, Xiang et al. 2009, Zhao, Huang et al. 2010). Moreover,
autophagy has been reported to play a crucial role in T-lymphocyte development (Pua, Guo
et al. 2009). The essential role of autophagy in maintaining T-lymphocyte survival and
proliferation has been demonstrated using mouse models with knockout of Atg5 or Beclin 1,
which possess a Bcl-2 homology 3 domain (BH3-only Atg6 in yeast). Loss of Atg5 or Beclin
1 results in T-lymphocyte deficiency and cell death, with disruption of T-cell proliferation
and a reduction in the number of mature total thymocytes and peripheral T and B
lymphocytes (Pua, Guo et al. 2009, Arsov, Adebayo et al. 2011).
Dysregulated autophagy has been linked to neurodegenerative diseases and cancer,
but the mechanisms are not well understood. Autophagy is thought to be essential for
healthy aging and longevity. The target of rapamycin (TOR) pathway, which regulates
autophagy, is one of three highly conserved signaling pathways that affect lifespan (Yen
and Klionsky 2008, Eisenberg, Knauer et al. 2009).
2.24 The Mammalian Target Of Rapamycin Signaling Pathway: (A) How does the
mTOR pathway work?
Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates
autophagy through its downstream effector, S6 kinase (Codogno and Meijer 2005, Kapahi,
Chen et al. 2010). mTOR phosphorylates S6K on T389, leading to S6K activation and the
phosphorylation of its downstream substrate, S6 (Figure 2.12). Generally, mTOR plays a
central role in the ability of cells to sense growth signals, amino acid levels, and energy
levels and make decisions that affect protein translation, cell growth, and proliferation
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(Mizushima and Levine 2010). The mTOR complex comprises mTORC1 and mTORC2,
which share three common subunits (mTOR, mlST8, DEPTOR). In addition, mTORC1 has
two specific proteins (RAPTOR and PRAS40), while mTORC2 has three specific proteins
(RICTOR, mSIN1, and Protor-1) (Shahbazian, Roux et al. 2006, Yang and Guan 2007).
Activation of mTORC1 leading to the phosphorylation of S6 kinase and the inactivation of
the translation repressor 4E-BP suppresses autophagy, activates mitochondrial
metabolism, and inhibits ESC differentiation (Tee and Blenis 2005, Shahbazian, Roux et al.
2006, Yang and Guan 2007). Rapamycin is a macrolide fungicide that inhibits mTOR
signaling by binding to FK506-binding protein 12 (FKBP12) (Hidalgo and Rowinsky 2000).
mTORC1 is more sensitive than mTORC2 to short-term exposure to rapamycin (Kapahi,
Chen et al. 2010).
Figure 2.12. The mTOR signaling pathway. TSC1/2 and Rheb are the major upstream
regulators of mTORC1. Several external signals affect the mTOR signaling pathway, such
as low energy. When the intracellular ATP concentration is low, AMPK becomes active and
subsequently activates the TSC2/1 complex and mTORC1 downstream pathway to
regulate cellular energy levels. This figure is modified from (Yang, Yang et al. 2008).
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The Mammalian Target Of Rapamycin Signaling Pathway: (B) How does the mTOR
pathway respond to and regulate energy demand?
The mTOR signaling pathway also cooperates with the tuberous sclerosis complex
(TSC) to control mitochondrial biogenesis and energy metabolism. TSC-mTOR is an
energy-sensing pathway. In ATP-depleted conditions, TSC2 forms a complex with TSC1
and activates AMP activated kinase (AMPK) to change the intercellular ATP/AMP ratio
(Zhou, Su et al. 2009).
One study has demonstrated that the TSC-mTOR pathway is essential for
maintaining quiescence and ROS production in HSCs (Chen, Liu et al. 2008). Depletion of
TSC1 in HSCs results in a shift from quiescent status to upregulation of mitochondrial
biogenesis and ROS production. Inhibition of mTOR expression by rapamycin leads to a
reduction in the interaction between mTOR, raptor, and the rapamycin-dependent
transcription factor ying-yang1 (YY1), which interacts with PGC1α (Cunningham, Rodgers
et al. 2007). Disruption of YY1 expression decreases mitochondrial-encoded gene
expression and respiration (i.e. oxygen consumption), which is consistent with decreased
PGC1α expression (Cunningham, Rodgers et al. 2007). During the reprogramming of
fibroblasts to cells with an ESC-like morphology by reprogramming factors, cells transition
to a state with a lower mitochondrial number and an immature mitochondrial structure and
shift from aerobic to anaerobic metabolism. It has been suggested that the mTOR signaling
pathway affects mitochondrial dynamics to segregate mitochondria by targeting defective
mitochondria to autophagosomes (mitophagy).
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The Mammalian Target Of Rapamycin Signaling Pathway: (C) How does the mTOR
pathway relate to mitochondrial biogenesis and stem cell behavior?
The mTOR signaling pathway regulates metabolism, cell growth, and ATP/ADP
status in cells. Inhibition of mTOR activity by rapamycin in either HEK 293 cells or mESCs
alters mitochondrial biogenesis to produce effects such as low expression of
mitochondrial-related phosphoproteins, low oxygen consumption, loss of mitochondrial
membrane potential, loss of ATP synthetic capacity, reduced lactate production, slower
rates of proliferation, and disruption of the Δψ (Schieke, Phillips et al. 2006, Schieke,
McCoy et al. 2008). When mESCs were sorted into two populations with low and high
mitochondrial membrane potential, the population with a low mitochondrial membrane
potential was found to reside in early G1 phase, whereas the population with high
mitochondrial membrane potential resided in late G1 phase and exhibited higher mTOR
expression, respiration, and oxidative capacity (Schieke, Ma et al. 2008). Indeed, the
mTOR signaling pathway plays a role in maintaining the self-renewal and pluripotency of
hESCs (Zhou, Su et al. 2009, Easley, Ben-Yehudah et al. 2010).
Inhibition of the mTOR pathway by rapamycin represses mTOR/S6k, which
enhances osteoblastogenesis, and promotes differentiation into mesoderm and endoderm
lineages (Zhou, Su et al. 2009, Lee, Yook et al. 2010). This indicates that activation of the
mTOR pathway can promote differentiation in hESCs. Disruption of S6K-mediated
transcription in the mTORC1 pathway by rapamycin in undifferentiated hESCs does not
alter cell viability or the expression of pluripotency markers, whereas p70S6K
overexpression in hESCs induces hESC differentiation (Easley, Ben-Yehudah et al. 2010).
The level of mTOR/p70S6K might be a crucial factor for maintaining pluripotency status in
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undifferentiated hESCs and for maintaining high levels of protein synthesis during
differentiation.
mTOR signaling allows cells to survive by increasing autophagy, thereby promoting
the degradation of internal proteins and organelles to generate the energy and raw
materials necessary for survival when extracellular nutrients are scarce. mTOR signaling
allows cells to grow when there are enough extracellular nutrients. Therefore, nutrient
depletion or inhibition of TOR with the drug rapamycin induces autophagy.
2.25 Selective Autophagy and Mitophagy
Mitophagy is an autophagic process that selectively degrades damaged
mitochondria (Kim, Lee et al. 2010). The selective degradation of mitochondria by
autophagosomes, called mitophagy, occurs in response to different conditions, such as
nutrient depletion, mitochondrial dysfunction, or red blood cell maturation. During nutrient
depletion or caloric restriction, which can increase longevity, Uth1p (Atg1) activates
autophagosome formation to break down unnecessary organelles for use as building
blocks and nutrients and to increase the removal of oxidatively damaged mitochondria and
mutated mtDNA (Camougrand, Kissova et al. 2004, Kissova, Deffieu et al. 2004, Keeney,
Quigley et al. 2009).
Mitochondrial damage or dysfunction is one of the main inducers of mitophagy.
Increased mitochondrial dysfunction might occur in the post-mitotic cells of aged organisms
that exhibit abnormal mitochondrial morphology (i.e. swelling, low number of cristae, and
destruction of inner membranes) (Ermini 1976, Beregi, Regius et al. 1988, Terman 1995).
Autophagic events decrease during aging; ATP production and respiration are lower in
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aged animals than in younger ones (Terman 1995). There is frequent mitochondrial
turnover in tissues such as the brain, heart, liver, and kidney (Menzies and Gold 1971).
Mitochondria are more vulnerable to oxidative damage and have less robust DNA repair
systems when compared with the nucleus (Yakes and Van Houten 1997, Gredilla, Bohr et
al. 2010). Damage to mtDNA could result in the synthesis of abnormal mitochondrial
proteins or disrupt synthesis altogether, which would exacerbate mitochondrial dysfunction.
In this process, damaged mitochondria (i.e. those with a compromised Δψ or
overproduction of ROS) release signals that promote uptake by autophagosomes,
triggering degradation (Sandoval, Thiagarajan et al. 2008). This provides a mechanism for
mitochondrial quality control and a general, controlled cytoprotective response.
In neurodegenerative diseases, for example, the cell benefits from mitophagy
through the selective removal of damaged and free radical-generating mitochondria.
Parkinson‘s disease (PD) is a common neurodegenerative movement disorder
characterized by tremors, muscular rigidity, bradykinesia, and postural instability
(Bossy-Wetzel, Schwarzenbacher et al. 2004). PD may be induced by an environmental
toxin, 1-methyl-4-phenylpyridinium (MPP+), an inhibitor of Complex I in the electron
transport chain. A precursor of MPP+, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP), can also induce Parkinsonism (Abou-Sleiman, Muqit et al. 2006), as can genetic
inheritance of PD-related genes (e.g. Parkin and PINK1), for example, in
autosomal-recessive juvenile-onset Parkinsonism (ARJP) (Bossy-Wetzel,
Schwarzenbacher et al. 2004, Choi, Woo et al. 2008, Harvey, Wang et al. 2008, Mellick,
Siebert et al. 2009). Studies of the mitochondrial proteins Parkin and PINK1 have
established a direct link between defective mitochondria and mitophagy (McBride 2008,
Narendra, Tanaka et al. 2008, Lutz, Exner et al. 2009). Parkin (encoded by PARK2) is an
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E3 ubiquitin ligase and is the first protein known to bind eight Zn2+ ions. Binding to zinc
through conserved cysteine-rich motifs is essential for structural maintenance, an
observation that explains the significance of the cysteine-based ARJP mutations found
throughout Parkin (Giasson and Lee 2001, Hristova, Beasley et al. 2009). Studies have
shown that loss of Parkin function results in vulnerability to oxidative damage,
dopaminergic neuron loss, and abnormal mitochondrial structure (i.e. swollen, distorted
mitochondrial morphology and fragmented cristae). Loss-of-function mutations in Parkin
decrease ATP production and enhance fission activity, resulting in cell death (Greene,
Whitworth et al. 2003, Whitworth, Theodore et al. 2005, Lutz, Exner et al. 2009). Loss of
Parkin activity also results in the loss of a cellular monitor of dysfunctional mitochondria,
whereas overexpression of Parkin could help to protect against toxicity and mark damaged
mitochondria for degradation (Petrucelli, O‘Farrell et al. 2002, McBride 2008, Narendra,
Tanaka et al. 2008).
Phosphatase and tensin homolog-induced putative kinase 1 (PINK1, also known as
PARK6) is a mitochondrial serine/threonine kinase that functions as a sensor of
mitochondrial or cellular stress and is thought to prevent mitochondrial dysfunction
(Bossy-Wetzel, Schwarzenbacher et al. 2004, McBride 2008). Studies have shown that
loss-of-function mutations in PINK1 result in defects in mitochondrial morphology,
dynamics, and function. This leads to an imbalance in mitochondrial fusion and fission in
Drosophila (Lutz, Exner et al. 2009). Moreover, high levels of PINK1 kinase activity are
important for the translocation of Parkin to mitochondria, whereas overexpression of Parkin
alone results in a cytosolic localization (Yang, Ouyang et al. 2008, Geisler, Holmstrom et al.
2010, Vives-Bauza, Zhou et al. 2010). Studies have demonstrated that Parkin targets
dysfunctional mitochondria and recruits PINK1 to damaged mitochondria with low
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membrane potential to promote autophagic degradation after treatment of cells with CCCP
(Narendra, Jin et al. 2010). The Parkin-PINK1 mechanism depends on voltage-dependent
inhibition in the clearance process. Through the proteolytic cleavage function of PINK1,
cleaved fragments are cleared by the proteasome (Narendra, Jin et al. 2010). Overall,
signals such as an increase in ROS production or a reduction in Δψ recruit the cytosolic
form of Parkin and PINK1 to fragmented mitochondria, which promotes the engulfment of
mitochondria by autophagosomes for degradation (McBride 2008, Narendra, Tanaka et al.
2008).
Mitophagy plays an essential role during differentiation when the elimination of
mitochondria is required, such as during the differentiation of reticulocytes into erythrocytes
(red blood cells). The autophagic gene Nix (also called Bnip3L) (Sandoval, Thiagarajan et
al. 2008) is upregulated in erythroid cells during terminal differentiation (Aerbajinai, Giattina
et al. 2003, Sandoval, Thiagarajan et al. 2008). Nix, a BH3-only member of the Bcl-2 family,
induces mitophagy by disrupting the Δψ(Sandoval, Thiagarajan et al. 2008). Therefore,
autophagy is important for maintaining homeostasis during development, differentiation,
and macromolecule synthesis and degradation (Mizushima and Levine 2010).
Mitophagy is also important for maintaining the number and function of mitochondria
during differentiation and de-differentiation. Knockdown of growth factor erv1-like (Gfer) in
ESCs upregulates dynamin-related protein 1 (DRP1) and results in decreased pluripotency
marker expression, smaller embryoid bodies (EBs), and loss of mitochondrial function (i.e.
mitochondrial membrane potential, cytochrome c expression). Knockdown of Gfer in ESCs
was also associated with fragmentation of mitochondria and an increase in apoptosis that
reduced the number of cells but did not affect differentiation ability (Todd, Damin et all.
2010) By contrast, overexpression of Gfer in ESCs reduced the expression of DRP1 and
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maintained pluripotency marker expression, the size of EBs, mitochondrial function (i.e. the
mitochondrial membrane potential), and mitochondrial morphology (elongated with
well-defined cristae). The findings suggest that Gfer modulates mitochondrial fission activity
to preserve mitochondrial function while maintaining pluripotency status in ESCs. Another
study investigated the response of ESCs to a reduction in mitochondrial membrane
potential induced by treatment with CCCP. Moreover, the study demonstrated that Parkin
was upregulated in clustered perinuclear mitochondria when mitochondrial function was
attenuated by CCCP treatment (Narendra, Tanaka et al. 2008, Vives-Bauza, Zhou et al.
2010, Mandal, Lindgren et al. 2011). These observations may suggest that mitophagy is
the mechanism that controls mitochondrial quality within hESCs. hESCs do not contain a
large number of mitochondria during the inner cell mass-derivation stage. In addition,
hiPSCs exposed to a reprogramming factor were reprogrammed and transformed into cells
with an hESC-like morphology from somatic cells. iPSCs and hESCs have similar
metabolic and mitochondrial properties.
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Chapter 3: Methods and Procedures
3.1 Cell Lines and Cell Cultures
All reagents were purchased from Invitrogen (Carlsbad, CA, USA) unless otherwise
stated. Human embryonic stem cell (hESC) lines were cultured on human foreskin
fibroblasts (hFFi) as a feeder and Matrigel with DMEM/F12 + 20% knockout serum
replacement (KOSR) + 1x L-glutamine solution (L-glut) + 1x non-essential amino acids
(NEAA) or conditioned medium (CM). HEK 293FT cells were cultured in DMEM with 10%
FBS for 2 to 3 days between passages. The hESC lines (Mel1, hES3, or H9) were cultured
using inactivated human foreskin fibroblasts as a feeder in DMEM/F12 with L-glut
supplemented with 20% KOSR, 1x NEAA, and 90 µM β-mercaptoethanol. For short-term
experimental purposes, hESC lines were maintained on Matrigel with conditioned medium,
which was collected from inactivated hFFi, and hESC culture medium. Cells were
passaged after the addition of 4 mg/ml collagenase I for 5 minutes and washed twice with
DMEM/F12. Cells were gently scraped to break colonies into clumps. Cell clumps were
centrifuged at 1,000 rpm for 3 minutes, resuspended in fresh KOSR medium, and gently
transferred to a new dish coated with hFFi cells at 7,000 cells/cm2 or Matrigel.
3.2 Harvesting and Passaging Cell Cultures:
(A) Human fibroblasts
At 80% confluence, cells were observed under microscope before splitting. Before
undergoing the splitting process, the culture medium, 1x PBS (wash solution) and TrypLE
(to detach cells from surface) were pre-warmed to 37°C and the culture medium in the cell
culture flask was suctioned off and discarded. Cells were then washed once with 1x PBS to
remove all remnants of the culture medium. The 1x PBS was removed through suction and
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TrypLE was added and incubated with the cells at 37°C for 10 minutes until the cells were
detached from the surface (checked by microscope). A fixed volume of additional fresh
culture medium was added to attenuate the activity of TrypLE by dilution. Cells were split
1:4 or 1:6 and transferred to a new flask together with the growth medium with the contents
described using: 500 µl for each well for a 24-well plate, 1.5 ml for each well in a 6-well
plate, 4 ml total for small flasks and 15 ml total for medium flaks. After each splitting, the
flask was marked with a new passage number.
(B) Human embryonic stem cells (hESCs) co-cultured with human feeder (hFFi) or
hESCs grown on Matrigel
At 80% confluence, cells were observed under microscope before splitting. Before
splitting, the culture medium, 1x PBS (wash solution) and Collagenase V (1 mg/ml) (to
detach cells from surface) were pre-warmed to 37°C and then the original culture medium
on the cell culture flask was suctioned off and discarded. Cells were then washed for one
time with 1x PBS to remove all the remnant of the culture medium. The 1x PBS was
removed and Collagenase V was added to the cells at 37°C for 3 minutes until the cells
were detached from the surface (checked by microscope). hESCs co-cultured with hFFi
with hESCs‘ culture medium (DMEM/F12 with L-Glut supplemented with 20% KOSR, 1x
NEAA and 90 µM β-Mercaptoethanol). hESCs grown on Matrigel with conditioned medium
(CM) (McElroy and Reijo Pera 2008). The preparation of CM was collected the hESCs‘
culture medium from the hFFi (20,000 cells/cm2) every day for one week and filtered with
0.22μm filter after adding an additional 4 ng/ml bFGF. The Matrigel-coated plates were
prepared by slowly thaw the BD Matrigel hESC-qualified Matrix (BD, #354277) stock
solution on ice at 4°C to avoid gel formation and dilute the Matrigel stock solution (1:15) in
cold DMEM/F12 medium. Incubate the Matrigel-coated plates for 1-2 hour(s) at room
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temperature or overnight at 4°C. A fixed volume of fresh culture medium was added to stop
the activity of Collagenase V. Cells were usually split into 1:4 or 1:6 and transferred to a
new growth flask together with the growth medium with the contents described as follows:
500 µl for each well of a 24-well plate, 1.5 ml for each well of a 6-well plate, 4 ml total for
small flasks and 15 ml total for medium flaks.
(C) Differentiation of hES Cell Cultures: Spontaneous-Differentiation
Spontaneous Differentiation: hESCs were either differentiated spontaneously or in
response to retinoic acid (RA, 10 µM; Sigma-Aldrich) and were allowed to precede for up
to 14 days in T-25 culture flasks (Figure 4.1). Retinoic acid (RA, 10 µM; Sigma-Aldrich)
induced experiment was applied from the method used in this published reference paper
(Parsons, Teng et al. 2011). For these experiments, cells were cultured in DMEM/F12 with
knockout serum replacement medium lacking bFGF and β-mercaptoethanol. DMSO
vehicle was added to the differentiation medium to serve as a negative control. Cells were
atmosphere with 5% CO2.
(D) Differentiation of hES Cell Cultures: Lineage-Specific Differentiation
Methods for the lineage-specific-differentiation of hESCs into ectoderm, endoderm,
mesendoderm, and cardiomyocytes (Fig 4.1).
Directed Ectoderm Differentiation: The differentiation of hESCs into neural lineages
was performed using the method of Briggs and Wolvetang et al (Briggs, Sun et al. 2013).
Briefly, hESCs were attached and transferred to a Matrigel-coated dish and cultured in
N2B7 media supplemented with 10 μM SB431542 (Calbiochem) and 5 μM Dorsomorphin
(DM, also known as Compound C, Sigma-Aldrich) for 6 days. After 6 days, the medium
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was replaced with N2B7 medium supplemented with 5 μM Dorsomorphin and cells were
cultured for additional 6 days to form neurospheres.
Definitive Endoderm Differentiation: The method for the differentiation of hESCs into
definitive endoderm was according to (D‘Amour, Agulnickk et al. 2005). Once the cells were
70-80% confluent, differentiation was initiated by washing with PBS on Matrigel-coated
dishes and then incubating in RPMI (Invitrogen) containing 100 ng/ml activin A and 25
ng/ml WNT3A (R&D) without FBS (Sigma-Aldrich) for 24 hours. On the second day, cells
were then treated with RPMI containing 0.2% FBS and 100 ng/ml activin A for 24 hours. For
the third day, cells were maintained in RPMI containing 0.5% fetal bovine serum and 100
ng/ml activin A. Cultured cells were characterized after 3 days of differentiation.
Mesendoderm and Cardiomyocyte Differentiation: The differentiation of hESCs into
mesendoderm was performed according to reference (Hudson, Titmarsh et al. 2012).
hESCs cultures were obtained from ASCC StemCore in a conditioned medium (CM) and
expanded by daily exchanging the medium until the colonies were ~90% confluent in the
centre of the wells. CM was then replaced with a basal medium comprised of RPMI 1640
medium supplemented with 2% B27 supplement and 1% penicillin/streptomycin (RPMI
B27). For primitive streak-like cell induction, 20 ng/ml bone morphogenetic protein-4
(BMP-4) and 6 ng/ml activin A were added to the basal medium. The basal medium was
then exchanged for every 3 days. The medium was then replaced by RPMI B27 only and
exchanged for every 2 days until day 16. After this time, cells were cultured in
un-supplemented RPMI B27 and the medium was exchanged for every 2 days. The
difference between the medium contents for mesendoderm differentiation and
cardiomyocyte differentiation is IWP-4, a small molecule inhibitor of Wnt production
(Hudson, Titmarsh et al. 2012). After 3 days of differentiation, the medium for
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cardiomyocyte differentiation RPMI B27 with 5 µM IWP-4 was added and the medium was
exchanged every 2 days until day 15.
3.3 Cryopreservation of Cell Cultures
Generally, the cells used in this thesis were cryopreserved by re-suspension the
cells in a cryopreservation medium (1 ml) followed by a stepwise freezing (~1°C/minute)
with a ―Mr Frosty‖ (Nalgene) in a -80°C freezer. After 3-5 days, the cryopreserved cells
were then storred in a liquid nitrogen tank until use.
Thawing medium (Fibroblasts: fibroblast DMEM culture medium; hESCs:
conditioned medium) was pre-warmed to 37°C first. The cell suspension of thawed cells (at
37°C) was transferred to a 4 ml of the thawing medium.
Additional thawing medium was then added drop by drop while agitating
continuously. After 500 μl of thawed medium was added the cell suspension was
centrifuged at room temperature at 1000 rpm for 5 minutes. The supernatant was discarded
and the cell pellet was resuspended in another 500 μl thawed medium and again
centrifuged as the condition described above. Lastly, the cell pellet was resuspended in
1000 μl culture medium and seeded in a 6-well plate.
3.4 Karyotypic Analysis
Cells were seeded in T25 flasks and cultured according to the conditions described
above. When reaching confluency, cells were treated with trypsin for 5 to 10 minutes at
37°C before harvest. The trypsin was then neutralized by the culture medium for further
karyotype analysis. Analysis of G-banded metaphase chromosomes was performed on
samples produced according to standard procedures from the School of Medicine at the
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University of Queensland. Karyotypes were assessed at the cytogenetic laboratory of the
School of Medicine at the University of Queensland.
3.5 Preparation of mtDNA-Depleted Primary Human Fibroblasts
Human fibroblast cells were grown in a complete DMEM medium (Dulbecco‘s
modified Eagle‘s medium supplemented with 50 μg/ml uridine (Sigma-Aldrich), 10% FBS, 1
mM sodium pyruvate (Gibco) and 1% penicillin/streptomycin (P/S) in the presence of 100
ng/ml Ethidium bromide (EtBr) or 100 μg/ml chloramphenicol (CAP) in a high-glucose
medium (4500 mg/ml) (Gibco). EtBr and CAP were kept in the media for 6 weeks. After 6
weeks cells were fed with complete DMEM media without EtBr or CAP for another 2 weeks.
Cells were kept between 30% and 80% confluence with excess fresh medium to ensure an
exponential growth. Cells were harvested followed by the extraction of genomic DNA and
RNA.
3.6 Production of Lentiviral Supernatants and Infection of Target Cells
Lentiviral vectors were produced in HEK293T cells, purchased from Invitrogen.
HEK293T cells are human embryonic kidney cell lines with a eukaryotic expression plasmid
encoding the temperature-sensitive mutation derived from the SV40 large T-antigen
(DuBridge, Tang et al. 1987). In this study, they served as a cell line for packaging and
producing of lentiviral particles. The produced viruses were tested in vitro by transducing to
different cell lines (Appendix Tables 2 and 10) and analysed by the methods described
below.
Infectious lentiviral particles are generally produced via three main lentiviral genes
that are encoded in the retroviral genome. Those include: (1) Gag: encoding a polyprotein
required for viral capsid assembly, (2) Pol: encoding four main enzymes, such as protease,
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integrase, reverse transcriptase and RNase H required for reverse transcription of the RNA
genome and finally (3) Env: encoding the necessary viral glycoproteins required for
budding at cellular membranes (Llano, Gaznick et al. 2009). Since pENTR-EF1α-TFAM
and pLKO-TFAM shRNAs lack all of the aforementioned viral structural genes, two
additional plasmids encoding the lentiviral structural genes were introduced.
Stocks of all viral plasmids were prepared using Nucleospin Plasmid
Midi-Preparation Kit and stock concentrations were adjusted to 1 μg/μl. Production of viral
particles was conducted over a period of five days according to the protocol elaborated as
follows:
Day 1: Plating the viral packaging cell line. The HEK293T cells were used as the
standard cell line for packaging and producing infectious viral particles. HEK293T
cells were plated at a density of 3x106 cells/flask in a T25 flask in 3 ml aliquots of
medium and were left to grow overnight at 37°C with atmosphere containing 5%
CO2.
Day 2: Transfection of HEK293T cells with viral plasmids: HEK293T cells were
transfected by all employed viral plasmids in the presence of the standard
Lipofectamin plus transfection reagent (Invitrogen). This is an organic polymer with a
high cationic-charge potential to facilitate binding to anionic surface receptors for
promoting the process of endocytosis (Kichler, Remy et al. 1995). To this end,
phenol red-free DMEM/F12 medium without serum, but with other additives were
used to prepare the transfection solution. The transfection solution mixed with
plasmids in each T25 flask was set up as shown in the Appendix Table 10.
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Meanwhile, all viral plasmids (i.e. viral plasmid or pCMV 8.2, VSVG together with the
Gag/Pol/Env plasmids) were also prepared in phenol red-free DMEM/F12 medium
containing DMEM/F12 medium (500 µl), plus reagent (4 µl), Lipofectamin transfection
regent (LTX, 8 µl). Total plasmid concentration per T25 flask contains 1 μg of viral plasmid,
2 μg of pCMV 8.2 (encoding for Env gene) and 1 μg of VSVG (encoding each for Gag and
Pol gene). The prepared transfection solution was then rapidly added to the viral plasmid
mixture and vortexes. The mixture was incubated at room temperature for 30 minutes. The
medium was then removed from the HEK293T cells and replaced with 4 ml of phenol
red-free DMEMF12 medium with 5% KSR. After 20 minutes, the 2 ml transfection mixture
was dropwisely added to the dish and incubated overnight at 37°C. Next day, the HEK293T
medium was replacd by KSR medium.
Day 5: Harvesting of viral particles and storage: HEK293T supernatants containing
infectious viral particles were filter-sterilized using a 0.45 um nitrocellulose filter. The
aliquots of the sterilized cell-free supernatants were stored at -80°C use.
3.7 Transduction (Infection) of Target Cells
In general, transduction or infection refers to the entry of the virus into the host cell
with certain exogeneous gene sequences being integrated into the cellular genome of the
host. Briefly, target cells (hESCs) were plated in 6-well cell culture plates at a density of
2x105 to 5x105 cells per well in 1 ml aliquots of medium and allowed to grow overnight. For
the second day, the medium of the target cells was removed and replaced with 2 ml of
undiluted viral stock solution containing 8 μg/ml polybrene and 10 ng/ml bFGF. For the
negative controls (i.e. un-transduced cells), viral particles were omitted. Cells were
incubated for additional 6 to 8 hours. Transduction was ultimately stopped by incubating
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cells in their own medium for 72 hours prior to the harvesting step and also further growing
the cells. To downgrade the transduced cells from Biosafety Level 2 to Biosafety Level 1,
culture medium of cultured transduced cells were harvested after 72 hours of transduction
and filter-sterilized using 0.45μm nitrocellulose filters. The cell-free filtrate was later
subjected to further analysis to assess the absence of virus particle production.
3.8 Cloning of Human EF1α-LC3-GFP and TFAM cDNA
EF1α-LC3-GFP construct, encoding LC3 (GenBank ID: NM_032514), was obtained
from Dr. Prescott as a kindly gift to establish hES3-LC-GFP single cells. The feature of
LC3-GFP plasmid was generated by Tim Tra using the following methods. The
LC3-GFP-pENTRTM-directional plasmid (10 fmole) and the pENTR-EF1α plasmid (10 fmole)
were simultaneously cloned into the pLenti6/R4R2/V5-DEST vector using LR Clonase II
Plus (MultiSite Gateway® Technology; Invitorgen) to generate the expression plasmid of
pLenti6-EF1α-TFAM. After proteinase K digestion, the plasmid was used to transform into
One Shot ® Stbl3 competent E.coli cells (Invitrogen) for plasmid purification and sequence
confirmation. Expression of the LC3-GFP was driven by the EF1α promoter using a
replication-incompetent lentivirus and the LC3-GFP lentivirus plasmid was amplified by
One Shot ® Stbl3 competent E.coli cells (Invitrogen).
The cDNA encoding human TFAM (GenBank: NM_003201) was obtained by
RT-PCR from human embryonic stem cells (hESCs) using TFAM-forward primer (5‘-
CACCATGGCGTTTCTCCGAAG-3‘) and TFAM-reverse primer (5‘-
TTAACACTCCTCAGCACCATATTTTCGTTG-3‘). TFAM cDNA was amplified by PCR.
PCR reactions were performed by using 0.5 μl pFusion high fidelity hot start enzyme with
10 μl of 5x pFusion buffer, 1.5 μl of 10mM dNTP, 1 μl of 10 μM of each pair of forward and
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reverse primers, 1 μl of 200 ng/μl cDNA and sterile water added to 50 μl total volume. PCR
reaction conditions were set as follows: 98°C for 1 minute, followed by 30 cycles at 98°C for
10 seconds, 56°C for 20 seconds, 72°C for 30 seconds and a final extension at 72°C, for 10
minutes before cooling down at 4°C. The TFAM amplification product was 741 base pairs
(bps) and was analysed by running in 1.5% agarose gel electrophoresis followed by UV
visualization. The product was purified with using the Agarose Gel DNA Extraction Kit
(Roche) and cloned into the pENTRTM-directional entry vector (Invitrogen). The sequence
of the TFAM cDNA was confirmed by PCR amplification and sequencing. EF1α (human
elongation factor-1α) promoter plasmid and EF1α-GFP expression plasmid were kindly
provided by members in the Wolvetang group. The TFAM-pENTRTM-directional plasmid (10
fmole) and the pENTR-EF1α plasmid (10 fmole) were simultaneously cloned into the
pLenti6/R4R2/V5-DEST vector using LR Clonase II Plus (MultiSite Gateway® Technology;
Invitorgen) to generate the pLenti6-EF1α-TFAM expression plasmid. After proteinase K
digestion, the recombination reaction was transformed into One Shot ® stbl3 competent
E.coli cells (Invitrogen) for further plasmid purification and sequence confirmation.
Expression of the TFAM cDNA was driven by the EF1α promoter while using a
replication-incompetent lentivirus.
3.9 shRNA Cloning
shRNA constructs were specific to TFAM to knockdown TFAM (termed TFAMi
shRNA) and a non-specific, control construct (termed ―targetless‖). Anneal 5 μl of 1 μg/μl of
double-stranded oligo (dsOligo) with 5 μl of 10x NEB buffer 2 and 35 μl of sterile water by
heating the sense and antisense sequence at 95°C for 4 minutes. Next step is the slow
cooling down at room temperature for 5 to 10 minutes. Serial dilution of dsOligo to 200 ng
mixed with T4 DNA ligase (New England Biolab) to further perform the ligation reaction with
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50 ng of pLKO-1 TRC-cloning vector (Addgene) digested by AgeI and EcoRI in order to
insert dsOligo at 95°C, overnight. Transform 5 μl of the ligation mix into XL1Blue competent
E. coli. The reaction mixture was then incubated in ice for 30 minutes and then heat
shocked for 30 seconds and then incubated in ice for another 30 minutes. Afterwards, the
reaction mixture was mixed with 1 ml of LB without antibiotics for 1 hour of shaking 50 to
100 μl of the mixure containing 100 μg/ml ampicillin was applied on LB agar plates.
PCR was used to screen all the possible positive clones which may have inserted
dsOligo. The thermal cycling profile of the PCR reaction was: 95°C for 4 minutes, 36 cycles
at 95°C for 30 minutes, 60°C for 30 minutes and 72°C for 30 minutes and finally extending
at 72°C for 5 minutes. Next, the plasmid DNA was prepared using the plasmid extraction kit
(Macherey) according to the manufacturer‘s instructions. Different clones were inoculated
in 5 ml LB with 100 μg/ml ampicillin overnight at 37°C shaking in the speed of 180 rpm.
They were then transferred to 100 ml liquid with 100 μg/ml ampicillin for 12 hours at 37°C,
180 rpm. The pellets were collected by centrifuging the bacteria supernatant in 50 ml tubes
(BD). A small amount of plasmid DNA with sequencing primer was prepared to sequence
the vector. Lastly, different clones TFAM shRNAs and pLKO-targetless were generated.
pLKO-targetless vector served as used as the negative control.
3.10 Plasmid DNA Purification:
(A) Minipreparation
E. coli cells from glycerol stocks (freeze cultures) containing plasmid were streaked
on an LBamp100 agar plate and grown overnight in a 37°C incubator. Stationary phase
cultures in LBamp100 agar were started by single colonies and incubated overnight while
vigorously shaking at 37°C. 4 ml of overnight bacterial culture was harvested by
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centrifugation at 200 rpm for 30 seconds at room temperature. The supernatant was
discarded. Plasmids were purified from stored E. coli culture pellets using NucleoSpin
Plasmid Column (Macherey), buffer A1 (resuspension buffer), A2 (lysis buffer), A3
(neutralization buffer), AW and A4 (wash buffers) and AE (eluation buffer). The protocol
followed the manufacturer‘s instructions. The pellets obtained in the previous step were
added to 250 µl of buffer A1 and inverted and vortexed until the pellet were completely
dissolved. Solution was then added 250 µl of buffer A2 and inverted before being incubated
at room temperature for 5 minutes. 300 µl of Buffer A3 was added and the solution was
inverted for several times to mix well before it was poured into a filter attached to a
collecting tube. The lysate was then transferred to the column and centrifuged at 200 rpm
for 5 minutes. The flow through was then removed and 500 µl of buffer AW preheated to
50°C was added to column and centrifuged at 200 rpm for 1 minute. 600 µl of buffer A4 was
added to the column and centrifuged at 200 rpm for 1 minute. The flow through was
discarded and re-centrifuged at 200 rpm for 2 minutes. The plasmid DNA was eluted with
50 µl of buffer AE at room temperature for 1 minute and centrifuged at 200 rpm for 1 minute.
The concentration of DNA was measured by Nano-drop ND-1000 (NanoDrop) before all the
samples were stored at -20°C seal.
(B) Midipreparation
E. coli cells from glycerol stocks (freeze cultures) containing plasmid were streaked
on an LBamp100 agar plate and grown overnight in a 37°C incubator. Stationary phase
cultures in LBamp100 agar were started by single colonies and incubated overnight by
vigorously shaking (200 rpm) it at 37°C. 1 ml of overnight bacterial culture was added into
each flask (100 ml of LBamp100). The flasks were then vigorously shaken at 180 rpm in a
37°C incubator overnight. The bacterial cells were harvested by centrifugation at 200 rpm
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for 15 minutes at 4°C. The bacterial pellet left on the bottom after centrifugation was stored
at -20°C until being used. The supernatant was discarded. Plasmids were purified from
stored E. coli culture pellets using NucleoBond Xtra Column (Macherey), buffer RES-EF
(resuspension buffer), LYS-EF (lysis buffer), NEU-EF (neutralization buffer), FIL-EF (QIA
filter wash buffer), EQU-EF (equilibration buffer), ENDO-EF (wash buffer) and ELU-EF
(elution buffer). The protocol followed the manufacturer‘s instructions. The pellet obtained
in the previous steps was added into 8 ml of buffer RES-EF and inverted and vortexed until
the pellet was completely dissolved. Solution was then added to the 8 ml of buffer LYS-EF
and inverted before it was incubated at room temperature for 5 minutes. Buffer NEU-EF
was pre-chilled before use: 8 ml was added and the solution was inverted for several times
to mix before it was poured onto a filter attached to a collecting tube. The lysate was then
incubated for 10 minutes on ice and centrifuged at 200 rpm for 10 minutes. 15 ml of buffer
EQU-EF was added to column and the column was slowly emptied by gravity flow. The
supernant of mixture was then added into the column and was allowed to bind to the resin
coated on the column by gravity flow. Several washing steps were conducted and the
following contents would elaborate on these steps. 5 ml of FIL-EF, 35 ml of ENDO-EF and
15 ml of WASH-EF were applied to wash the column slowly through gravity flow. The
plasmid DNA was eluted with 5 ml of buffer ELU-EF. The DNA was then precipitated by
adding 3.5 ml room temperature isopropanol. The solution was mixed and centrifuged at
200 rpm for 60 minutes at 4°C. Supernatant was carefully decanted and discarded. The
pellet obtained after centrifugation was washed with 2 ml 70% room temperature ethanol
and centrifuged at 200 rpm for 60 minutes at 4°C. Supernatant was carefully decanted and
discarded. The pellet was left to air-dry for 10 minutes before being re-dissolved in
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autoclaved water. Concentration of DNA was measured with Nano-drop ND-1000
(NanoDrop) before samples were stored at -20°C until being used.
3.11 Sequencing of cDNA
The dideoxynucleotide sequencing method was applied to confirm the sequence of
the interested gene. Sanger sequencing was performed using BigDye Terminator reagents
(Applied Biosystems). The sequencing primer was used at a concentration of 10 nM. The
sequencing product was purified by plasmid DNA before sending to the Australian Genome
Research Facility (AGRF) for analysis performed by the chromatograph. The
chromatograph was viewed using Chrome software.
3.12 Genomic DNA Isolation
Total genomic DNA was isolated from cultured human cells using a commercial kit
(Qiagen) and the concentration was determined by NanoDrop 1000 spectrophotometer
(Thermo Scientific). Mitochondrial DNA (mtDNA) copy number was determined by real-time
PCR using an ABI Prism 7500Fast thermocycler (Applied Biosystems). The -ACTIN gene
was simultaneously amplified as a normalization control standard. Amplification was
performed as the procedures described as follows: 95°C for 1 minute, 40 cycles at 95°C for
30 seconds, 52°C for 30 seconds; and final dissociation to generate a melting curve for
verification of amplification product specifically. During PCR amplification, the concentration
of amplified products was measured continuously by measuring fluorescence emission
strength. Then gDNAs were used for quantitative real-time PCR (qPCR) analysis.
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3.13 RNA Extraction & cDNA Generation
Total RNAs were extracted using Qiagen RNA extraction kit (Qiagen) followed by
DNase treatment (Qiagen) and the same RNA sample was used for quantitation by
real-time PCR, or cDNA cloning. The cDNAs were then synthesized as follows: in a single
reaction, 1 μg of RNA quantified by NanoDrop, (NanoDrop 1000, Thermo Scientific) was
mixed with RNase-free water and 1 μl of oligo d(T)20 (500 μg/ml) (Geneworks) to reach the
final volume of 12 μl, then it was incubated at 65°C for 5 minutes. After incubation, the
reaction was placed on ice before the addition of 4 μl of 5x first-strand buffer and 2 μl of 0.1
M DTT (Invitrogen) and then incubated at 37°C for 2 minutes. Finally, 1 μl (200 U) of
M-MLV reverse transcriptase (Invitrogen) was added into the mixture and was incubated at
37°C for 50 minutes followed by an extension period for 15 minutes at 70°C.The cDNA
generated in previous steps was then used for quantitative real-time PCR (qPCR) analysis.
3.14 Quantitative Real-time PCR (qPCR)
The expression of gene was quantitated using the ABI 7500 real-time PCR detection
system (Applied Biosystem) which uses fluorescein as an internal passive reference dye for
the purpose of normalization of well-to-well optical variation. PCR amplifications were
carried out in a total volume of 10 μl, containing 5 μl of 2x SYBR Green supermix (Applied
Biosystem), 0.2 μl of 10 μM primers and 0.2 μl of cDNA samples mixed with sterile water.
The reaction was initiated at 95°C for 2 minutes, followed by 40 three-step amplification
cycles consisting of 30 seconds denaturation step at 95°C, 30 seconds annealing step at
52°C and 30 seconds extension step at 72°C. The final dissociation stage was run to
generate a melting curve for verifying the specificity of amplification. Real-time qPCR was
monitored and analysed by the ABI 7500 fast optical system software (Applied Biosystem).
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Single primer in each sample was carried out in triplicate. Relative mRNA levels were
calculated using the comparative Ct method (Applied Biosystem instruction manual) and
presented by their ratio to their biological controls. The fold change in expression of each
target mRNA relative to -ACTIN was calculated as 2-Δ(ΔCt), where ΔCt=Ctgenes-ΔCt -ACTIN.
-ACTIN transcript levels were confirmed to correlate well with total RNA amounts and
therefore were used for normalization throughout. All primer pairs used were confirmed to
approximately double the amount of product within one cycle and to yield a single product
of the predicted sizes. Primers were designed by Seqtool
(http://seqtool.sdsc.edu/CGI/BW.cgi#) or (http://pga.mgh.harvard.edu/primerbank/). At the
end of PCR reactions, the specificity of the PCR products was confirmed after observing
the presence of a single peak while using 2% gel electrophoresis stained with EtBr and
visualized under UV light. For the calculation of PCR efficiencies, standard curves were
generated from assays made with serial dilutions of cDNA from experiments with triplicate,
which enabled the determination of the Ct values and PCR efficiencies of each individual
assay and the calculation of the correlation relation between them. The PCR efficiency (E)
was calculated according to the equation E= (10 [1/-slope] -1). The E is around 110% to 90%
when the slope falls between -3.1 to -3.6. The slope was calculated by plotting the dilution
fold of cDNA versus their Ct values
(http://www.stratagene.com/techtoolbox/calc/qpcr_slope_eff.aspx).
3.15 Cell-Staining protocols
(A) Live-cell staining protocols
The live-cell staining experiments were separated into two parts: (1) JC-1 staining:
Cells were grown in wells with normal culture media. The culture medium was removed
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and cells were washed with PBS twice for 5 minutes at room temperature. The
mitochondrial membrane potential was determined by using 5, 5‘, 6, 6‘-tetrachloro-1, 1‘, 3,
3‘-tetraethylbenzimidazolylcarbocyanine iodide (JC-1, Sigma-Aldrich). JC-1 is a
fluorescent dye with cationic and lipophilic properties that accumulates in mitochondria
depending on differential membrane potentials. High membrane potential is indicated as
JC-1 aggregates and low membrane potential is indicated as JC-1 monomers. Studies
have shown that JC-1 is the better fluorescent dye compared with other (Salvioli, Ardizzoni
et al. 1997). JC-1 monomers and JC-1 aggregates were emitted at 488 nm (green) and
561 nm (red), respectively. Next, a probe was diluted to the optimal concentration for
working in a buffer (200 µl for 1 well of a chamber slide) and incubated for 30 minutes at
37˚C. Then the cells were washed with PBS twice for 5 minutes at room temperature. The
DNA was counterstained with 10 μM of Hoechst 33258 in PBS and the cells were covered
and incubated for 5 minutes. The cells were then washed with PBS twice for 5 minutes at
room temperature. Images were acquired via a 10x objective lens with a fluorescence
microscope. Each sample was compared with a control. (2) hES-LC3-GFP cells staining
with MitoTracker staining: Cells were grown in wells with normal culture media. The culture
medium was removed and washed with PBS twice for 5 minutes at room temperature.
Mitochondria were marked and determined using MitoTracker Deep Red dye (Invitrogen).
Next, the probe was diluted in a blocking buffer and incubated at 37°C for 30 minutes. The
cells were washed with PBS twice for 5 minutes at room temperature. Images were
acquired through a 10x objective lens with a fluorescence microscope.
(B) Timelapse Image Analysis of Mitophagy in Live hESCs
The hES-LC3-GFP cell line was manually cut and seeded on Matrigel in 6 well
plates and grown to ~80% confluency for 2 days prior to initiating the experiments of
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phase-contrast timelapse microscopy. The required agent (200 nM rapamycin, 50 µM
CCCP, 100 ng/ml EtBr; Sigma-Aldrich) was added to the medium and cells were
incubated under 5% CO2
induced by withdrawing bFGF for 7 or 14 days, or induced by RA (Sigma-Aldrich). The
cells were washed with PBS, stained with 100 nM MitoTracker Red (Invitrogen) in PBS for
30 minutes at 37°C and then washed again. Finally, the hESC medium was added. The
experiment was continued and each one of the serial images was acquired for every 1
710). Each sample was compared with undifferentiated hESCs. Images were analysed
using Zen 2008 Light Edition software (Carl Zeiss).
(C) Immuno-Staining Protocols: Fixed-cell staining
The cells were grown on coverslips with normal culture media. The culture medium
was removed and washed with PBS twice for 5 minutes at room temperature. The cells
were then fixed with 4% paraformaldehyde solution in PBS for 15 to 20 minutes. They
were removed from the fixation solution and washed with PBS twice for 5 minutes at room
temperature. (When staining nuclear markers, cells require permeabilization while
incubated in 0.1% Triton X-100 in PBS/TBS for 20 minutes at room temperature.) The cells
were washed with PBS twice for 5 minutes at room temperature. The non-specific binding
was blocked by using a sufficient volume of 4% goat serum solution in PBS (200 µl for 1
well of a chamber slide). The cells were incubated for 30 minutes at room temperature.
Next, the primary antibodies were diluted to working concentration with blocking buffer and
added to the cells for 1 to 2 hour(s) at room temperature or 4oC overnight (Appendix Table
9). Then the cells were washed with PBS twice for 5 minutes at room temperature. Next,
the secondary antibodies were diluted to working concentration with blocking buffer before
105
added to the cells for 1 hour at room temperature. The cells were then washed with PBS
twice for 5 minutes at room temperature. The DNA was counterstained with 1 μg/ml of
DAPI in PBS and the cells were covered and incubated for 5 minutes. Then the cells were
washed with PBS twice for 5 minutes at room temperature. The slides were mounted using
Vectashield or ProLong Gold following the manufacturers‘ instructions. Images were
acquired via a 10x objective lens with a fluorescence microscope. Each sample was
compared with the control.
(D) Immuno-Staining hES-LC3-GFP Fixed-Cell Staining
hES-LC3-GFP cells were grown on coverslips in normal culture medium or
differentiation medium. They were fixed with 2% (w/v) paraformaldehyde at room
temperature for 20 minutes and then stained with antibodies to pluripotency markers
POU5F1 (Santa-Cruz Biotechnology) and tumor recognition antigens (TRA-1-60.
Millipore). After incubation with the primary antibody, the cells were washed three times in
phosphate-buffered saline (PBS) and incubated with the appropriate secondary antibody
(1:2,000, goat anti-mouse Alexa 488 (or goat anti-rabbit Alexa 568)-conjugated IgG,
Invitrogen) at room temperature for 30 minutes (Appendix Table 9). The cells were then
washed with PBS and mounted in mounting medium containing DAPI (Sigma-Aldrich).
microscope (Zeiss LSM 710).
3.16 Transmission Electron Microscopy (TEM)
Electron photomicrographs for mitochondrial morphology of hESCs were prepared
as described previously (Graham and Orenstein 2007). In brief, cell pellets were washed
in PBS buffer and fixed with the final concentration of 2.5% glutaraldehyde in PB buffer for
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15 minutes. Pellets were then post-fixed in osmium tetroxide (OsO4) and dehydrated
through a series of acetone solutions and embedded in epoxy. Thin sections were stained
with uranyl acetate (Fahmy‘s) and lead citrate. Images were acquired by using a
JEOL1010B microscope (Japanese Electron Optics Laboratories, Peabody, MA).
3.17 Flow Cytometry General Protocol
Cells were stained using the following general protocol. The specific protocol is
indicated in the relevant chapter. All flow cytometry analysis was conducted by either an
LSRII or ACCURI flow cytometer (BD Biosciences) with (generally) 10,000 counts taken
from per sample. Negative control was indicated by staining with secondary antibodies for
all analysis to distinguish the signal form noisy background and non-specific binding.
Fluorescence gates for positive cells were set to give false positive rates of <1%.
(A) Live-imaging Flow Cytometry and Data Acquisition
The hES-LC3-GFP cell line was seeded on Matrigel in T-25 flasks and grown for 2
days until ~80% confluent. The desired agent (200 nM rapamycin, 50 µM CCCP, 100
ng/ml EtBr; Sigma-Aldrich) was added to the medium and cells were incubated under 5%
CO2 at 37°C for 2 hours. Differentiation was either spontaneously induced by withdrawing
bFGF for 7 or 14 days or induced by RA (Sigma-Aldrich). The cells were then trypsinized
using TrypLE (Invitrogen) and collected into 15-ml centrifuge tubes. The cells were
washed with PBS, stained with 100 nM MitoTracker Deep Red (Invitrogen) for 30 minutes
at 37°C and then washed again. They were analysed by flow cytometry at 488 nm with
simultaneous fluorescence image captured at 633 nm using the ImageStream system
(Amnis Corporation, Seattle, WA). Data represents the results of triplicate analyses. For all
samples, populations were gated for both GFP and single cells and processed further by
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spot counting. Once cells of interest were gated, the feature of spot count analysis could
be performed. Spot counts of LC3 punctuate of representative cells were then applied to
the default mask for autophagosome analysis using ImageStream Data Exploration and
Analysis Software (IDEAS) v3.0.
(B) Flow Cytometry for Live-cell Staining
Cells were harvested in a single cell-suspension using trypsin. The suspension was
gently and repeatedly suctioned in and out to break up the aggregates. The cells were
centrifuged for 5 minutes at 1000 rpm and supernatant was removed and resuspended in 1
ml blocking buffer. The non-specific binding was blocked by sufficient volume of 500 µl of
4% goat serum solution in PBS and incubating for 15 minutes at room temperature. Next,
the primary antibodies were diluted in blocking buffer and incubated on ice for 20 minutes
(Appendix Table 9). For controls, only secondary antibodies or vehicles were used. The
cells were washed by centrifuging them for 3 minutes at 1000 rpm and the supernatant was
removed and resuspended in 1 ml of PBS twice for 3 minutes at room temperature. Next,
the secondary antibodies were diluted to working concentration in a blocking buffer and
incubated on ice for 20 minutes (Appendix Table 9). The cells were washed by centrifuging
them for 3 minutes at 1000 rpm and the supernatant was removed and resuspended in 250
µl of PBS for twice. The cells were transferred to flow cytometry tubes on ice and analysis
was conducted by the flow cytometer. Each sample would be compared with a control.
Each sample, representing 10,000 events of counting, was analysed using appropriate
software and measured in triplicate. The flow cytometry data are all the averages of
triplicate experiments.
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(C) Flow Cytometry for Mitochondrial Staining of Living Cells
The cells were harvested in a single cell-suspension approach using trypsin. The
suspension was gently and repeatedly suctioned in and out to break up aggregates. The
cells were centrifuged for 5 minutes at 1000 rpm and the supernatant was removed and
resuspended in a 1 ml working solution of probes such as MitoTracker Deep Red dyes
(Invitrogen) or 5, 5‘, 6, 6‘-tetrachloro-1, 1‘, 3, 3‘-tetraethylbenzimidazolylcarbocyanine
iodide (JC-1, Sigma-Aldrich) (Appendix Table 9). JC-1 is a fluorescent dye with cationic and
lipophilic properties and it accumulates in mitochondria depending on differential
membrane potentials. A high membrane potential is indicated by JC-1 aggregates and a
low membrane potential is indicated by JC-1 monomers. Studies have shown that JC-1 is
the better fluorescent dye compared with others (Salvioli, Ardizzoni et al. 1997). JC-1
monomers and JC-1 aggregates were emitted at 488 nm (green) and 561 nm (red),
respectively. Next, the probe was diluted into blocking buffer and incubated at 37°C for 30
minutes. The cells were washed by centrifuging them for 3 minutes at 1000 rpm and the
supernatant was removed and resuspended in 250 µl of PBS for twice. The cells were
transferred to flow cytometry tubes on ice and analysis was conducted by the flow
cytometer. Each sample was compared with the corresponding control. Each sample,
representing 10,000 events of counting, was analysed by using appropriate software and
measured in triplicate. The flow cytometry data are all the averages of triplicate
experiments.
(D) Flow Cytometry of Fixed-Cells
The cells were harvested in a single cell-suspension approach using trypsin. The
suspension was gently and repeatedly suctioned in and out to break up aggregates. The
109
cells were centrifuged for 5 minutes at 1000 rpm and the supernatant was removed and
resuspended in 1 ml 2% paraformaldehyde solution in PBS for 20 to 30 minutes with
occasional mixing motion. The cells were washed by centrifuging them for 5 minutes at
1000 rpm. The supernatant was removed and resuspended in the blocking buffer. (This
was repeated to wash away excess paraformaldehyde if necessary). The non-specific
binding was blocked by using sufficient volume of 500 µl of 4% goat serum solution in PBS
and incubating for 15 minutes at room temperature. (If it is staining nuclear markers, the
cells require permeabilisation by being incubated in 0.1% Triton X-100 in PBS incubates for
5 minutes at room temperature.) The cells were washed by centrifuging them for 3 minutes
at 1000 rpm and the supernatant was removed and resuspended in 1 ml of PBS twice. Next,
the primary antibodies were diluted in a blocking buffer for 1 hour at room temperature or
4oC overnight. For controls, only secondary antibodies or vehicles were used. The cells
were washed by centrifuging them for 3 minutes at 1000 rpm and the supernatant was
removed and resuspended in 1 ml of PBS twice for 30 minutes at room temperature. Next,
the secondary antibodies were diluted to appropriate working concentration in a blocking
buffer before added to the cells for 30 minutes at room temperature. The cells were washed
by centrifuging them for 3 minutes at 1000 rpm and the supernatant was removed and
resuspended in 250 µl of PBS for twice. The cells were transferred to flow cytometry tubes
for further analysis undergone by the flow cytometer. Each sample was compared with its
corresponding control. Each sample, representing 10,000 events of counting, was analysed
using proper software and measured in triplicate. The flow cytometry data are all the
averages of triplicate experiments.
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3.18 Lactate Production and Glucose Consumption
In order to assess lactate production and glucose consumption, 1x105 cells were
seeded for 24 hours before medium collection and filtration through a 0.22um filter.
Triplicate samples were then analysed by using YSI Glucose/Lactate Analyser (YSI 2300
STAT Plus). L-lactate and glucose consumption per cell were calculated according to the
standard curve and converting their concentration (mg/l) to mM by dividing with their
molecular weight (i.e. molecular weight of L-lactate and glucose are 90.08 and 180.15588,
respectively). Cell medium only and normalised control are all subtracted from the
calculation values.
3.19 Detection of Intracellular Total and Oxidised Glutathione (GSH) in Cells
For quantification of the ratio between oxidized glutathione (GSSG) and reduced
glutathione (GSH), cells were trypsined and collected in tubes for counting processes. Cells
were lyzed by adding 100 ul of 5% metaphosphoricacid (MPA, 2.5 g/50 ml) and sonciated
for 10 seconds with 10 seconds of break on ice. Supernatant of each sample was placed on
ice after centrifugation. Aliquots of each sample and standards were then incubated with 40
mM of 4-vinylpyridine at room temperature for 1 hour. Kinetic monitor of optical density was
carried out following the addition of reaction mix that contained 5,5‘-dithiobis-2-nitrobenzoic
acid. The sulfhydryl group of GSH reacted with DTNB (5,5‘-dithiobis-2-nitrobenzoic acid,
Ellman‘s reagent) to produce a yellow colored 5-thio-2-nitrobenzoic acid (TNB) that would
absorbed the fluorescence at 405 nm. The rate of TNB production was directly proportional
to the concentration of glutathione in the samples. Changes in the OD 405 nm values per
minute were converted into the total oxidized glutathione concentrations using the GSH
standard curve (Griffith 1980) (Assay designs).
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3.20 Intracellular ATP and ADP Concentrations of Mitochondrial Depleted Cells
ATP was measured using bioluminescence based on the procedures of the
luciferin-luciferase reaction (Higashi, Isomoto et al. 1985). The reaction was catalyzed by
luciferase and would emit 450 nm light. Experiments were carried out in triplicate in opaque
384-well tissue culture opaque plates. First, trypsinised cells were collected in tubes and
resuspended in 200 µl PBS. Then is sonicated twice for 10 seconds for each plate and
placed on ice. Cells were centrifuged before beginning the ATP/ADP (EnzyLight ADP/ATP
Ratio Assay Kit, Bioassay Systems, Hayward, CA) and Bradford protein concentration
(Sigma-Aldrich) assays. In 384-well plates, 10 µl of samples or standard, respectively
(Sigma-Aldrich) in the 24.4 µl of the reaction mixture, includes 0.3 µl substrate and 0.3 µl
ATP enzyme. Samples were mixed and incubated at room temperature for 10 minutes. The
ATP signal was allowed to undergo spontaneous decay for 10 minutes in order to reach a
steady state and then measured by a SpectraMax M5 microplate spectrophotometer
(Molecular Devices, Australia) to express by values of relative light units (RLU). After 10
minutes, the ADP in the well was converted to ATP after addition of 2.5 µl ADP converting
reagent. The 2.25 µl of the reaction mixture consisted of 0.25 µl of ADP enzyme. After
10-minute incubation, RLUs were recorded and converted to the amount of ADP with an
ADP standard curve. Under optimal conditions and at concentrations of ATP and ADP from
0 to 30 µM, the RLUs were directly proportional to the amount of ATP and ADP presents
(data not shown). The mean value and standard deviation (SD) of the triplicate samples
were recorded by the SoftMaxPro software and then calculated and normalised to the
protein concentration by Excel (Microsoft).
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3.21 Statistical Analysis
Data are presented in the form of the mean ± standard deviation (SD) or standard
error (SEM). The statistical differences between two groups were tested by Student‘s t-test.
The statistical differences among more than two groups were tested by two way ANOVA
or student t-test. P value less than 0.05 was considered to show significant differences in
all experiments for multiple comparisons (n=3). Analysis of the data was done using
GraphPad software (version 5, University of Queensland). The plotting of the figures was
done using SigmaPlot or Excel software (Version 8.0, Chicago, IL, U.S.A).
113
Chapter 4: Results
Objective 1: Mitochondrial-Encoded and Mitochondrial Biogenesis-Related Gene
Expression During Lineage-Specific Differentiation of Human Embryonic Stem
Cells
4.1 Phase I: Mitochondrial Changes in Differentiating hESCs
For this study, hESCs (cell line hES3) were allowed to differentiate spontaneously by
withdrawing the pluripotency maintenance factor (basic fibroblast growth factor, bFGF)
from the culture medium or by adding retinoic acid (RA) at the time points shown in Figure
4.1. Differentiation of ESCs is characterized by the loss of pluripotency and upregulation of
lineage-specific transcription factors. Here, cell differentiation was monitored using the
surface antigen marker (or a pluripotency marker) TRA-1-60 (Table 4.1). Flow cytometric
analysis showed a significant decrease in the expression of TRA-1-60 in cells treated with
RA or upon withdrawal of bFGF (Figure 4.2), indicating that the cells underwent
differentiation (Figure S1).
Since it is known that mitochondria provide the energy required for cell differentiation,
their mass is expected to increase during differentiation. We determined the mitochondrial
mass by flow cytometry using fluorescent MitoTracker Red (a dye specific to mitochondria)
and demonstrated a significant increase in mitochondrial mass at day 7, but not at day 14,
during the differentiation (P < 0.05 compared with control, Figure 4.2 and Table 4.1). These
data indicated that there is a temporary increase in mitochondrial mass in the early stage of
spontaneous differentiation (day 7) and a decrease at day 14 of spontaneous
differentiation.
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Quantitative real-time PCR (qPCR) was then conducted to determine whether and how
the differentiation process (spontaneous and RA-treated) affected mitochondrial gene
expression. We showed that POU5F1 (a pluripotency gene) expression was dramatically
reduced after 7–14 days of spontaneous differentiation and after 3 days of RA treatment
(Figure 4.3A and Table 4.2). Interestingly, mtDNA copy number, determined using D-loop
as a template for PCR, was elevated at day 7, but decreased at day 14 (Figure 4.3 and
Table 4.2). Such profile changes, however, were not seen in cells treated with RA for 3
days, suggesting that the underlying mechanisms of action of these two commonly used
techniques to induce ESC differentiation are quite different.
To test whether the profile changes in mitochondrial genes are dependent on cell line
during differentiation, we next examined two different stem cell lines, hES3 and Mel 1, to
determine whether the effect of differentiation on mitochondrial gene expression could be
duplicated between these two cell lines.
First, we observed that the expression patterns between the mitochondrial genes, ND1
and CYTB, were quite similar in differentiated hES3 cells (Figure 4.3A and Table 4.2). At
day 7 of spontaneous differentiation, the expression level of ND1 remained the same as
that of the control. The level of CYTB in the hES3 cells was 1.36-fold higher (P < 0.05) than
that in the control (Figure 4.3A and Table 4.2). At day 14, the expression of ND1 and CYTB
was almost identical to that of controls. At day 3 of RA treatment, ND1 expression was not
significantly different to that in the control, while CYTB expression showed a 4-fold
reduction (P < 0.05). From our results, the mitochondrial responses of hESCs to
spontaneous differentiation differed widely depending on the hESC lines being used.
115
At day 7 of spontaneous differentiation, ND1 expression in the cell line Mel 1
decreased 2.5-fold relative to that in the control (P < 0.05), but this reduction was not
observed for CYTB (Figure 4.3A and Table 4.2). At day 14 of spontaneous differentiation,
the expression of ND1 was not significantly different from the control, but there was a 3-fold
increase in the expression of CYTB (P < 0.05). At day 3 of RA treatment, the expression of
ND1 was reduced by 10-fold (P < 0.05) in contrast to that of CYTB, which showed a 10-fold
increase (P < 0.05) compared with the control. Our data indicated that mitochondrial gene
expressions may be varied in response to differentiation as well as to the differentiation
method employed. There are differences between cell lines and between differentiation
protocols in terms of mtDNA gene expression.
To examine the genes that regulate mitochondrial biogenesis, expression levels of
TFAM, NRF1, and POLG were assessed (Figure 4.3A and Table 4.2). In the hES3 cell line,
at day 7 of spontaneous differentiation, the expression levels of TFAM, NRF1, and POLG
were reduced by 1.3-, 7-, and 6-fold (P < 0.05), respectively, relative to that in the control
(Figure 4.3A and Table 4.2). At day 14, TFAM expression was reduced by 4-fold (P < 0.05),
and NRF1 and POLG expression reduced by 8- and 7-fold, respectively (P < 0.05). At day 3
of RA treatment, TFAM expression was reduced by 2-fold (P < 0.05), whereas expression
of NRF1 and POLG was not significantly reduced. Despite the inconsistent pattern of
mitochondrial-encoded gene expression, the expression of transcription factors involved in
mitochondrial biogenesis seemed to be steadily decreasing
In Mel 1, the expression level of TFAM remained the same as that of control at day 7 of
spontaneous differentiation, while the expressions of NRF1 and POLG showed a 10-fold
decrease (P < 0.05) (Figure 4.3A and Table 4.2). At day 14, TFAM and NRF1 expression
remained the same as that in the control, but POLG expression increased by 1.1-fold (P <
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0.05). After three days of RA treatment, TFAM and NRF1 expression remained the same
as that in the control and POLG expression decreased by 3-fold (P < 0.05). Collectively
these data indicated that the expression of known mitochondrial biogenesis-related
transcriptional factors do not necessarily coincide with changes in mitochondrial mass,
mitochondrial copy number, and mitochondrial-encoded genes.
PGC1α is a master regulator in mitochondrial biogenesis and energy expenditure, and
in most cell types, it facilitates the differentiation process by positively regulating the
expression of TFAM and NRF1 (Goffart and Wiesner 2003, Scarpulla 2011). To determine
the role of PGC1α expression during differentiation, hES3 and Mel 1 cell lines were initially
used for the study. In hES3 cells, the expression level of PGC1α showed 14.3- and 7-fold
increase at days 7 and 14, respectively (Figure 4.3A and Table 4.2). After RA treatment,
the expression of PGC1α was markedly elevated by 7.5-fold. In Mel 1 cells, the expression
level of PGC1α mRNA was also significantly elevated at days 7 and 14 during spontaneous
differentiation (1.7- and 2.7-fold, respectively) relative to that in the control (Figure 4.3A and
Table 4.2). At day 3 of RA treatment, the expression level of PGC1α was slightly elevated
(1.2-fold) relative to that in the control. Collectively, these data indicated that mitochondrial
mass and PGC1α expression were transiently elevated during the early stages of hESC
differentiation, suggesting that differentiation is highly dependent on energy consumption.
Surprisingly, PGC1α affects mitochondrial biogenesis in hESCs without influencing TFAM
levels. Thus, PGC1α expression may be an early indicator of mitochondrial biogenesis
during cell differentiation.
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Morphological Changes in Mitochondria during Differentiation
Changes in mitochondrial morphology during cell differentiation remain mostly
unknown thus far. By TEM, we examined the morphology of hESCs (Mel 1) during
differentiation. Figure 4.3B reveals that mitochondria exhibited an immature morphology
with only few cristae in the cells before differentiation (Figure 4.3B). They possessed more
mature cristae with an orthodox morphology at day 14 of the differentiation process (Figure
4.3C), indicating that mitochondria mature morphologically during spontaneous
differentiation.
We hypothesized that an increase in cellular energy may drive an initial differentiation
of mitochondria or alternatively, it may simply be a consequence of differentiation. One of
the interesting findings in murine erythroleukemia cell differentiation is that the
mitochondrial membrane potential appears to be important in the control of the
differentiation (Levenson, Macara et al. 1982). This finding shows that mitochondria may
regulate cytoplasmic calcium levels at the initiation of differentiation. Therefore, we
attempted to measure the mitochondrial membrane potential in order to address the
hypothesis mentioned above. In general, an accumulation of ratiometric fluorescent dye,
JC-1, within the mitochondrial matrix can be used to assess membrane potential. When the
potential is high, JC-1 aggregates and emits fluorescence in red. In contrast, under low
potential, JC-1 forms monomers and emits fluorescence in green (Salvioli, Ardizzoni et al.
1997, Mandal, Lindgren et al. 2011). In undifferentiated hESCs, we showed that the
mitochondrial membrane potential was low and generally clustered near the center of
hESC colonies (Figure 4.3D). Interestingly, after spontaneous differentiation for 14 days,
cells exhibited a marked increase in membrane potential on the edges of the hESC
colonies with low membrane potential scattered throughout the colonies (Figure 4.3E). The
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observation suggests that mitochondria become depolarized during the differentiation
process. The mitochondrial transmembrane potential of a subset of differentiated hESCs
has been changed during the differentiation. It is clear that additional work will be required
before achieving a complete understanding of the changes in membrane potential.
Phase II: Mitochondrial Biogenesis during Lineage-Specific Differentiation
During early embryogenesis, stem cells are exposed to different growth factor regimes
to induce the growth of primitive mesendodermal, endodermal, and ectodermal precursor
cells (Thomson, Itskovitz-Eldor et al. 1998). In phase II of the study, we attempted to
evaluate whether mitochondrial-encoded and mitochondrial biogenesis-related
transcription factors are correlated with various between differentiations into the three germ
layers. A general experimental design was carried out with the time points given in Figure
4.1. Expression of cell type-specific genes was used to track the lineage differentiation as
previously described (Reubinoff, Pera et al. 2000).
Ectoderm, endoderm, and mesoderm differentiation were monitored by determining
the expression of POU5F (pluripotency), paired box 6 (PAX6, ectoderm), SRY-box 17
(SOX17, endoderm), insulin-like growth factor 2 (IGF2, endoderm), GATA binding protein 4
(GATA4, endoderm), mix paired-like homeobox (MIXL1, mesoderm), and brachyury (BRY
mesoderm).
Ectoderm Differentiation
When stem cells differentiate into ectoderm cells, they are initially processed from
neural progenitor cells into the neurosphere. The expression of neural-specific markers
during ectoderm differentiation has been previously reported (Briggs, Sun et al. 2013).
Figure 4.4 shows that the mRNA level of PAX6 (neuronal biomarker) was dramatically
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elevated after 8 and 12 days of differentiation by 517- and 357-fold, respectively, relative to
the control (Figure 4.4 and Table 4.3), a result which is consistent with our previous finding
(Briggs, Sun et al. 2013). The expression of a pluripotency marker, POU5F1, was
attenuated at days 8 and 12 by 4.5- and 16.7-fold, respectively. Mitochondrial-encoded
ND5 and COX3 were transcriptionally upregulated 12-fold and 13-fold, respectively, at day
8 of neural induction (P < 0.05), but returned to baseline levels 4 days after neurosphere
formation. The expression of genes involved in mitochondrial biogenesis, TFAM and NRF1,
were not significantly affected. However, POLG was upregulated 1.6-fold (P < 0.05) at day
8, but its expression returned to baseline levels 4 days after neurosphere formation.
PGC1α mRNA was upregulated 7.8-fold and 2-fold at days 8 and 12, respectively, during
neuronal differentiation. Because gene expression increased and subsequently returned to
baseline, it is suggested that mitochondrial-encoded genes and mitochondrial
biogenesis-related transcription factors are transiently increased during neuronal
differentiation.
Endoderm Differentiation
To induce endoderm differentiation, activin A and bone morphogenetic protein 4
(BMP4) were added into hESCs culture (D‘Amour, Agulnick et al. 2005). We showed that
the mRNA level of SOX17, a member of the SOX family of transcription factors that
contains a high-mobility DNA binding domain, was markedly elevated from days 1 to 3,
whereas expression of POU5F1, a marker for pluripotency, decreased over time (Figure
4.5 and Table 4.4). The expression of genes encoded from mtDNA was either slightly
attenuated (ND5) or remained unchanged (COX3). This finding suggests that SOX17, a
transcription factor encoded by nuclear DNA, plays a role in or its expression may serve as
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a pivotal marker for endoderm differentiation. The expression of mitochondrial
biogenesis-related genes does not change during definitive endoderm differentiation.
Mesendoderm Differentiation
To investigate the changes in gene expression during mesoderm differentiation,
expression of markers of mesoderm (MIXL1 and BRY), endoderm (IGF2 and GATA4), and
ectoderm (PAX6) were monitored (Figure. 4.6 and Table 4.5). First, we showed that the
levels of mRNA of the mesoderm marker MIXL1 was significantly raised at days 3 and 5
(40- and 7-fold, respectively) and returned to baseline level from days 7 to 16. BRY mRNA
expression was markedly elevated in the early stage at days 3 and 5 (15- and 13-fold,
respectively) and its level returned to baseline value from days 5 to 16. Surprisingly,
expression of endoderm markers (IGF2 and GATA4) increased substantially throughout the
16 days of differentiation, with the levels being much greater than those of mesoderm
markers. The expressions of the latter two genes also lasted longer for up to 16 days. No
changes were found in the ectoderm marker PAX6. The data suggest that stem cells may
differentiate into a mixture of mesodermal and endodermal cell types under our
experimental condition.
Expression of POU5F1 was significantly decreased over the 16 days of
differentiation. On the other hand, ND1, encoded by mtDNA, was upregulated after 7 days
of mesendoderm differentiation (2.2-fold). Most interestingly, the expression of genes
involved in mitochondrial biogenesis (TFAM, NRF1 and POLG) was not affected by the
differentiation into mesoderm.
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Cardiomyocyte Differentiation
The expression of cardiac-specific markers during cardiomyocyte differentiation
have been previously reported (Hudson, Titmarsh et al. 2012). We investigated the
changes in mitochondrial-related gene expression during cardiac-specific differentiation.
Figure 4.6 shows the expression of genes encoded by mtDNA, in which ND5 was
upregulated 2-fold at day 7, but its expression decreased 10-fold at day 16. In contrast,
COX3 expression did not change significantly during the 16 days of cardiomyocyte
differentiation. Transcription of the mitochondrial biogenesis gene TFAM was significantly
reduced throughout 16 days of cardiomyocyte differentiation. Interestingly, PGC1α mRNA
expression was dramatically elevated (252-fold) after 16 days of cardiomyocyte
differentiation (Figure 4.7 and Table 4.6). These results suggest that ND5 and PGC1α may
primarily affect the early stages of mesoderm differentiation and suggest that PGC1α is a
driver of cardiomyogenesis, which is accompanied by an early upregulation of ND5.
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Objective 2: Regulation of Mitochondrial Biogenesis in Chloramphenicol- and
EtBr-treated Primary Human Fibroblasts
4.2 Morphology and Karyotyping of Mitochondria-Depleted Cells
Human fetal fibroblasts were cultured for six weeks in the presence of 100 ng/mL EtBr,
an agent that inhibits the replication of mtDNA, or 100 µg/mL chloramphenicol, a widely
used antibiotic that inhibits mitochondrial protein synthesis. The fibroblast culture exhibited
a large amount of dead cells in the presence of EtBr or chloramphenicol within six weekss.
After recovery for two weeks, the proliferation rate of EtBr-treated fibroblasts recovered
significantly more slowly relative to the chloramphenicol-treated cells. Both EtBr- and
chloramphenicol-treated fibroblasts displayed normal karyotypes (Figure 4.8).
Expression of Mitochondrial-Encoded and Mitochondrial Biogenesis-Related Genes
of Mitochondria-Depleted Cells
Human fetal fibroblasts were cultured for six weeks in the presence of EtBr to block
DNA synthesis or in the presence of chloramphenicol to inhibit mitochondrial protein
synthesis, which mimics the six-week antibiotics treatment. To evaluate the ability of
primary human fibroblasts to recover from these mitochondrial insults, fibroblasts were then
cultured under standard conditions for an additional two weeks following the removal of the
drugs. Samples were collected for analysis after six weeks of treatment and after two
weeks of recovery to determine the effect on the expression of mtDNA-encoded genes
posed by EtBr and chloramphenicol, performed via qPCR. As shown in Figure 4.9 and
Table 4.7, both EtBr and chloramphenicol treatments inhibited transcription of
mitochondrial genes, but to various extents. qPCR analysis of the D-loop was used to
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estimate the mtDNA copy number (i.e., the number of mtDNA molecules per cell). The
analysis revealed that mtDNA was almost completely abolished in EtBr-treated cells,
whereas the mtDNA copy number in chloramphenicol-treated fibroblasts was not
significantly affected.
After cells were treated with EtBr for six weeks, expressions of ND1 and ND4 (both
encoded by mtDNA) were undetectable (Figure 4.9 and Table 4.7). After two weeks of
recovery, however, the expression of these genes rose slightly to 0.07 and 0.11 times that
of untreated control levels, respectively (Figure 4.9 and Table 4.7). Similarly, expression of
CYTB was undetectable after six weeks of EtBr treatment, but increased to only 0.08 times
that of control levels after two weeks of recovery. Expression of COX2 was undetectable
after six weeks of EtBr treatment, but rose to 0.36 times that of control levels after two
weeks of recovery. Following chloramphenicol treatment for six weeks, expressions of ND1
and ND4 were both undetectable. After two weeks of recovery, however, expression levels
of these genes rose to 0.35 and 1.81 times that of untreated controls, respectively (Figure
4.9 and Table 4.7). Expression of CYTB decreased to 0.50 times that of the control levels
after six weeks of chloramphenicol treatment, but did not change after two weeks of
recovery. Expression of COX2 decreased to 0.02 times that of control levels after six weeks
of chloramphenicol treatment, but rebounded to 1.34 times that of control levels after two
weeks of recovery (Figure 4.9 and Table 4.7).
Taken together, these results showed that both EtBr and chloramphenicol lead to a
virtual cessation of mitochondrial gene transcription. The only exception was for CYTB, the
expression of which dropped by only 50% in response to chloramphenicol. After two weeks
of recovery from EtBr treatment, expressions of ND1, ND4, CYTB, and COX2 were still
considerably lower than the corresponding experimental control group. In contrast, after
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two weeks of recovery from chloramphenicol treatment, expressions of ND1 and CYTB
recovered by 50%. Expression of ND4 and COX2 increased relative to control levels after
recovery from chloramphenicol treatment. It was concluded that inhibition of both protein
synthesis and mtDNA replication would effectively eliminate mtDNA-derived gene
expression with the exception of CYTB in the chloramphenicol treatment condition.
Following the removal of the drug from the medium, mRNA levels began to recover,
although the rate of recovery was highly variable. These findings led us to test how these
different treatments and recoveries were related to mitochondrial biogenesis responses in
the nucleus.
To this end, qPCR was used to evaluate the expression of nuclear-encoded
mitochondrial biogenesis genes, including TFAM, NRF1, and POLG. Expression levels
were determined after six weeks of EtBr or chloramphenicol treatment, and after two weeks
of recovery (Figure 4.9 and Table 4.7), TFAM was chosen because it regulates the
transcription of genes within the mitochondrial genome and interacts with other
transcription factors. TFAM is also a direct transcriptional target of NRF1. After six weeks of
EtBr treatment, TFAM expression levels decreased to 0.42 times that of untreated control
levels, but rebounded to 5.09 that of control levels following two weeks of recovery.
Expression of NRF1 decreased to 0.19 times that of control levels after six weeks of EtBr
treatment, but increased to 2.92 times that of control levels after two weeks of recovery.
Expression of POLG decreased to 0.10 times that of control levels after six weeks of EtBr
treatment, but increased to 1.15 times that of control levels after two weeks of recovery.
After cells were treated with chloramphenicol for six weeks, expression of TFAM decreased
to 0.53 times that of control levels, but increased to 3.53 times that of control levels after
two weeks of recovery. Expression of NRF1 decreased to 0.02 times that of control levels
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after six weeks of chloramphenicol treatment, but increased to 0.83 times that of control
levels after two weeks of recovery. Expression of POLG decreased to 0.34 times that of
control levels after six weeks of chloramphenicol treatment, but increased to 2.02 times that
of control levels after two weeks of recovery.
Collectively, these data showed that mRNA levels of TFAM, NRF1, and POLG were
downregulated in response to EtBr or chloramphenicol treatment. Recovery from EtBr
treatment, however, involved the upregulation of TFAM and NRF1, whereas recovery from
chloramphenicol treatment involved the upregulation of TFAM and POLG. Thus, primary
human fibroblasts may employ different strategies of mitochondrial biogenesis to recover
from EtBr or chloramphenicol treatment.
Lactate Production, Glucose Consumption, Redox Status, Oxygen Consumption,
and Energy Production
Next, we determined whether the two distinct recovery responses were correlated with
specific metabolic or cellular parameters. Effects of EtBr and chloramphenicol treatment on
mitochondrial function in primary human fibroblasts were investigated with respect to
lactate production and glucose consumption. We showed that the lactate production and
glucose consumption were 2.38 × 10–8 µmmol/cell/h and 1.36 × 10–8 µmmol/cell/h,
respectively, at 48 h (Figures 4.10A, 4.10B and Table 4.8). Reduced GSH and GSSG
levels were 8.46 × 10–4 pmol/cell and 7.46 × 10–5 pmol/cell, respectively (Figure 4.10C and
Table 4.8). EtBr treatment increased lactate production (3.43 × 10–8 µmmol/cell/h), glucose
consumption (2.07 × 10–8 µmmol/cell/h), and the GSSG/GSH ratio. In contrast,
chloramphenicol treatment did not significantly affect the production of lactate (2.49 × 10–8
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µmmol/cell/h), glucose metabolism (1.25 × 10–8 µmmol/cell/h), or the GSSG/GSH ratio
(Figure 4.10C and Table 4.8).
The next aim was to determine the respiratory activity in control, EtBr-treated, and
chloramphenicol-treated fibroblasts. Figure 4.10D shows that EtBr-treated cells displayed a
90% reduction in oxygen consumption, whereas chloramphenicol-treated cells showed only
approximately a 20% reduction. To compare the effects of EtBr and chloramphenicol
treatment on mitochondrial function in primary human fibroblasts, the ratio of ATP/ADP was
measured. This difference in mitochondrial energy production level directly reflected the
energy status of the cells, with EtBr-treated cells displaying a 76% reduction in the
ATP/ADP ratio and chloramphenicol-treated cells exhibiting a more modest 30% reduction
in the ATP/ADP ratio (Figure 4.10E and Table 4.8). These results suggest that
mitochondrial dysfunction is higher in fibroblasts treated with EtBr than in those treated with
chloramphenicol and are consistent with the observation that EtBr results in a greater redox
imbalance and severely alters glycolytic flux.
Mitochondrial Membrane Potential (Δψm)
Δψm was visualized using JC-1 staining and quantified via FACS analysis. In fibroblast
treated with EtBr, red fluorescence associated with JC-1 (high Δψm) was decreased to 0.68
times that of the control value, whereas green fluorescence (low Δψm) was similar to control
levels. Neither high nor low IMM potentials were significantly affected in
chloramphenicol-treated cells. These data indicated that EtBr treatment reduced the
number of mitochondria with a high Δψm (Figure 4.11 and Table 4.9). Such effect was not
observed in cells treated with chloramphenicol.
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Transmission Electron Microscopy of Mitochondria
Both EtBr- and chloramphenicol-treated fibroblasts displayed normal karyotypes and
morphologies (Figure 4.8). At the ultra-structural level, however, differences between EtBr-
and chloramphenicol-treated cells were observed. Transmission electron microscopy
indicated that control cells contained homogeneous, elongated mitochondria with an oval,
budding, or spindle appearance. In addition, wild-type mitochondria had transverse or
oblique parallel cristae. In contrast, EtBr-treated fibroblasts contained round, fragmented
mitochondria and were very few in number. Cristae within these mitochondria were either
absent, generating ghost-like mitochondrial scaffolding, or formed a disorganized whorled
pattern. Chloramphenicol-treated cells possessed a normal number of mitochondria, but
they seemed immature with morphologically abnormal cristae (Figure 4.12) (King, Godman
et al. 1972). Collectively, these data indicated that EtBr treatment caused oxidative stress,
loss of mitochondria with high Δψm, and morphological impairments to mitochondria. The
effects of EtBr treatment were much more severe than those observed after exposure to
chloramphenicol.
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Objective 3: Manipulation of TFAM Expression in Human Embryonic Stem Cells
4.3 TFAM overexpression in hESC lines
hESCs were engineered to overexpress recombinant TFAM in ESCs (Norrman,
Fischer et al. 2010) using human elongation factor-1α (EF1α) as a promoter. This process
was achieved by constructing TFAM cDNA with the pLenti6 vector. hESCs expressing
green fluorescent protein (GFP) served as vehicle controls. hESCs were transduced with
these lentiviruses and selected for stable overexpression of TFAM by culturing in medium
containing 1 μg/mL blasticidin for one week. qPCR was then used to measure both
endogenous and exogenous TFAM mRNA levels in blasticidin-resistant clones of hESCs.
In three distinct hES cell lines (Mel 1, hES3, and H9), the EF1α-TFAM construct
increased TFAM expression (2.06-, 1.71-, and 1.65-fold, respectively) (Figure 4.13, Table
4.10 and Figure S2). In addition, the mtDNA copy number determined using D-loop as a
template increased 28-fold in hES3 and 5-fold in H9 compared with controls, but did not
increase in Mel 1 cells. Essentially, overexpression of TFAM did not affect the morphology
of hESCs, but resulted in increased cell proliferation in all three cell lines. However, these
cells had low viability after being frozen for storage. During the first 2–3 passages, the
plating efficiency was reduced to <3%. The sensitivity of the TFAM-overexpressing hESCs
in the condition of freezing and/or thawing created a substantial technical barrier for the
study.
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TFAM repression in hESC Line: Development of cell line with constitutive repression
of TFAM
Lentiviral transduction was used to assess the effects of both constitutive and inducible
repression of TFAM in hESCs (Figure S9). Preliminary data demonstrated that TFAM
expression was repressed at different levels by different shRNAs. Unexpectedly, the
expression of TFAM was elevated 2.5-fold and 2-fold when targeted by shRNA to the
sequence 266–286 and 591–611 in Mel 1 cells, respectively (Figure 4.14). When the
position 618–638 was targeted, the expression of TFAM was reduced by 20% (Figure 4.14).
Moreover, constitutive knockdown of TFAM in hESCs substantially induced cell death,
making subsequent experimentation difficult. Inducible knockdown of TFAM expression in
hESCs was used to circumvent these difficulties.
TFAM Repression in hESC Lines: Development of cell lines with inducible repression
of TFAM
Genetic knockout of Tfam resulted in mouse embryonic death (Larsson, Wang et al.
1998). In addition, constitutive knockdown of TFAM in hESCs appeared to affect their
survival and pluripotency, making it difficult to analyze these cells. To circumvent these
difficulties, an inducible system was used to suppress TFAM expression in hESCs (Figure
4.15, Table 4.11 and Figure S3). With this strategy, transfected cell lines were apparently
stable. To induce knockdown, the cells were treated with doxycycline (4 μg/mL) for three
days. Cells without doxycycline treatment were used as a control. Vector transformation
was selected using 2 μg/mL puromycin for one week. The results of qPCR analysis
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indicated that all hESC clones selected by the antibiotic expressed the TFAM knockdown
construct (Figure 4.15 and Table 4.11).
To assess the efficiency of the shRNA-mediated TFAM knockdown, hESCs were
exposed to doxycycline and the levels of TFAM mRNA were determined. The effects were
different in the three hESC lines. In addition, different target sites within the TFAM mRNA
resulted in different levels of repression. When exon 5 was targeted (TFAMi-3), TFAM
expression was repressed 5-, 1.3-, and 1.8-fold in Mel 1, hES3, and H9 cells, respectively
(Figure 4.15A and Table 4.11). Targeting exon 7 (TFAMi-5) resulted in 30- and 2-fold
repression in Mel 1 and H9 cells, respectively.
Flow cytometry was also used to determine the levels of TFAM in these cells.
Targeting exon 5 reduced the TFAM levels 7-fold in Mel 1 cells, 1.8-fold in hES3 cells and
2-fold in H9 cells (Figure 4.15B-C and Table 4.11). Targeting exon 7 reduced the TFAM
protein levels 6-fold in Mel 1 cells and 2-fold in H9 cells.
The effects of reduced TFAM expression on the morphology of hESCs were also
examined. The morphologies of Mel 1 cells were not affected by reduced TFAM expression
(Figure 4.16). The proliferation rates appeared to be similar between the control without
virus transduction and the virus-transduced cell lines without any addition of doxycycline.
Next, the proliferation rates between virus-transduced cell lines in the presence and
absence of doxycycline were mutually compared. The proliferation rate was slower in
virus-transduced cells in the presence of doxycycline compared with those in the absence
of doxycycline.
More extensive reduction in the expression of TFAM was seen in H9 cells than in the
two other cell lines (Figure 4.16). A similar observation was made when TFAM was
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inducibly repressed via exon 7 targeting. These inducible knockdown lines were easier to
grow after being frozen in contrast to those constitutively expressed for TFAM. However,
high levels of cell death were still seen in these TFAM knockdown cells in culture. Once
again, this result created a considerable technical barrier for the study on the effect of
TFAM downregulation on cell differentiation.
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Objective 4: Mitophagy in Human Embryonic Stem Cells
4.4 Characterization of Autophagosome in GFP Reporter Cell Line
The establishment of an hESC line expressing the LC3-GFP fusion protein has been
previously reported (Tra, Gong et al. 2011) and yet, it was difficult to recover the
hES-LC3-GFP cells from the frozen storage. A new hES3 cell line that expresses the
LC3-GFP fusion protein was therefore prepared and adapted in our study to allow single cell
growth as described in the Materials and Methods. Punctuated green dots within these cells
represent autophagosomes (Figure 4.17A). Under normal culture conditions, this
hES-LC3-GFP line exhibited a diffused distribution of GFP fluorescence throughout the
cytosol with typically one or two fluorescence punctates (Figure 4.17A). The expression of
pluripotency markers POU5F1 and TRA-1-60 in these cells is also shown (Figure 4.17B and
Figures S4 and S5)
Mitophagy in hESCs by Time-Lapse Microscope
Since mitochondrial turnover remained to be investigated, levels of autophagy and
mitophagy in hESCs were assessed next. To determine the extent of mitophagy, the
hES-LC3-GFP reporter line was labeled with MitoTracker Red and the extent of
co-localization of GFP and MitoTracker Red was analyzed. First, hES-LC3-GFP cells were
treated with reagents known to induce mitochondrial damage, such as rapamycin (an
inducer of autophagy), carbonyl cyanide m-chlorophenyl hydrazone (CCCP, a mitochondrial
membrane potential uncoupler) (Figure S6), and EtBr (a mtDNA replication inhibitor).
Time-lapse microscopic examination of MitoTracker-labeled hES-LC3-GFP cells treated
with CCCP or EtBr revealed that these agents triggered mitophagy in an asynchronous
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manner within the individual cells from 0 to 10 minutes followed by changes in the colonies
(Figure 4.18A–D, Videos 4.1–4.4 and Figures S7 and S8). The occurrence of mitophagy
during differentiation was examined. Mitophagic events were increased between 0 to 20
minutes of differentiation compared with the control (Figure 4.18E, Video 4.5 and Figures S7
and S8). Therefore, mitophagy appears to be a dynamic process.
The biological significance of mitophagy taking place in hESCs was further
investigated. First, a time course analysis of EtBr treatment was conducted in hESCs. EtBr
triggers widespread and rapid mitophagy across the colonies of the cells within the first 16
hours (Figure 4.18F, Video 4.6 and Figures S7 and S8). After one week of exposure to 100
ng/ml EtBr, cell death occurred in a significantly higher number of hESCs than the controls
(Figure 4.18G-H, Video 4.7-4.8 and Figures S7 and S8).
Quantification of Mitophagy Using Live Cell Cytometry
The number of autophagosomes was subsequently determined using AMNIS
live-imaging flow cytometry in order to quantify mitophagic events. Compared with that in the
DMSO-treated control, the number of autophagosomes was clearly higher in hESCs treated
with rapamycin, CCCP, or EtBr (Figures 4.19A and 4.20 and Table 4.12). Because it is
technically difficult to determine spot count using the AMNIS live cytometer (as a result of
the two fluorescence images), >500 images captured under each experimental condition
were manually scored. The number of autophagosomes that were positively stained for
MitoTracker was determined. In control samples, hESCs possessed an average of one
autophagosome and one mitophagosome per cell. The majority of the cells contained zero
to two autophagosomes, whereas some cells exhibited as many as five or more
autophagosomes (Figures 4.19B and 4.20 and Table 4.12). In samples treated with
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rapamycin, CCCP, or EtBr, the number of autophagosomes increased to one to two per cell.
In addition, the average number of mitophagosomes also increased to one to two per cell (P
< 0.05, Figures 4.19B and 4.20 and Table 4.12). In samples treated with rapamycin, CCCP,
or EtBr, the majority of cells possessed two to three autophagosomes, whereas some cells
exhibited up to fifteen autophagosomes. The average number climbed to three to four per
cell (P < 0.05, Figures 4.19B and 4.20 and Table 4.12). Therefore, induced mitochondrial
dysfunction (i.e., dysfunction brought about by the addition of rapamycin, CCCP, or EtBr)
resulted in an enhanced autophagic process, which was related to mitophagy.
As indicated above, control levels of autophagosomes and mitophagosomes within
hESCs were 0.69 and 0.65 per cell, respectively (Figure 4.19B and 4.20 and Table 4.12). In
RA-treated or spontaneously differentiated cells, at days 7 and 14, the average number of
autophagosomes was 1.6, 1.4, and 0.83 per cell and that of mitophagosomes was 1.4, 1.2,
and 0.8 per cell, respectively (Figure 4.19B and 4.20 and Table 4.12). For autophagosomes,
at days 7 and 14, most cells exhibited one to two autophagosomes, whereas some cells
possessed up to five autophagosomes per cell. Average values climbed to 3.1, 3.0, and 2.1,
respectively (Figure 4.19B and 4.20 and Table 4.12). The number of autophagosomes
ranged from two to three. These data demonstrated that during the early stages of hESC
differentiation, both autophagosome formation and mitophagy increased. TEM analysis
showed that hESCs treated with rapamycin, CCCP, or EtBr (24 hours) had fragmented
mitochondria with very few cristae (Figure 4.19C). Furthermore, more mitochondria inside
the autophagic vacuoles were detected in these cells as compared with controls (Figure
4.19C).
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Chapter 5: Discussion
Objective 1: Mitochondrial-Encoded and Mitochondrial Biogenesis-Related Gene
Expression During Lineage-Specific Differentiation of Human Embryonic Stem
Cells
The levels of mtDNA can affect oocyte maturation, which occurs after fertilization,
and embryo differentiation (Spikings, Alderson et al. 2007). A number of studies have
shown that mitochondrial maturation is involved in the differentiation process (Cho, Kwon et
al. 2006). However, the role of mitochondrial-encoded genes and genes related to
mitochondrial biogenesis remains mostly unknown. To the best of our knowledge, our study
is the first to describe the expression profiles of these genes during spontaneous
differentiation, lineage-specific differentiation (in the ectoderm, endoderm, and mesoderm),
and specific cell-type differentiation (neuronal and cardiomyocytic). The first objective was
to determine the gene expression patterns of mitochondrial-encoded and mitochondrial
biogenesis-related genes during spontaneous and directed lineage-specific differentiation.
MtDNA Copy Number and mtDNA-Encoded Gene Expression during Spontaneous
Differentiation
Dynamic changes (e.g. downregulation of pluripotency genes, upregulation of
differentiation genes and a shift to a different set of metabolic enzymes) in the early phase
of cellular differentiation have been recently observed (Prigione and Adjaye 2010, Prigione,
Lichtner et al. 2011). Yet, differentiation and changes in mitochondrial gene expression
may not always occur concurrently (Facucho-Oliveira, Alderson et al. 2007). We have
shown that the mtDNA copy number was significantly elevated at the 7th day of
spontaneous differentiation and gradually reduced by the 14th day (Figure 4.3). In addition,
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the expression of the pluripotency markers TRA-1-60 and POU5F1 was downregulated
during differentiation, thus suggesting that the expression of mitochondrial-related genes
may be transient. We studied the expression of two mitochondrial-encoded genes, ND1
and CYTB, and found that ND1 expression was significantly downregulated at day 7 in
spontaneous and RA-induced differentiation of the Mel 1 cell line; however, there was no
significant subsequent change in its expression at day 14. The level of CYTB, however,
was significantly upregulated at day 14 (Figure 4.3 and Table 4.2). This result suggests that
the expression of mitochondrial-related genes may be transient.
It has been reported that different cell lines at various stages of pluripotency may show
similar gene expression (Giritharan, Ilic et al. 2011). Our finding, however, has shown that
different hES cell lines exhibited different mitochondrial-encoded gene expression at the
same time point during differentiation. Different copy numbers of mtDNA were observed
when the cells were differentiated using different induction protocols. Amplification of the
D-loop region also seemed to trigger transcription of genes encoded by mtDNA. It was
surprising to find that dynamic changes in gene expression were seen in different cell lines
at various time points. We further showed that the apparent discrepancy of mtDNA copy
number obtained by using different treatments might be due to the level of differentiation
obtained with the different treatments at various time points, which affected the level of
mitochondrial replication. However, there is limited information attributed to this finding;
hence, additional work is required to achieve a complete understanding of this result.
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Expression of Mitochondrial Biogenesis-Related Genes during Spontaneous
Differentiation
In recent years, several reports have described mitochondrial biogenesis-related gene
expression during differentiation of hESCs. When hESCs are allowed to differentiate
spontaneously (by removing the basic fibroblast growth factor 2 (bFGF2), KOSR and the
feeder layer), the expression of both mtDNA-encoded and mitochondrial biogenesis-related
genes is upregulated (Cho, Kwon et al. 2006). However, our results suggest that the
expression of genes encoding mitochondrial biogenesis-related transcription factors (TFAM,
NRF1 and POLG) may not coordinate with the expression of mitochondrial
biogenesis-related transcription factors upon spontaneous differentiation. Undoubtedly, it
remains to be determined whether the difference is due to the use of different cell lines or
differentiation protocols.
The changes in gene expression at various time points during spontaneous hESC
differentiation remain elusive. The differentiation of mESCs is initially associated with
decreased mtDNA copy number, but this number gradually increases as pluripotency
markers are downregulated (Facucho-Oliveira, Alderson et al. 2007). Our data are
consistent with the notion that there may have been a difference in mitochondria number in
undifferentiated and differentiated stem cells. The transcription levels of TFAM, NRF1 and
POLG, which may not be the main mitochondrial biogenesis-related transcription factors,
were not increased during hESC differentiation.
Cellular differentiation is often accompanied by changes in gene expression and
enzymatic activities associated with the mitochondrial oxidative phosphorylation process
(Wieckowski, Giorgi et al. 2009). The results obtained in the present study further
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demonstrated that mitochondria in undifferentiated hESCs exhibit an immature morphology.
In addition, differentiated hESCs possessed more JC1 aggregates than undifferentiated
cells, indicating a higher mitochondrial membrane potential in the differentiated cells. These
results suggest that mitochondrial differentiation is associated with early cellular
differentiation; this could indicate that energy produced from mitochondria is needed during
the differentiation process, as proposed in the study by Tormos et al (Tormos, Anso et al.
2011). Genes involved in mitochondrial biogenesis (e.g. polymerase RNA (POLRMT),
mitochondrial transcription termination factor (MTERF), TFBM1/2, NRF1/3 and UCP2/4)
are downregulated upon differentiation (Zhang, Khvorostov et al. 2011). Our results
demonstrated that mitochondrial-content increases in response to early spontaneous
differentiation and that the master of mitochondrial biogenesis-related genes (PGC1α) is
also responsible for early spontaneous differentiation.
Lastly, ROS can function as a signal that triggers adipogenesis (Tormos, Anso et al.
2011). During the early stages, adipocyte differentiation, oxygen consumption and ATP
production were upregulated and a PPAR-gamma-dependent transcriptional cascade was
initiated. This implied that changes in mitochondrial metabolism, which occurred during
differentiation, might have resulted from the evolving energy demands or from the
production of ROS; this could lead to characteristics of aging as a result of ROS
overproduction (i.e. the mitochondrial theory of aging) (Adhya, Mahato et al. 2011, Yamada,
Furukawa et al. 2011). Therefore, the increase in ROS production upon differentiation may
be implied.
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MtDNA-Encoded and Mitochondrial Biogenesis-Related Gene Expression during
Lineage-Specific Differentiation
Mitochondrial properties are altered during the process of lineage-specific
differentiation in early gastrulation (Baker 1964) and neurogenesis (Cordeau-Lossouam,
Vayssiere et al. 1991). The increase in mitochondrial mass that accompanies each step of
neurogenesis produces large variations in mitochondrial protein levels (Cordeau-Lossouam,
Vayssiere et al. 1991). In this section, the results of the study on lineage-specific
differentiation help to understand that the mitochondrial biogenesis also requires precise
coordination between the mitochondrial and nuclear genomes.
Neural differentiation:
Neurons are metabolically active cells with high energy demands, but the expression
profiles of mitochondrial-encoded and mitochondrial biogenesis-related genes remain to be
determined. Our results revealed that the expression of genes, which were involved in
mitochondrial biogenesis, was elevated after 8 days of neurosphere formation, but reduced
at day 12. First, we showed that the differentiation may result in an increase in
mitochondrial-encoded gene expression, suggesting that the mitochondrial genome has
been replicated. Second, one of the most significantly upregulated mitochondrial
biogenesis-related genes, which we identified as PGC1α, had elevated expression after 8
days of neuronal-specific differentiation, whereas the expression of TFAM, NRF1 and
POLG was not significantly upregulated when compared to PGC1α. The latter result is
similar to that reported by Marchetto et al. in 2010 (Marchetto, Carromeu et al. 2010), which
revealed that the expression levels of TFAM, NRF1 and POLG either remained the same or
decreased. The results suggest that PGC1α is probably one of the early genes potentially
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involved in the upstream regulation of mitochondrial biogenesis upon neuronal
differentiation. However, PGC1α expression was upon neural differentiation, and therefore,
further investigation is needed to study the relative ROS production, antioxidant-related
gene expression, and protein expression. Our data indicated that PGC1α could be a driver
for mitochondrial biogenesis-related gene expression in mitochondrial regulation during
neural differentiation.
Endoderm differentiation
Next, we demonstrated that mitochondrial-encoded and mitochondrial
biogenesis-related genes played important roles in the early differentiation of hESCs into
the endoderm. TFAM, NRF1, POLG and PGC1α have been reported to be involved in and
be responsible for the distinct regulation of endoderm differentiation (Kanai-Azuma, Kanai
et al. 2002, Grapin-Botton and Constam 2007, Kim, Yoon et al. 2011, Spence, Mayhew et
al. 2011, Teo, Arnold et al. 2011). The results of our study are similar to those of the study
by Spence et al. which showed low TFAM, NRF1, POLG and PGC1α expression upon
activin-induced definitive endoderm formation and mimicking embryonic intestinal
development (Spence, Mayhew et al. 2011). As shown in previous studies, mRNAs derived
from mtDNA (e.g. CYB and ND1) are expressed throughout the small intestine and colonic
epithelium in humans (Macpherson, Mayall et al. 1992) and expression levels of
mitochondrial rRNAs and mRNAs in these tissues are somewhat similar to those in
immature enteroblasts. Interestingly, some researchers also reported higher levels of
mitochondrial mRNA and TFAM expression in immature enteroblasts as compared with the
mature cells of the villus (D‘Amour, Agulnick et al. 2005). We observed a different result in
our study of the early endoderm-specific differentiation (Figure 4.5); this discrepancy is due
to the fact that the maturation of villus cells occurs only 50–60 days after differentiation. In
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addition, mitochondrial biogenesis-related gene expression patterns within specific
endodermal cell types were not analyzed. Therefore, in future studies, the duration of
endoderm differentiation could be extended in order to further understand the mechanism
involved in enterocyte maturation related to mitochondrial biogenesis.
Mesendodermal or cardiomyocyte differentiation
The heart is a very important organ that is heavily dependent on oxidative energy
generated from mitochondria. Distorted structure and dysfunction of the mitochondria are
found in patients associated with cardiovascular diseases. The heart is also the first organ
to mature in an embryo. For this reason, it was our strategy to utilize differentiating
cardiomyocytes in this study to investigate the expression of mitochondrial-encoded and
mitochondrial biogenesis-related genes in order to understand the role of mitochondria in
cell differentiation. In addition, the study may provide an insight for regenerative medicine
since cardiomyocytes cannot be reproduced in patients with ischemic damage. Our results
are consistent with those obtained by Salomonis et al. (Salomonis, Nelson et al. 2009) that
showed that the expression of PGC1α was markedly upregulated, whereas that of TFAM
and NRF1 was is downregulated upon mesendoderm and cardiomyocyte differentiation.
On the other hand, there were no significant changes in the expression of POLG. PGC1α is
known to be a key factor involved in the induction of mitochondrial biogenesis in many
tissues (Kelly and Scarpulla 2004) and its expression is upregulated after osteogenic
differentiation of human mesenchymal stem cells or after cardiomyocytic differentiation of
mESCs (Pietila, Lehtonen et al. 2010). PGC1α is proposed to be acting, upstream of both
TFAM and NRF1, as a master genetic switch. However, our study showed that upregulation
of PGC1α expression was not accompanied by increased TFAM and NRF1 expression,
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suggesting that PGC1α utilizes an alternative signal transduction pathway, which is
independent of TFAM and NRF1, to upregulate mitochondrial activity.
During cellular differentiation, mitochondria must also differentiate and mature via
processes involving both structural and functional changes, such as changes in the
mitochondrial membrane potential. In developing cardiomyocytes, changes in
mitochondrial activity occur at both the pre- and post-natal stages of development, which
are associated with the upregulation of oxidative phosphorylation and an increase in
mtDNA copy number. We showed that the mitochondrial-encoded genes (ND1 and ND5)
were upregulated at the 7th day of mesendoderm and cardiomyocyte differentiation. Here,
we attempted to study the correlation between mitochondrial-encoded genes and
mitochondrial biogenesis-related transcription factors in cardiomyocyte differentiation.
We examined several genes encoded by mtDNA and genes related to mitochondrial
biogenesis. It was surprising to find that, during hESC differentiation, the expression levels
of several mitochondria-related genes did not correlate with levels of PGC1α. Interestingly,
PGC1α is consistently upregulated in spontaneous, RA-induced, ectoderm-specific, or
cardiomyocyte-specific differentiation, but not in endoderm-specific differentiation.
Differences in the temporal expression patterns during the transition of undifferentiated to
differentiated hESCs might have been the result of the variable half-lives of mitochondrial
mRNAs (Nagao, Hino-Shigi et al. 2008). Different lineage-specific differentiations, such as
ectoderm-specific and cardiomyocyte-specific differentiations, displayed different patterns
in mitochondrial biogenesis. In summary, we conclude that the specific nucleus-encoded
mitochondrial biogenesis-related transcription factors have different gene expression
patterns upon lineage differentiation. Therefore, in future studies, we will focus primarily on
evaluating the feasibility of changes in mitochondrial biogenesis upon differentiation of
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hESCs in a time manner and gain a better understanding of the role of mitochondria during
differentiation.
This study did not address the molecular mechanism explaining how mitochondrial
maturation is associated with differentiation (St John, Ramalho-Santos et al. 2005, Cho,
Kwon et al. 2006, St John, Amaral et al. 2006). Many researchers indicated their
observation of mitochondrial maturation upon differentiation. In this study, we measured
gene expression levels, but we were unable to find enough evidence to explain the
molecular mechanism of role of mitochondrial maturation in differentiation. Therefore,
additional studies will need to be carried out to determine the possible effect of mtDNA
copy number and to explore whether mitochondrial maturation is responsible for the
mitochondrial activity in different hESC lines. First, the protein levels of the pluripotency
markers (TRA-1-60 and POU5F1), lineage-specific biomarkers, mitochondrial-encoded
genes, and mitochondrial biogenesis-related genes should be analyzed using a western
blot to determine their significance in the process of differentiation. Another consideration is
to examine mitochondrial functions by looking into mitochondrial respiration, membrane
potential, energy metabolism (ATP/ADP, glucose uptake and lactate production), and the
level of reactive oxygen species (DCF or mitoSOX red staining or GSH/GSSG) in order to
gain an insight on mitochondrial metabolism during differentiation. Finally, the molecular
mechanism of PGC1α in the mitochondrial biogenesis signaling pathway (Scarpulla 2008)
could be investigated to explain how it could respond to lineage-specific differentiation.
PGC1α seems to be an early indicator of the upregulation of mitochondrial biogenesis in
the lineage-specific differentiation of hESCs. Overall, a combination of these experimental
results would provide unique and valuable insights into the regulation of mitochondrial
biogenesis in stem cell differentiation.
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Objective 2: Regulation of Mitochondrial Biogenesis in Chloramphenicol- and
EtBr-treated Primary Human Fibroblasts
Defects in mitochondrial function due to mtDNA mutations or chemicals that affect
mitochondrial protein function lead to a range of pathologies, often serious (Zeviani and
Antozzi 1997, Lane 2006). The exact mechanism by which mitochondrial impairments
cause the wide range of observed pathologies remains to be determined. Some studies
have shown that, during the early preimplantation stage, there is limited or no active
mitochondrial replication (Cao, Shitara et al. 2007, Wai, Ao et al. 2010). A minimum mtDNA
copy number threshold is required before the cells of the fertilized oocyte can differentiate
into various tissues. The second objective was to investigate the effects of mitochondrial
depletion on human fibroblasts and hESCs. The novel finding in this study was that there is
compensation in the expression of mitochondrial-encoded and mitochondrial
biogenesis-related genes after removing the treatments.
In the present study, mitochondrial functions were impaired by treating the cells with
EtBr (an inhibitor of mtDNA replication) or chloramphenicol and the impacts of these two
drugs on mitochondrial function and nuclear expression of mitochondrial biogenesis
regulators was compared. Both drugs impaired mitochondrial function, albeit to a different
extent, and led to the differential expression of both mtDNA-encoded genes and nuclear
transcription factors that control mitochondrial biogenesis. This study is the first to
demonstrate that established human fibroblast cell lines devoid of characteristic mtDNA
markers can be obtained from cell populations exposed to EtBr for a long time. Each cell
may contain different mitochondrial content, and each mitochondrion may contain 1–10
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mtDNA. The mitochondrial mass or content of a cell will affect its mtDNA copy number.
Here we report the generation of characterized primary human fibroblasts, which exhibited
a substantial reduction in mtDNA levels and a concomitant reduction in respiratory-chain
enzyme activities.
The data derived in this study showed that the inhibition of protein synthesis (by
chloramphenicol) or of mtDNA synthesis (by EtBr) caused severe reductions in the
transcription of mtDNA genes, with the exception of CYTB, after chloramphenicol exposure.
After two weeks of recovery from chloramphenicol treatment, some genes encoded by the
mitochondrial genome (ND1 and CYTB) exhibited partial recovery of expression. Other
mtDNA genes (ND4 and COX2) exhibited an overcompensation (i.e. expression levels
were higher after recovery than those before the treatment). However, the cells only
showed a minor recovery from mitochondrial genome depletion by EtBr treatment. EtBr and
chloramphenicol, therefore, had different effects on mitochondrial transcription in human
fibroblasts. In response to both treatments, TFAM expression was upregulated. In contrast,
POLG expression was upregulated by chloramphenicol and NRF1 expression was
upregulated by EtBr. We do not know why the cells choose to employ these different
compensatory mechanisms. Seeking to identify cellular parameters underlying these
different responses, we found that EtBr-treated cells exhibited a reduced Δψm, an
increased lactate/glucose ratio, morphologically abnormal mitochondria, and increased
oxidative stress-effects that were generally not observed in chloramphenicol-treated cells
(Soslau and Nass 1971, Storrie and Attardi 1973, Stuchell, Weinstein et al. 1975, Lipton
and McMurray 1977, Wiseman and Attardi 1978). The EtBr data closely resembles that
previous studies performed with HeLa and C2C12 cells (Hayashi, Ohta et al. 1991, Joseph,
Rungi et al. 2004), which showed that the elimination of mtDNA causes deficiencies within
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the electron transport chain, an increase in intracellular ROS, a metabolic shift towards
glycolysis, increased lactate production, reduced ATP synthesis and lower Δψm. These
changes collectively resulted in the upregulation of NRF1 and TFAM expression. It should
be noted, however, that increases in NRF1 and TFAM mRNA expression do not
necessarily affect protein levels (Davis, Ropp et al. 1996, Miranda, Foncea et al. 1999).
TFAM may be less stable in the absence of mtDNA (Miranda, Foncea et al. 1999).
Replication and transcription of mtDNA depend on proteins encoded by genes on the
nuclear genome, such as NRF1 and TFAM (Scarpulla 2006, Scarpulla 2008). In agreement
with this notion, disruption of NRF1 or TFAM in mice leads to reduced levels of mtDNA and
lethality during early stages of embryogenesis (Larsson, Wang et al. 1998, Huo and
Scarpulla 2001).
Chloramphenicol inhibits mitochondrial protein synthesis and delays muscle
regeneration (Wagatsuma, Kotake et al. 2011) and in a reversible manner (Rebelo,
Williams et al. 2009). Surprisingly, the data obtained in this study revealed, after the
chloramphenicol treatment was stopped, NRF1 was not up-regulated during recovery.
Rather, this recovery process involved POLG and TFAM, suggesting that the inhibition of
mitochondrial protein synthesis elicits a different retrograde signal than that triggered by
mtDNA depletion. The data in this study suggest that the chloramphenicol-based signal
does not involve the ATP/ADP ratio, the Δψm, altered glycolytic flux, or altered oxygen
consumption, as these parameters were not affected by chloramphenicol treatment.
As the mtDNA copy number goes down, so do respiratory chain activities. In
EtBr-treated fibroblasts, respiratory activity dropped more rapidly than in
chloramphenicol-treated ones, possibly because of the toxic side effects associated with
EtBr. Similar decreases in respiratory function (including complex activities and oxygen
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consumption) have been observed in ρ° cells (King and Attardi 1989). EtBr-treated cells
exhibited lower levels of mtDNA expression and respiratory activity. Once mtDNA levels
dropped beneath a certain level, the cell‘s bioenergetic status rapidly deteriorated. This
result suggests that each cell has a mtDNA threshold beneath which it cannot maintain a
sufficient level of energy production. It is likely that cells have a surplus of mtDNA
molecules that extend well above this mtDNA threshold, which could protect the cell from
sporadic mutations to the mtDNA. The results in this study have shown that reductions in
Δψm, together with the elevation in lactate formation, suggested oxidative phosphorylation
(OXPHOS) impairment. However, there was a clear decrease in the red/green fluorescence
ratio in the treated group, and some cells failed to show a lower Δψm after EtBr treatment.
Interestingly, some cells with ρ° status maintained a normal Δψm independent of impaired
OXPHOS. Despite the apparent impairment in OXPHOS in the treated groups, the amount
of mtDNA per cell did not decrease over time with EtBr treatment (Buchet and Godinot
1998). According to King and Attardi (1996) (King and Attardi 1996), it is impossible to
isolate ρ° derivate of all human cell lines using EtBr. Our results suggest that the
mitochondrial content may not be erased completely and some mitochondrial activity
remains in some cells.
Depletion of mtDNA genes resulted in the reduction of mitochondrial-encoded gene
expression. In the present objective, the fibroblasts initially showed a decrease in the
amount of mtDNA when treated with EtBr, but they subsequently regained normal levels of
mtDNA after being withdrawn from treatments. The amount of mtDNA was increased in
treated cells compared with controls, as exemplified by a lower ΔΨm and higher levels of
lactate production. The higher levels of lactate production might correspond to a cellular
alternative for the generation of ATP by the glycolytic process. In both treatments, the
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mtDNA replication might be due to the failure of RNA polymerase to transcribe mtDNA,
leading to a decline in Δψm. Finally, the data in this thesis could provide insights into the
response of human cells to mtDNA depletion
In future, additional studies should be carried out to determine if other effects on the
depletion of mtDNA copy number are observed with different dosages of EtBr and
chloramphenicol in different cell lines; these studies should include the use of two or more
fibroblasts and two or more hES cell lines. A higher resolution of mitochondrial
morphologies examined by TEM would also benefit us with more information in this study.
Recently, few practitioners and researchers have become aware that the mutant
mitochondrial DNA can carry over and affect the differentiation of embryonic stem cells into
various cell types, a discovery that can possibly help prevent the transmission of
mitochondrial diseases to one‘s offspring (Tanaka, Sauer et al. 2013).
In summary, the results presented in this thesis demonstrated that treatment with EtBr
and chloramphenicol affect the mtDNA replication to different degrees. By characterizing
the mitochondrial-encoded and the nucleus-encoded mitochondrial biogenesis-related
genes in mitochondria-depleted cells subjected to two different treatments, it is shown that
chloramphenicol interferes with mtDNA replication and results in a moderate depletion of
mtDNA copy number and function to a larger extent as compared with EtBr. The
observations guide us to understand that the reduction in mitochondrial gene expression
could be a signal of the retrograde recovery of mitochondrial biogenesis-related
transcription factors as an example of altered nuclear gene expression. However,
additional mechanisms of retrograde mitochondrial signaling, such as mitochondrial
autophagy, remain to be investigated.
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Objective 3: Manipulation of TFAM Expression in Human Embryonic Stem Cells
When generatingrho-minus cells with which to investigate how the mtDNA copy
number affects hESC survival, one of the main challenges is that cells cannot survive
without mitochondria. The third objective waso establish hESC lines in which TFAM was
overexpressed or knocked down and to characterize the effects of TFAM expression on
mitochondrial biogenesis during the proliferation of hESCs. The purpose of this study was
to test the hypothesis that TFAM expression is associated with mtDNA copy number and
cell proliferation in hESCs. The study was the first to manipulate TFAM expression in
hESCs. An expression vector was used to overexpress TFAM, while TFAM-specific short
hairpin RNAs (shRNAs) were used to suppress TFAM expression in three hESC lines (Mel
1, hES3, and H9).
We observed different expression levels of TFAM in various hESC lines, suggesting
that these cells use different mechanisms to regulate mtDNA. The results presented here
showed that TFAM overexpression results in an increase in mtDNA copy number to various
degrees in different hESC lines. EF1α-TFAM hESCs exhibited higher rates of proliferation
relative to control cells. Previous studies have shown that the overexpression of TFAM in
other cell types resulted in an increase in mtDNA copy number (Larsson, Wang et al. 1998,
Ekstrand, Falkenberg et al. 2004, Kanki, Ohgaki et al. 2004), whereas the knockdown of
TFAM resulted in a decrease in mtDNA. Interestingly, it has been reported in the study by
Ylikallio et al. (2010) that the high mtDNA copy number is associated with cell death
(Ylikallio, Tyynismaa et al. 2010) which is possibly, because C-MYC (MYC) binds to the
TFAM promoter (Li, Wang et al. 2005) to regulate cell proliferation and energy metabolism
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by regulating glycolysis (Zeller, Jegga et al. 2003). This result suggests that MYC may
upregulate TFAM expression in hESCs, thereby acting as a master switch to control
mitochondrial biogenesis and to couple the metabolic needs of a cell to its growth and
proliferation.
This observation is supported by a previous demonstration that the C-terminal tail of
TFAM is critical for binding to the light-strand and heavy-strand promoters (Malarkey,
Bestwick et al. 2012). TFAM that lacks a C-terminal tail retains the ability to bind DNA, but
does not activate transcription (Pastukh, Shokolenko et al. 2007). Overexpression of TFAM
restores memory impairment (including working memory and hippocampal long-term
potentiation), attenuates the mitochondrial production of ROS and limits mtDNA damage in
aged mice (Hayashi, Yoshida et al. 2008). The optimal stoichiometry is five molecules of
TFAM per copy of mtDNA. However, TFAM-mediated transcriptional activity is reduced or
eliminated if the TFAM:mtDNA ratio exceeds five (Garrido, Griparic et al. 2003, Kanki,
Nakayama et al. 2004) Both endogenous and exogenous TFAM bind mtDNA to form a
mitochondrial-nucleoid complex (kanki, Nakayama et al. 2004). Consequently, a decrease
in TFAM expression may lead to reduced mtDNA copy number, which may disrupt
oxidative phosphorylation in organs. This result may explain that the overexpression of
TFAM in hESCs was not compatible with survival well after thawing. It is possible that
overexpression of TFAM stimulates cell proliferation, which may lead to insufficient levels of
ATP production. However, EF1α-TFAM hESCs typically had low survival rates during the
first 24 hours for 1–2 passages. hESCs that overexpress TFAM could grow, but they could
not be re-thawed; this may have been a result of increased levels of ROS during the early
stages of plating. Additional experiments are needed to determine the level of cell
proliferation and the rate of ATP and ROS production before and after freezing.
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Because TFAM may affect mitochondrial transcription and replication, inhibition of
TFAM would result in some mitochondrial dysfunction. The extent of the damage caused by
mitochondrial dysfunction can vary greatly depending on the type of tissue or cell, but
muscle and heart tissues are particularly sensitive to this effect. In mice, homozygous
TFAM knockout embryos exhibited severe mtDNA depletion with abolished oxidative
phosphorylation and died before complete embryo development at day 7.5, whereas the
heterozygous mice exhibited reduced mtDNA copy number and respiratory chain
deficiency in the heart (Larsson, Wang et al. 1998). Our preliminary data indicated that
downregulation of TFAM affected the in vitro survivability and proliferation of hESCs.
Several factors could have also influenced the outcome of this study (cell death
induced after knockdown of TFAM). The experimental design in the present study revealed
that there are some discrepancies in comparison with previous reports. Therefore, in the
future, some additional studies should be carried out:
(1) To analyze and determine the mechanisms by which constitutive and inducible
repression of TFAM results in cell death, although there are no studies documenting
cell death in TFAM knockout stem cells;
(2) To use other strategies for manipulating TFAM expression in different hESC lines in
order to determine mtDNA copy number, mitochondrial gene transcription,
mitochondrial respiration, cell proliferation, cell survival, and pluripotency on
differentiated cells;
(3) To ultimately determine any other possible changes in mitochondrial function (such as
changes in mitochondrial respiration and membrane potential), energy metabolism
(ATP/ADP, glucose uptake and lactate production), and ROS level (DCF, mitoSOX red
staining, or GSH/GSSG). Moreover, higher resolution investigations of cell and
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mitochondria morphologies should be conducted using light and transmission electron
microscopy.
The importance and role of an upregulated PGC1α expression should be evaluated in
the lineage-specific differentiation of hESCs. Manipulation of PGC1α expression in these
cells may provide insights into the mechanism underlying the involvement of mitochondria
in the differentiation of hESCs into specific cell lineages.
In summary, the results of this study revealed that downregulation and upregulation of
TFAM expression decreased and increased mtDNA expression, respectively, suggesting
that TFAM must be expressed at tightly controlled levels for cell survival and to maintain
mitochondrial functions.
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Objective 4: Mitophagy in Human Embryonic Stem Cells
The autophagic process breaks down cellular components and plays a particularly
important role during periods of starvation or when organelles are damaged.
Autophagosomes allow each cell to maintain a critical balance between the synthesis,
degradation, and recycling of cellular structures. Therefore, it is not surprising that
autophagy plays a significant role in cell growth and homeostasis during development. It is
thought that autophagy is essential for healthy aging and longevity. The last objective was
to compare mitochondrial turnover (mitophagy) following exposure to mitochondrial toxins
and during the differentiation of hESCs. This study investigated the processes that occur
during differentiation- and toxin-induced mitophagy. The primary aims were: (1) to establish
the LC3-GFP system as a means to measure mitophagy quantitatively; (2) to understand
the dynamics of mitophagy when cells are challenged with toxins that affect mitochondrial
function; and (3) to understand the dynamics of mitophagy during hESC differentiation.
The fourth objective was to determine whether mitochondrial turnover is essential in
maintaining optimal cellular function for cell differentiation. In regards to mitochondrial
turnover, there is little evidence illustrating the mechanism by which it occurs. In this study,
differentiation and toxic stimuli were used to demonstrate that mitochondrial turnover in
hESCs occurs via the uptake of mitochondria into autophagosomes, a process called
mitophagy. To this end, a sensitive method using live-imaging flow cytometry (AMNIS) was
used for the determination of mitophagy. First, we showed that mitophagy in hESCs is
triggered by interfering mtDNA replication and mitochondrial membrane potential. Second,
increased levels of mitophagy were found to be associated with hESC differentiation, either
spontaneous or RA-induced. Spontaneous differentiation was initiated by removing
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pluripotency maintenance factors from the culture medium. At day 7 of spontaneous
differentiation, mitophagy levels were elevated, but they returned to baseline by day 14. The
short window of mitophagy (during a time of differentiation and mitochondrial biogenesis)
reflects the removal of mitochondrial components contaminated by free radicals in order to
ensure that a healthy population of mitochondria is present for cell differentiation.
The high rate of mitophagy found in our study seemed to contradict some
characteristics of differentiated hESCs with elevated mitochondrial gene expression,
increased mtDNA copy numbers and a more mature mitochondrial morphology. Autophagy
plays an important role in the degradation of maternal RNA and proteins during mammalian
embryonic development (Mizushima and Levine 2010). Mitochondria with low membrane
potentials or excessive ROS production could trigger mitophagy. Furthermore,
differentiation-induced mitophagy may selectively cull the germ line copies of mtDNA that
contain mutations, as reported in mouse models (Yang, Przyborski et al. 2008). Here, our
preliminary data showed that, during the early stage of differentiation, hESCs may
selectively eliminate defective mitochondria via mitophagy; however, the detailed
limitations of the mechanisms involved in the initiation of mitophagy during cell
differentiation remain to be addressed as follows:
We need to show the pluripotency status (the loss of stem cell markers POU5F1 and
TRA-1-60) of the undifferentiated hESCs before and after EtBr and rapamycin
treatments and the early differentiation of hESCs subjected to retinoic acid treatment.
It is necessary to further test whether the upregulation of autophagic or mitophagic
events are equally associated with treatments and/or retinoic acid treatments, as
indicated in the early differentiation phase, in relation to time and dosage.
Mitophagy-related markers, such as PINK1 and PARK and autophagy markers need to
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be established in future studies to understand specific mitophagic mechanisms.
Since CCCP triggers an increase in the number of autophagosomes and in mitophagic
activity, we need to use CCCP to depolarize mitochondrial membrane potential and
fragments, as described by (Narendra, Tanaka et al. 2008), to further explore
whether mitophagy plays a role in scavenging the damaged mitochondria and, thus,
in maintaining the quality of hESCs. Furthermore, it is important to examine whether
mitophagy precedes the expansion of mitochondria upon differentiation and
selectively removes damaged mitochondria to prevent the accumulation of mtDNA
mutations. Interestingly, PTEN-induced putative kinase 1 (PINK1) has been shown to
be selectively accumulated in damaged mitochondria that possess a low membrane
potential due to depolarization] caused by CCCP (Acton, Jurisicova et al. 2004).
Parkinson protein 2 (PARK2/PARKIN) ubiquitinates the voltage-dependent anion
channel 1 (VDAC1) and plays a role in generating mitochondrial outer membrane
potential, and recruits P62, an autophagy receptor, to the mitochondria damaged by
autophagy. P62 interacts with microtubule-associated protein 1/light chain 3 alpha
(MAP1LC3A/LC3) on the phagophore surface, leading to engulfment by the
autophagosomal structure and subsequent lysosomal degradation (i.e. mitophagy).
The mechanism of mitophagy still remains to be determined and reinforced in which
an early mitochondrial turnover could associate with the mechanism of
PINK1/PARK2/P62/LC3-dependent mitophagy upon differentiation of hESCs.
One of the strategies in this study was to expose hESCs to EtBr for various time
intervals (short-term exposure, such as 20 minutes, two-hour exposure followed by
observation for 16 hours, and long-term exposure). Images acquired by time-lapse
microscopy showed that the early stage of mitophagic events occur within 20 minutes to
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two hours of EtBr treatment, while a significant upregulation of mitophagic events occurred
within 16 hours. Eventually, the images revealed that there was significant cell death after
EtBr treatment for one week. Such mitophagy may be responsible for the high rate of cell
death observed in this study.
The identification of mitophagy in hESCs with the modulation of mitochondrial turnover
in either hESCs exposed to toxins or upon hESC differentiation represents the first steps in
understanding the role of mitophagy and mitochondria in hESC differentiation.
Nevertheless, we have shown that: (1) all autophagy in hESCs with EtBr or
CCCP-treatment appears to be mitophagy, (2) rapamycin could induce mitophagy (Figure
4.18) and (3) mitophagy increases during hESC differentiation, which may be either
spontaneous or retinoic acid-induced. The conclusion of this study is that the differentiation
of hESCs was accompanied by an increase in both mitochondrial biogenesis and
mitochondrial turnover. In future, some experiments are required to determine whether
other factors may interfere with mitophagy and indirectly affect mitochondrial quality. It is
also worth mentioning here that we demonstrated the up-regulation of the expression of
mitochondrial and autophagy markers in differentiated hESCs. The use of knockdown
technique to measure expression levels of mitophagy-associated genes (e.g., PARK2 or
PINK1) could be considered in the future in a parallel study of autophagy-associated genes
(e.g., Atg5 or LC3). These studies will help to further delineate the similarities and
differences between mitophagy and autophagy. To this end, we could selectively
manipulate mitophagy that would be relevant to clinical applications using hESCs or human
inducible pluripotent stem cells (hiPSCs), particularly the applications involving iPSCs
obtained from older individuals who have accumulated mtDNA damage.
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This study demonstrated for the first time that mitophagy occurs in hESCs. It also
provided evidence that during or immediately preceding hESC differentiation, mitophagy is
involved in the selective turnover of mitochondria, which may represent a mechanism of
optimizing or controlling mitochondrial quality within differentiating hESCs. A better
understanding of the molecular mechanisms that control mitophagy in hESCs may facilitate
the manipulation and improve the quality of hESCs that can be used in regenerative
medicine.
158
Chapter 6: Final Conclusion
While most studies of human pluripotent stem cells have focused on changes in the
expression of nuclear genes during differentiation (Cho, Kwon et al. 2006, Saretzki, Walter
et al. 2008), relatively little attention has been paid to mitochondrial gene expression during
this process. mRNA changes do not necessarily result in protein expression changes
because protein levels are also controlled by mRNA translation, protein stability, and protein
turnover. In the present study, we investigated some of the changes in mitochondrial gene
expression in stem cells differentiating into three specific lineages, namely, ectoderm,
endoderm, and mesoderm.
Our results revealed that different hESC lines (hES3 and Mel 1) possess different
differentiation capabilities and mechanisms for regulating mtDNA expression. We further
found that the transcription of peroxisome proliferator-activated receptor gamma
coactivator 1 alpha (PGC1α) gene is upregulated as the cells differentiate into ectoderm or
mesoderm (Watkins, Basu et al. 2008). Changes in mitochondrial gene expression
observed during lineage-specific differentiation possibly reflect changes necessary for the
metabolic maturation of mitochondria.
The main finding in the second study was the apparent and different gene expression
patterns before and after treatmnt in human of fibroblasts with agents that interefere with
mitochondrial translation or DNA replication. Inhibition of mtDNA or protein synthesis
resulted in altered expression of both the mtDNA-encoded proteins and the nuclear
transcription factors that control mitochondrial biogenesis, and the existence of different
compensatory mechanisms.
The main discovery in the third study was that the TFAM expression affects the
survival of hESCs. No study on manipulating the mtDNA copy number or TFAM expression
159
in hESCs and mouse embryonic stem cells (mESCs) exists in the literature. In our work, we
demonstrated that TFAM expression level has a positive effect on mtDNA copy number.
Furthermore, these preliminary data suggest that artificially increasing/decreasing mtDNA
copy number influences cell survival. This information is relevant for developing gene
therapy approaches for regenerative medicine of mtDNA related diseases.
Finally, the main finding in the fourth study was that mitophagy is a possible
mechanism for regulation of mitochondrial turnover during rapamcyin, CCCP or EtBr
treatment or early differentiation in hESCs. The data also suggest that mitophagy may be
responsible for the selective removal and quality control of mitochondria in hESCs upon
differentiation. No study has investigated any of the differences and the mechanisms of
mitophagy in hESCs in detail. A better understanding of the molecular mechanisms that
control mitophagy in hESCs may allow manipulation of this process for minimizing the
number of dysfunctional mitochondria in long-term cultured hESCs for use in
regenerative medicine.
Overall, the data in this thesis provide information on the interplay between
mtDNA copy number, cell-specific differentiation and cell response upon alteration of the
mtDNA copy number and the expression of nuclear regulators of mitochondrial
biogenesis. Moreover, these data may provide valuable information to fully understand
mitochondrial diseases and might be useful for regenerative medicine approaches in the
future.
160
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Tables
Objective 1: Mitochondrial-Encoded and Mitochondrial Biogenesis-Related Gene
Expression During Lineage-Specific Differentiation of Human Embryonic Stem Cells
Table 4.1 Comparison of TRA-1-60 and mitochondrial staining in undifferentiated and
differentiated hESCs.
Markers for pluripotency (TRA-1-60) and mitochondria (MitoTracker Red) were analyzed in
hESCs under the indicated treatment conditions. Histogram is also shown in the Figure 4.2. Data
reflect the mean ± standard deviation (SD, n = 3). *P<0.05 as compared to control. Abbreviations:
Undiff, undifferentiated hESCs cultured in conditioned medium; Spd. diff-day 7, hESCs cultured
without basic fibroblast growth factor for 7 days; Spd. diff-day14, hESCs cultured without basic
fibroblast growth factor for 14 days; RA-day 3, hESCs cultured with retinoic acid (RA) for 3 days.
Undiff Spd. Diff-day 7 Spd. Diff-day 14 RA-day 3
Intensity of
TRA-1-60
staining
164,124 ± 7110
(1.00 ± 0.04)
168,146 ±
18,775
(1.01 ± 0.16)
91,091 ± 4406 *
(0.56 ± 0.05 *)
14,841 ± 188 *
(0.09 ± 0.01 *)
Intensity of
MitoTracker
staining
6,518,461 ±
189,787
(1.00 ± 0.03)
11,216,946 ±
1,410,269 *
(2.01 ± 0.16 *)
757,6781 ±
181,829
(1.16 ± 0.02)
6,577,975 ±
121,149 „
(1.01 ± 0.02)
190
Table 4.2 Changes in gene expression upon hESC differentiation.
RT-PCR analysis was used to determine gene expression levels within undifferentiated and
differentiated hESCs. Both the Mel 1 and hES3 cell lines were examined. Expression levels were
normalized to β-ACTIN (ACTB). Histogram is also shown in the Figure 4.3. Data were the mean ±
SD. *P<0.05 as compared to control. Abbreviations: POU5F1, POU class 5 homeobox 1; ND1,
NADH dehydrogenase 1; CYTB, cytochrome b; TFAM, mitochondrial transcription factor A; NRF1,
nuclear respiratory factor 1; POLG, DNA polymerase subunit gamma; PGC1α, peroxisome
proliferator-activated receptor gamma coactivator 1 alpha; β-ACTIN(ACTB), beta-actin; D-loop,
displacement loop.
191
Mel 1 line Undiff. Spd. diff.-day 7 Spd. diff.-day 14 RA-day 3
POU5F1 1.00 ± 0.10 0.01 ± 0.01 **** 0.01 ± 0.01 **** 0.01 ± 0.001
****
ND1 1.00 ± 0.10 0.38 ± 0.01 * 1.11 ± 0.85 0.01 ± 0.001
****
CYTB 1.00 ± 0.13 0.97 ± 0.03 3.32 ± 0.09 **** 2.79 ± 0.07
****
TFAM 1.00 ± 0.88 0.60 ± 0.10 1.18 ± 0.16 0.14 ± 0.04
NRF1 1.00 ± 0.04 0.08 ± 0.07 * 0.37 ± 0.58 0.34 ± 0.02
POLG 1.00 ± 0.01 0.05 ± 0.05 **** 1.11 ± 0.02 ** 0.30 ± 0.01
****
PGC1α 1.00 ± 0.13 1.67 ± 0.20 * 2.66 ± 0.80 ** 1.20 ± 0.32 *
D-loop 1.00 ± 0.51 2.61 ± 0.66 * 1.30 ± 0.27 N/A
hES3 line Undiff. Spd. diff.-day 7 Spd. diff.-day 14 RA-day 3
POU5F1 1.00 ± 0.62 0.21 ± 0.10 0.19 ± 0.02 0.06 ± 0.02 *
ND1 1.00 ± 0.50 1.10 ± 0.30 0.66 ± 0.46 1.29 ± 0.54
CYTB 1.00 ± 0.03 1.36 ± 0.04 * 0.99 ± 0.06 0.66 ± 0.19 *
TFAM 1.00 ± 0.15 0.63 ± 0.03 ** 0.25 ± 0.02 **** 0.48 ± 0.03 ***
NRF1 1.00 ± 0.03 0.14 ± 0.09 ** 0.12 ± 0.04 ** 0.59 ± 0.34
POLG 1.00 ± 0.25 0.16 ± 0.17 ** 0.14 ± 0.13 ** 0.67 ± 0.29
PGC1α 1.00 ± 0.27 14.25 ± 1.40 **** 6.98 ± 0.27 *** 7.47 ± 1.32 ***
D-loop 1.00 ± 0.23 31.49 ± 2.47 **** 2.11 ± 0.12 0.46 ± 0.05
192
Table 4.3 Changes in gene expression associated with ectoderm-specific differentiation of hESCs.
Expression and qPCR expression fold changes were shown for gene associated with
pluripotency (POU5F1), ectoderm-differentiation (PAX6), genes encoded by mitochondria (ND5
and COX3) and mitochondrial biogenesis–related genes (TFAM, NRF1, POLG and PGC1α). Data
were normalised to β-Actin (ACTB). Histogram is also shown in the Figure 4.4. Data were the mean
± SD. *P<0.05 as compared to control. Abbreviations: PAX6, paired box 6; ND5, NADH
dehydrogenase 5; COX3, Cytochrome oxidase 3.
Genes Day 0 Day 8 Day 12
PAX6 1.00 ± 0.39 517.19 ± 116.64 *** 357.55 ± 9.77 **
POU5F1 1.00 ± 0.12 0.22 ± 0.04 **** 0.06 ± 0.01 ****
ND5 1.00 ± 0.08 11.69 ± 0.92 **** 1.33 ± 0.13
COX3 1.00 ± 0.05 12.74 ± 0.40 **** 1.45 ± 0.13
TFAM 1.00 ± 0.12 1.10 ± 0.17 0.86 ± 0.09
NRF1 1.00 ± 0.21 1.59 ± 0.68 0.93 ± 0.08
POLG 1.00 ± 0.02 1.56 ± 0.27 * 0.61 ± 0.04
PGC1α 1.00 ± 0.16 7.76 ± 0.32 **** 2.03 ± 0.25 **
193
Table 4.4 Changes in gene expression associated with endoderm-specific differentiation of hESCs.
Expression and qPCR expression fold changes were shown for gene associated with
pluripotency (POU5F1), endoderm differentiation (SOX17), genes encoded by mitochondria (ND5
and COX3) and mitochondrial biogenesis–related genes (TFAM and PGC1α). Data were normalised
to β-Actin (ACTB). Histogram is also shown in the Figure 4.5. Data were the mean ± SD. *P<0.05
as compared to control. Abbreviations: SOX17, SRY-box 17.
Genes Day 0 Day 1 Day 2 Day 3
SOX17 1.00 ± 0.14 5.57 ± 0.48 * 12.95 ± 2.43 **** 21.09 ± 0.85 ****
POUT5F1 1.00 ± 0.22 0.54 ± 0.13 * 0.63 ± 0.05 * 0.40 ± 0.03 **
ND5 1.00 ± 0.16 0.45 ± 0.03 *** 0.54 ± 0.01 *** 0.52 ± 0.01 ***
COX3 1.00 ± 0.02 0.92 ± 0.03 0.95 ± 0.08 0.89 ± 0.04
TFAM 1.00 ± 0.06 0.64 ± 0.07 * 0.69 ± 0.21 * 0.57 ± 0.01 **
PGC1α 1.00 ± 0.39 0.56 ± 0.11 0.85 ± 0.21 0.42 ± 0.08
194
Table 4.5 Changes in gene expression associated with mesendoderm-specific differentiation of
hESCs.
qPCR analysis was used to determine expression fold changes for genes associated with
pluripotency (POU5F1), ectoderm differentiation (PAX6), mesoderm differentiation (BRY and
MIXL1), endoderm differentiation (IGF2 and GATA4) and mitochondrial biogenesis (TFAM, NRF1
and POLG). A gene encoded by mtDNA (ND1) was also examined. Data were normalised to
β-Actin (ACTB). Histogram is also shown in the Figure 4.6. Data were the mean ± SD. *P<0.05 as
compared to control. Abbreviations: BRY, Brachyury; MIXL1, Mix paired-like homeobox; IGF2,
Insulin-like growth factor 2; GATA4, GATA binding protein4.
Genes Day 0 Day 3 Day 5 Day 7 Day 16
PAX6 1.00 ± 0.45 0.60 ± 0.15 0.32 ± 0.30 0.32 ± 0.38 3.07 ± 0.55 ***
BRY 1.00 ± 0.30 40.38 ± 0.22 ****
7.19 ± 0.40 **** 0.90 ± 0.14 0.27 ± 0.25
MIXL1 1.00 ± 0.23 14.70 ± 0.08 ****
13.35 ± 0.06 ****
7.83 ± 0.10 ****
2.86 ± 0.28 ****
IGF2 1.00 ± 0.09 27.94 ± 0.21 ****
305.36 ± 0.11 ****
924.35 ± 0.09 ****
2210.79 ± 0.18 ****
GATA4 1.00 ± 0.24 510.59 ± 0.13 ****
133.68 ± 0.22 ****
173.61 ± 0.12 ****
485.15 ± 0.15 ****
POU5F1 1.00 ± 0.05 0.50 ± 0.25 ** 0.14 ± 0.10 **** 0.03 ± 0.06 ****
0.00 ± 0.08 ****
ND1 1.00 ± 0.29 0.45 ± 0.20 N/A 2.21 ± 0.61** 1.02 ± 0.27
TFAM 1.00 ± 0.24 0.39 ± 0.58 0.46 ± 0.35 0.46 ± 0.59 0.30 ± 0.52
NRF1 1.00 ± 0.34 0.64 ± 0.24 0.57 ± 0.41 0.78 ± 0.22 0.64 ± 0.20
POLG 1.00 ± 0.08 0.86 ± 0.27 1.21 ± 0.07 1.68 ± 0.22 ** 1.13 ± 0.14
195
Table 4.6 Changes in gene expression associated with cardiomyocyte-specific differentiation of
hESCs.
qPCR analysis was used to determine expression fold changes for genes encoded by mtDNA
(ND5 and COX3) and genes related to mitochondrial biogenesis (TFAM and PGC1α). Data were
normalised to β-Actin (ACTB). Histogram is also shown in the Figure 4.7. Data were the mean ± SD.
*P<0.05 as compared to control.
Genes Day 0 Day 3 Day 7 Day 16
ND5 1.00 ± 0.22 0.92 ± 0.05 1.97 ± 0.22 *** 0.02 ± 0.01 ***
COX3 1.00 ± 0.54 0.95 ± 0.08 0.80 ± 0.10 0.61 ± 0.14
TFAM 1.00 ± 0.06 0.58 ± 0.07 *** 0.56 ± 0.11 *** 0.02 ± 0.01 ****
PGC1α 1.00 ± 0.59 1.01 ± 0.24 1.22 ± 0.21 252.75 ± 63.97 ****
196
Objective 2: Regulation of Mitochondrial Biogenesis in Chloramphenicol- and EtBr-treated
Primary Human Fibroblasts
Table 4.7 Gene expression and qPCR expression fold changes of mitochondria-encoded genes,
transcription factors, and mtDNA copy number in control, ethidium bromide– or
chloramphenicol-treated cells after treatment for 6 weeks and release from treatment for 2 weeks.
Expression of mitochondria-encoded genes (ND1, ND4, CYTB and COX2) and mitochondrial
biogenesis transcription factors (TFAM, NRF-1 and POLG) in each sample was normalised to
β-Actin (ACTB). Histogram is also shown in the Figure 4.9. Data were the mean ± SD. *P<0.05 as
compared to control. Abbreviations: ND4, NADH dehydrogenase subunit IV; COX2, cytochrome c
oxidase subunit II; control, human fibroblasts; EtBr-treated, human fibroblasts treated with 100
ng/ml ethidium bromide; and CAP-treated, human fibroblasts treated with 100 µg/ml
chloramphenicol.
197
Genes Control-6w EtBr-treated-6w CAP-treated-6w
ND1 0.12 ± 0.04 (1.00) 3.50x10
-6 ± 1.50x10
-6 *
(0.00) 8.33x10
-4 ± 1.49x10
-4 * (0.01)
ND4 0.16 ± 0.04 (1.00) 4.00x10
-7 ± 1.00 x10
-7 *
(0.00) 3.11x10
-5 ± 4.80x10
-6 * (0.00)
CYTB 0.91 ± 0.04 (1.00) 6.52x10
-4 ± 4.79x10
-5 *
(0.00) 0.45 ± 0.11* (0.50)
COX2 0.10 ± 0.02 (1.00) 6.00x10
-7 ± 3.00x10
-7 *
(0.00) 2.08x10
-3 ± 2.56x10
-4 * (0.02)
TFAM 1.70x10
-6 ± 8.00
x10-7
(1.00)
7.00x10-7
± 4.00x10-7
(0.42) 9.00x10
-7 ± 4.00x10
-7 (0.53)
NRF1 2.95x10
-6 ± 5.30
x10-6
(1.00)
5.60x10-6
± 8.00x10-7
* (0.19)
5.00x10-7
± 1 x10-8
* (0.02)
POLG 2.13x10
-4 ± 5.76 x10
-5
(1.00)
2.16x10-5
± 9.50x10-6 *
(0.10) 7.33x10
-5 ± 1.83x10
-5 *
(0.34)
D-loop 0.21 ± 0.11 (1.00) 5.28x10
-4 ± 3.60x10
-4 *
(0.00) 0.18 ± 0.03 (0.28)
Control-6+2w EtBr-treated-6+2w CAP-treated-6+2w
ND1 0.63 ± 0.12 (1.00) 0.04 ± 0.01 * (0.06) 0.19 ± 0.02* (0.30)
ND4 0.27 ± 0.03 (1.00) 0.02 ± 0.08 * (0.08) 0.42 ± 0.05 (1.56)
CYTB 0.04 ± 0.01 (1.00) 0.01 ± 0.001 * (0.06) 0.02 ± 0.01 (0.44)
COX2 0.35 ± 0.01 (1.00) 0.10 ± 0.03 * (0.28) 0.40 ± 0.18 (1.15)
TFAM 0.01 ± 0.001(1.00) 0.01 ± 0.001 * (4.05) 0.01 ± 0.001 * (3.09)
NRF1 0.01 ± 0.001 (1.00) 0.01 ± 0.001 * (2.29) 0.01 ± 0.001 (0.71)
POLG 0.01 ± 0.001 (1.00) 0.01 ± 0.001 * (0.92) 0.01 ± 0.002 * (1.77)
D-loop 0.91 ± 0.09 (1.00) 0.09 ± 0.03 * (0.10) 0.39 ± 0.04 * (0.42)
198
Table 4.8 Relative amounts of glucose, lactate, GSH, and GSSG, and intracellular ATP, ADP
concentrations in control, ethidium bromide– or chloramphenicol-treated cells after treatment for 6
weeks and release from treatment for 2 weeks.
The quantities of intracellular ATP concentration, intracellular ADP concentration, and
intracellular ATP/ADP ratio per cell were expressed in mg. The rate of lactate production or
glucose consumption per cell was expressed in pmol/cell. The background was subtracted from all
measurements, which were performed in triplicate. The quantities of GSH and GSSG per cell were
expressed in fmol/cell. Histogram is also shown in the Figure 4.10. Data were the mean ± SD.
*P<0.05 as compared to control.
Control EtBr-treated CAP-treated
Total GSH (fmol/cell) 0.92 ± 0.05 0.55 ± 0.04 * 3.84 ± 0.03 *
Oxidized GSSG (fmole/cell) 0.07 ± 0.01 0.54± 0.03 * 0.05
± 0.02
Reduced GSH (fmol/cell) 0.85± 0.03 0.02 ± 0.002 * 3.78 ± 0.03 *
Oxidized GSSG / reduced GSH (ratio)
0.09 35.00 * 0.01
ATP avg (uM) / protein (mg) 80.08 ± 36.82 1.20 ± 0.59 * 3.47 ± 1.26 *
ADP avg (uM) / protein (mg) 44.90 ± 14.88 2.75 ± 0.46 * 2.76 ± 0.55 *
ATP / ADP (ratio) 1.78 0.44 * 1.26 *
199
Table 4.9 Comparison of the mitochondrial membrane potential in control, ethidium bromide– or
chloramphenicol-treated cells after treatment for 6 weeks and release from treatment for 2 weeks.
Mitochondrial membrane potential staining by JC1: JC1-monomers and JC1-aggregates
indicate low and high mitochondrial membrane potential, respectively. The fluorescence intensity
of JC1-monomers and JC1-aggregates was indicated in mean ± SD. *P<0.05 as compared to
control. Histogram is also shown in the Figure 4.11.
Avg ± SD (Folds) JC1-monomers JC1-aggregates
Control 1892.00 ± 228.33 (1.00) 681.43 ± 130.90 (1.00)
Ethidium bromide-treated 1669.77 ± 98.98 (0.88) 397.20 ± 23.32 * (0.58 *)
Chloramphenicol-treated 2124.97 ± 39.65 (1.12) 577.30 ± 18.10 (0.85)
200
Objective 3: Manipulation of TFAM Expression in Human Embryonic Stem Cells
Table 4.10 Levels of TFAM expression in hESC lines (gain-of-function).
(A) TFAM expression and (B) mtDNA copy number (D-loop) were normalised to β-Actin (ACTB).
(C) TFAM protein levels were determined by flow cytometer, and were normalised to the negative
control. Histogram is also shown in the Figure 4.13. Data were the mean ± SD. *P<0.05 as
compared to control.
(A) TFAM mRNA expression
Mel 1 hES3 H9
hESCs 1.00 ± 0.10 1.00 ± 0.03 1.00 ± 0.14
hESC-Vector 0.32 ± 0.10 *** 0.76 ± 0.15 ** 0.95 ± 0.20
hESC-EF1α-TFAM 2.06 ± 0.03 **** 1.71 ± 0.15 * 1.65 ± 0.43
(B) D-loop expression
hESCs 1.00 ± 0.29 1.00 ± 0.10 1.00 ± 0.11
hESC-Vector 0.69 ± 0.07 0.67 ± 0.10 0.86 ± 0.15
hESC-EF1α-TFAM 1.42 ± 0.32 28.34 ± 0.51 **** 5.44 ± 0.50 **
(C) TFAM protein expression (fluorescence intensity)
hESCs N/A 22894.95 ± 4675.06 29,186.07 ± 5223.61
hESC-Vector N/A 4186.15 ± 1093.36 * N/A
hESC-EF1α-TFAM N/A 50394.71 ± 8123.81 ** 114737.50 ± 28940.68 *
201
Table 4.11 Levels of TFAM expression in hESC lines (loss-of-function).
(A) TFAM mRNA levels were normalised to β-Actin (ACTB). (B) TFAM protein levels were
determined via flow cytometer, and were normalised to the negative control. Histogram is also
shown in the Figure 4.15. Data were the mean ± SD. Abbreviations: dox, doxycycline. *P<0.05 as
compared to control.
TFAM mRNA expression
Mel 1 hES3 H9
(A) Without dox
With dox Without dox
With dox Without dox With dox
hESCs 1.00 ± 0.07
0.60 ± 0.02 1.00 ± 0.34 1.23 ± 0.11 1.00 ± 0.07 3.22 ± 0.64 *
TFAMi-3
1.00 ± 0.09
0.17 ± 0.02 *
1.00 ± 0.23 0.84 ± 0.18 *
1.00 ± 0.19 0.55 ± 0.19 *
TFAMi-5
1.00 ± 0.39
0.03 ± 0.01 *
N/A N/A 1.00 ± 0.25 0.17 ± 0.04 *
TFAMi-6
N/A N/A 1.00 ± 0.01 0.92 ± 0.02 1.00 ± 0.15 1.22 ± 0.21
TFAM protein expression (fluorescence intensity)
Mel 1 hES3 H9
(B) Without dox
With dox Without dox
With dox Without dox With dox
hESCs 4.17x104
± 1.27x10
3
5.50x104
± 8.05x103
1.97x104
± 7.46x102
2.01x104
± 3.27x102
1.58x104
± 1.23x103
1.58x104
± 8.05x102
TFAMi-3
6.77x104
± 2.28x10
3*
9.76x103
± 5.06x102
*
1.11x105
± 1.30x103
7.39x104
± 4.44x103
*
8.31x103
± 1.30x102
7.01x103
± 2.93x102
*
TFAMi-5
2.82x104
± 2.62x10
3*
5.16x103
± 1.71x102
*
N/A N/A 1.28 x104
± 5.03x102
7.62x103
± 4.65x102
*
TFAMi-6
N/A N/A 2.16x104
± 5.72x102
2.21x104
± 3.28x102
8.25x103
± 6.63x101
8.25x103
± 2.31x102
202
Objective 4: Mitophagy in Human Embryonic Stem Cells
Table 4.12. The number of autophagosomes and mitophagy events per hES cell.
Following the indicated treatment, the number of autophagosomes was calculated either from
the statistical data with the ANOVA or from images of single cells, which were exported from
AMNIS, respectively. The average number of mitophagy events was calculated manually from
live-cell flow cytometry images. Histogram is also shown in the Figure 4.20. *P<0.05 as compared
to control. Abbreviations: control, hES-LC3-GFP cells treated with DMSO; Rap, hES-LC3-GFP
cells treated with rapamycin; CCCP, hES-LC3-GFP cells treated with carbonyl cyanide
3-chlorophenylhydrazone; EtBr, hES-LC3-GFP cells treated with ethidium bromide; RA-3,
hES-LC3-GFP cells culture treated with retinoic acid for 3 days; Spd. Diff-7, hES-LC3-GFP cells
culture without basic fibroblast growth factor for 7 days; Spd. Diff-14, hES-LC3-GFP cells culture
without basic fibroblast growth factor for 14 days.
Number of autophagosomes
per cell was counted from
statistical data, exported from
AMNIS.
Number of autophagosomes and mitophagic
events per cell was manually counted from
images of single cell, exported from AMNIS.
Treatment Sample
size
Autophagosomes
(mean SEM)
Sample
size
Autophagosome
s (mean SEM)
Mitophagosomes
(mean SEM)
Control 995 1.87 1.00 * 270 0.69 0.02 * 0.65 0.02 *
Rap 1300 3.65 0.07 * 322 1.63 0.01 * 1.46 0.01 *
CCCP 1156 3.64 0.07 * 481 1.49 0.01 * 1.30 0.01 *
EtBr 1453 3.42 0.07 * 474 1.43 0.01 * 1.30 0.01 *
RA-3 1560 3.06 0.07 * 693 1.59 0.01 * 1.42 0.01 *
Spd. Diff-7 1906 3.02 0.06 * 613 1.37 0.02 * 1.24 0.02 *
Spd. Diff-14 209 2.08 0.12 * 86 0.83 0.03 0.81 0.03 *
203
Figures
Objective 1: Mitochondrial-Encoded and Mitochondrial Biogenesis-Related Gene Expression
During Lineage-Specific Differentiation of Human Embryonic Stem Cells
Figure 4.1. Schematic drawing of the experimental design for spontaneous and lineage-specific
differentiation in hESCs.
Time courses are shown for the protocols to induce (A) spontaneous differentiation, (B)
ectoderm-, (C) endoderm-, and (D) mesendoderm- and cardiomyocyte-differentiation in days.
204
* * *
*
205
Figure 4.2 Comparison of TRA-1-60 and mitochondrial staining in undifferentiated and
differentiated hESCs.
(A) Markers for pluripotency (TRA-1-60) and (B) mitochondria (MitoTracker Deep Red) were
analyzed in live hESCs. Analyzed samples included undifferentiated (control), 7 and 14 days of
spontaneous differentiation, and 3 days of RA treatment. Dot plots were analyzed by ACCURI
software. Black and red lines represent unstained and stained samples, respectively. (C) Graph
represents the fluorescence intensity associated with TRA-1-60 immunoreactivity (black) and
MitoTracker Red (grey). Data were normalised to the control, and reflect the mean ± standard
deviation (SD, n = 3). *P<0.05 as compared to control.
206
B
D
C
E
207
Figure 4.3 Characterization of mitochondria in undifferentiated and differentiated hESCs. D-loop
expression in Mel 1 and hES3.
(A) Gene expression of pluripotency (POU5F1), mtDNA-encoded genes (ND1 and CYTB), and
nuclear-encoded mitochondrial biogenesis genes (TFAM, NRF1, PLOG and PGC1α) in Mel 1(bar
without diagonal lines) and hES3 (bar with diagonal lines) cell lines was subjected to qPCR
analyzes both before and after differentiation, respectively. Analyzed samples of hESCs include
undifferentiated (white), 7 days of spontaneous differentiation (light grey), 14 days of spontaneous
differentiation (dark grey), and 3 days of RA treatment (black). Gene expression levels were
normalised to β-Actin (ACTB). Gene expression values are also shown in the Table 4.2. Data reflect
the mean ± SD. *P<0.05 as compared to control.
TEM analysis of mitochondria (M) in (B) undifferentiated and (C) 14 days of spontaneously
differentiated hESCs. JC-1 staining was used to assess mitochondrial-membrane potential of (D)
undifferentiated and (E) 14 days of spontaneously differentiated hESCs. Low and high membrane
potentials are indicated by green and red staining, respectively. Arrows indicate JC-1 staining.
Representative images are shown. Scale bars represent either 500 nm (B, C) or 100 m (D, E).
Abbreviations: POU5F1, POU class 5 homeobox 1; ND1, NADH dehydrogenase 1; CYTB,
cytochrome b; TFAM, mitochondrial transcription factor A; NRF1, nuclear respiratory factor 1;
POLG, DNA polymerase subunit gamma; PGC1α, peroxisome proliferator-activated receptor
gamma coactivator 1 alpha; β-Actin (ACTB), beta-actin; D-loop, displacement loop.
208
Figure 4.4 Mitochondrial biogenesis profiles during ectoderm differentiation.
A time course of ectoderm differentiation is shown. Undifferentiated hESC (white), day 8 (light
grey), and day 12 (black) of differentiation are shown. Gene expression values are also shown in the
Table 4.3. Analyzed genes included an ectoderm marker (PAX6), a pluripotency marker (POU5F1),
mtDNA-encoded genes (ND5 and COX3), and mitochondrial biogenesis genes (TFAM, NRF1,
POLG and PGC1α). All qPCR values were normalised to β-Actin (ACTB). Gene expression values
are also shown in the Table 4.3. Data are the mean ± SD. *P<0.05 as compared to control.
Abbreviations: PAX6, Paired box 6; ND5, NADH dehydrogenase 5; COX3, Cytochrome oxidase 3.
209
Figure 4.5 Mitochondrial biogenesis profiles during endoderm differentiation.
Time courses of ectoderm and endoderm differentiation are shown. Undifferentiated hESC
(white), day 1 (light grey), day 2 (dark grey), and day 3 (black) of differentiation are shown.
Analyzed genes included a gene associated with endoderm (SOX17), a pluripotency marker
(POU5F1), mtDNA-encoded genes (ND5 and COX3), and mitochondrial biogenesis genes (TFAM
and PGC1α). All qPCR values were normalised to β-Actin (ACTB). Gene expression values are also
shown in the Table 4.4. Data are the mean ± SD. *P<0.05 as compared to control. Abbreviations:
SOX17, SRY-box 17.
210
Figure 4.6 Mitochondrial biogenesis profiles during mesendoderm differentiation.
Time courses of ectoderm and mesendoderm differentiation are shown. Undifferentiated hESC
(white), day 3 (light grey), day 5 (dark grey), day 7 (black), and day 16 (white with diagonal lines)
of differentiation are shown. Analyzed genes included genes associated with the primitive streak
(BRY and MIXL1), an ectoderm marker (PAX6), endoderm markers (IGF2 and GATA4), a
pluripotency marker (POU5F1), a mtDNA-encoded gene (ND1), and mitochondrial biogenesis
genes (TFAM, NRF1 and POLG). All qPCR values were normalised to β-Actin (ACTB). Gene
expression values are also shown in the Table 4.5. Data are the mean ± SD. *P<0.05 as compared to
control. Abbreviations: BRY, Brachyury; MIXL1, Mix paired-like homeobox; IGF2, Insulin-like
growth factor 2; GATA4, GATA binding protein 4.
211
Figure 4.7 Mitochondrial biogenesis profiles during cardiomyocyte differentiation.
Time courses of cardiomyocyte differentiation are shown. Undifferentiated hESC (white), day
3 (light grey), day 7 (dark grey), and day 16 of differentiation (black) are represented. Analyzed
genes included mtDNA-encoded genes (ND5 and COX3), and mitochondrial biogenesis genes
(TFAM and PGC1α). All qPCR values were normalised to β-Actin (ACTB). Gene expression values
are also shown in the Table 4.6. Data are the mean ± SD. *P<0.05 as compared to control.
212
Objective 2: Regulation of Mitochondrial Biogenesis in Chloramphenicol- and EtBr-treated
Primary Human Fibroblasts
Figure 4.8 Morphology and karyotypes of human rho minus cells.
(A and B) Image of control and (E and F) ethidium bromide-treated and (I and J)
chloramphenicol-treated cells were taken by inverted microscopy (Olympus) with a 10x objective
lens. (C and D) G-band karyotypes of control and (G and H) ethidium bromide-treated and (K and L)
chloramphenicol-treated cells. Abbreviations: control, human fibroblasts; Ethidium bromide-treated,
human fibroblasts treated with 100 ng/ml ethidium bromide; and Chloramphenicol-treated, human
fibroblasts treated with 100 µg/ml chloramphenicol.
213
214
Figure 4.9 Relative expressions of mitochondrial-encoded and mitochondrial biogenesis-related
genes after treatment for 6 weeks (6w) and release from treatment for 2 weeks (6+2w, bar with
diagonal lines).
Mitochondria-encoded genes (ND1, ND4, CYTB and COX2), mitochondrial biogenesis
transcription factors (TFAM, NRF1 and POLG), and mitochondrial content (D-loop) in control
(black), EtBr-treated (grey), and CAP-treated fibroblasts (black) at 6 weeks (6w) and release from
treatment for 2 weeks (6+2w). Gene expression of each sample was normalized to β-ACTIN (ACTB).
Gene expression values are also shown in the Table 4.7. Data are the mean ± SD. *P<0.05 as
compared to control. Abbreviations: ND4, NADH dehydrogenase subunit 4; COX2, cytochrome c
oxidase subunit 2.
215
A B
C D
E
* *
*
216
Figure 4.10 Metabolic effect of ethidium bromide and chloramphenicol treatment on human
fibroblasts after treatment for 6 weeks and release from treatment for 2 weeks.
(A) The lactate production, (B) glucose consumption, and (C) ratio of oxidized GSSG to
reduced GSH after treatment for 6 weeks and release from treatment for 2 weeks. The background
was subtracted from all measurements, which were performed in triplicate. (D) Comparison of the
effect of ethidium bromide and chloramphenicol treatment on oxygen consumed by human
fibroblasts. The plot shows the oxygen concentration in the cell culture medium with time. The
decrease in oxygen concentration with time seen in the chloramphenicol-treated samples was due to
oxygen uptake by the cells. No oxygen uptake by cells treated with ethidium bromide (red) was
detectable with this assay. Red line indicates ethidium bromide-treated fibroblasts, and blue line
indicates chloramphenicol-treated fibroblasts. (E) Intracellular ATP/ADP ratio. The values are also
shown in the Table 4.8. Data are the mean ± SD. *P<0.05 as compared to control.
217
C
218
Figure 4.11 Mitochondrial membrane potential in ethidium bromide– or chloramphenicol-treated
cells after treatment for 6 weeks and release from treatment for 2 weeks.
Low membrane potential is indicated by JC-1 monomers (green), and high membrane potential
is indicated by JC-1 aggregates (red). (A) Fluorescent image of control, ethidium bromide-treated
and chloramphenicol-treated human fibroblasts cells. Images were taken with a 20× objective lens
with an Olympus IX81 microscope and analyzed with Cell^ software (Olympus). (B) Mitochondrial
membrane potential was analyzed by WEASEL software. (C) Histogram represents mean intensity
of JC1-monomers and -aggregates in control (white bar), ethidium bromide- (black bar) or
chloramphenicol-treated cells (grey bar). The values are also shown in the Table 4.9. Data are the
mean ± SD. *P<0.05 as compared to control.
219
Figure 4.12 Assessment of mitochondrial ultrastructure transmitted by electron microscopy after
treatment for 6 weeks and release from treatment for 2 weeks (Figure S10). Ultrastructure of
mitochondria of (A) control, (B) ethidium bromide-treated and (C) chloramphenicol-treated human
fibroblasts cells. The original magnification was 12 kV.
A: Control B: EtBr-treated C: CAP-treated
220
Objective 3: Manipulation of TFAM Expression in Human Embryonic Stem Cells
221
Figure 4.13 Levels of TFAM expression in hESC lines (gain-of-function).
(A) TFAM gene expression and mtDNA copy number (D-loop) for Mel 1 (white), hES3 (grey),
and H9 (black) single cells. Data were normalised to β-ACTIN (ACTB). (B) TFAM protein levels
were determined by flow cytometer. Data were normalised to the negative control. Data are the
mean ± SD. Gene expression values are also shown in the Table 4.10. Data are the mean ± SD.
*P<0.05 as compared to control. Abbreviations: Ctrl, hESCs without viral transduction; vector,
hESCs transduced with the vector backbone; TFAM, hESC transduced with EF1α-TFAM viral
particles.
222
Figure 4.14 Constitutive knockdown of TFAM levels in hESCs (Figure S9).
Three TFAM shRNA (TFAM-shRNA-1, TFAM-shRNA-2, and TFAM-shRNA-3) are targeted
to position 266-286, 591-611 and 618-638, respectively. (A) Western blot analysis of TFAM
expression in the control and lentivirus-transduced hESCs was performed using antibodies against
the proteins indicated on the left. β-ACTIN (ACTB) was included as a loading control. Untreated
hESCs were analyzed as a negative control. (B) TFAM expression in hESCs was quantified by
phosphor-imager analysis of the western blots.
223
*
*
* * * *
* *
*
* * *
* *
* *
*
224
Figure 4.15 Levels of TFAM expression in hESC lines (loss-of function, Figure S9).
(A) Levels of TFAM mRNA were determined for each sample using PCR. (B) Levels of
TFAM protein were determined for each sample using flow cytometry. (C) TFAM was analyzed by
CFLOW software. Different cell lines (Mel 1, hES3 or H9) transfected by different TFAM shRNA
(control: black; TFAMi-3: red; TFAMi-5: blue; TFAMi-6: green) are shown. mRNA levels (both
endogeneous and exogenous) were normalised to β-ACTIN (ACTB), whereas protein levels were
normalised to the negative controls. Gene expression values are also shown in the Table 4.11. Data
are the mean ± SD. *P<0.05 as compared to control. Abbreviations: dox, doxycycline; 1-TFAMi-3,
Mel 1 single cells transduced with TFAMi-3; 3-TFAMi-3, hES3 single cells transduced with
TFAMi-3; 9-TFAMi-3, H9 single cells transduced with TFAMi-3; 1-TFAMi-5, Mel 1 single cells
transduced with TFAMi-5; 9-TFAMi-5, H9 single cells transduced with TFAMi-5; 1-TFAMi-6,
Mel 1 single cells transduced with targetless shRNA; 3-TFAMi-6, hES3 single cells transduced
with targetless shRNA; 9-TFAMi-6, H9 single cells transduced with targetless shRNA.
225
Figure 4.16 Morphological phenotypes associated with TFAM loss- and gain-of function in hESC
lines.
Images represent hESCs incubated in the presence or absence of doxycycline (controls). TFAM
knockdown (TFAMi-3 of targeting exon 5; TFAMi-5 of targeting exon 7; TFAMi-6 of targetless) is
shown for Mel 1, and H9 cells. Images were acquired using inverted microscopy (Olympus) and a
10 x objective.
226
Objective 4: Mitophagy in Human Embryonic Stem Cells
Figure 4.17 Characterization of the hES-LC3-GFP reporter cell line.
(A) LC3-GFP localisation within hESCs. (B) Localisation of POU5F1 (left, red) and TRA-1-60
(right, green) within the hES-LC3-GFP cell line. Nuclei are counterstained with DAPI (blue).
Representative images are shown. Scale bars represent 50 m.
227
A Control
B Rap
C CCCP
D EtBr
E Spd. Diff-7
F EtBr-16
hours
G EtBr-1 week
H Control
228
Figure 4.18 Mitochondrial toxin and differentiation-induced mitophagy in hES-LC3-GFP cells.
Images represented from left to right in time manner. Images are combination of LC3-GFP
signal (green), and MitoTracker Red signal (red) channels. Treatment conditions are listed at left: (A)
control (DMSO), (B) rapamycin (positive control, 200 nM) in 0, 5 and 10 minutes, respectively, (C)
CCCP (mitophagy induction, 50 µM) in 0, 7 and 15 minutes, (D) EtBr (mitophagy induction, 100
ng/ml) treatment, (E) 7 days of bFGF withdrawal (spontaneous differentiation) in 0, 10 and 20
minutes, (F) EtBr (100 ng/ml) treatment in 0, 8 and 16 hours, (G) EtBr (100 ng/ml for 1 week)
treatment in 0, 8 and 16 hours, and (H) control in 0, 8, 16 hours, respectively. Representative images
from supplementary videos and Appendix Figures 7 and 8 are shown and mitophagy events are
circled by red circles. Scale bars represent 50 m. Abbreviations: control, hES-LC3-GFP cells
treated with DMSO; Rap, hES-LC3-GFP cells treated with Rapamycin; CCCP, hES-LC3-GFP cells
treated with carbonyl cyanide 3-chlorophenylhydrazone; EtBr, hES-LC3-GFP cells treated with
ethidium bromide; Spd. Diff-7, hES-LC3-GFP cells culture without basic fibroblast growth factor
for 7 days.
229
A. Control
B. Rapaymin
C. CCCP
D. EtBr
E. RA-3
F. Spd. Diff-7
230
C. Rapamycin-induction CCCP-induction EtBr-induction
231
Figure 4.19 Quantification of mitophagy in hES-LC3-GFP cells using live-imaging flow-cytometer.
(A) Representative images of single cells are shown. From left to right, images are bright-field,
LC3-GFP signal (green), MitoTracker Red signal (red), and combined green and red channels. (B)
The number of autophagosomes per cell was determined for each treatment condition. Data were
displayed as a histogram. Compared to control cells, each treatment condition resulted in a
statistically significant increase in autophagic events (p < 0.05). Treatment conditions are listed at
left and included control (DMSO, blue), rapamycin (positive control), CCCP (mitophagy induction),
EtBr (mitophagy induction), 3 days of retinoic acid (RA) treatment (induced differentiation), 7 days
of bFGF withdrawal (spontaneous differentiation), and 14 days of bFGF withdrawal (spontaneous
differentiation), (C) TEM images of mitochondrial ultrastructure following treatment with
rapamycin, CCCP, or EtBr. Autophagic vacuoles containing mitochondria (black arrows) were
found for all treatment conditions. Scale bars represent 1 m. Abbreviations: control,
hES-LC3-GFP cells treated with DMSO; Rap, hES-LC3-GFP cells treated with rapamycin; CCCP,
hES-LC3-GFP cells treated with carbonyl cyanide 3-chlorophenylhydrazone; EtBr, hES-LC3-GFP
cells treated with ethidium bromide; Spd. Diff-7, hES-LC3-GFP cells culture without basic
fibroblast growth factor for 7 days; Spd. Diff-14, hES-LC3-GFP cells culture without basic
fibroblast growth factor for 14 days; RA-3, hES-LC3-GFP cells culture treated with retinoic acid
for 3 days.
232
Figure 4.20 Quantification of autophagosomes and mitophagosomes in hESCs.
The average number of autophagosomes per cell was calculated automatically using the
ImageStream system software (white), the average number of autophagosomes (grey) and the
average number of mitophagosomes (black) were calculated manually from live-cell flow cytometry
images. The values are also shown in the Table 4.12. Data are the mean ± SD. *P<0.05 as compared
to control.
* * * * * * * * * * * * * *
233
Videos:
Video 4.1: Live-imaging of mitophagy in hES-LC3-GFP.
Representative video of combination of LC3-GFP signal (green), and MitoTracker Red signal
(red) is shown. Images were captured every 1 minute for 10 cycles. Scale bars represent 50 m.
Video 4.2: Live-imaging of mitophagy in hES-LC3-GFP with 200 nM rapamycin.
Representative video of combination of LC3-GFP signal (green), and MitoTracker Red signal
(red) is shown. Images were captured every 1 minute for 10 cycles. Scale bars represent 50 m.
Video 4.3: Live-imaging of mitophagy in hES-LC3-GFP with 50 µM CCCP.
Representative video of combination of LC3-GFP signal (green), and MitoTracker Red signal
(red) is shown. Images were captured every 1 minute for 15 cycles. Scale bars represent 50 m.
Abbreviation: CCCP, carbonyl cyanide 3-chlorophenylhydrazone
Video 4.4: Live-imaging of mitophagy in hES-LC3-GFP with 100 ng/ml EtBr.
Representative video of combination of LC3-GFP signal (green), and MitoTracker Red signal
(red) is shown. Images were captured every 1 minute for 20 cycles. Scale bars represent 50 m.
Abbreviation: EtBr, ethidium bromide.
Video 4.5: Live-imaging of mitophagy in spontaneously differentiated hES-LC3-GFP without basic
fibroblast growth factor for 7 days.
234
Representative video of combination of LC3-GFP signal (green), and MitoTracker Red signal
(red) is shown. Images were captured every 1 minute for 20 cycles. Scale bars represent 50 m.
Video 4.6: Live-imaging of mitophagy in hES-LC3-GFP with 100 ng/ml EtBr.
Representative video of combination of LC3-GFP signal (green), and MitoTracker Red signal
(red) is shown. Images were captured in 0, 8th
and 16th
hour.
Video 4.7: Live-imaging of mitophagy in hES-LC3-GFP with 100 ng/ml EtBr for one week.
Representative video of combination of LC3-GFP signal (green) and MitoTracker Red signal
(red) is shown. Images were captured in 0, 8th
and 16th
hour.
Video 4.8: Live-imaging of mitophagy in hES-LC3-GFP for one week.
Representative video of combination of LC3-GFP signal (green) and MitoTracker Red signal
(red) is shown. Images were captured in 0, 8th
and 16th
hour.
235
Appendix
Appendix Table 1. List of Abbreviations
Units
h Hours
w Weeks
bp Base pairs
SDS Sodium dodecyl sulphate
SD Standard deviation
rRNA Ribosomal RNA
tRNA Transfer RNA
Β-ACTIN (ACTB) Actin, beta
GAPDH Glyceraldehyde-3-phosphate dehydrogenase
Dox Doxycyclin
VSV-G Vesicular stomatitis virus glycoprotein G
VP Viral particles
WPRE Woodchuck hepatitis virus post-transcriptional regulatory element
PB Phosphate buffer
PBS Phosphate-buffered saline
DMEM Dulbecoo‟s Modified Eagle‟s Medium
EGFP Enhanced green fluorescent protein
GFP Green fluorescent protein
HEK 293 Human embryonic kidney cell line
Stem cells
dpc Days post coitum
ASC Adult stem cells
ASCC Australian Stem Cell Centre
236
b-FGF Basic Fibroblast Growth Factor
CM Conditioned medium
E Embryonic day
EC Embryonal carcinoma
EBs Embryonic bodies
hESCs Human embryonic stem cells
hiPSCs Human induced pluripotent stem cell
HSCs Haemoatopoietic stem cells
HTERT Human telomerase reverse transcriptase
ICM Inner cell mass
ID Inhibitor of differentiation
IVF In vitro fertilization
KSR Knockout serum replacement
NANOG Nanog homeobox
NEAA Non-Essential Amino Acids
NFκB Nuclear Factor Kappa B
MESC Mouse embryonic stem cell
PGCS Primordial germ cells
PSCS Pluripotent stem cells
POU5F1 or OCT4 POU class 5 homeobox 1
SCID Severe combined immunodeficient
SOX2 SRY (sex determining region Y)-box 2
SSEA Stage-specific embryonic antigen
TOR The Target Of Rapamycin
Mitochondrion
ΔΨ Mitochondrial membrane potential
ADP Adenosine diphosphate
237
ATP Adenosine triphosphate
ATP6 ATP synthase subunit VI (Complex V)
ATP8 ATP synthase subunit VIII (Complex V)
CAP Chloramphenicol
CCCP Carbonyl cyanide 3-chlorophenylhydrazone
COX1-3 Cytochrome c oxidase subunit I-III (Complex IV)
CYTB Cytochrome b (Complex III)
D-loop Displacement loop
ER Endoplasmic reticulum
EtBr Ethidium bromide
ETC Electron transport chain
HMG High-mobility group
IMM Inner mitochondrial membrane
JC1 5, 5‟, 6, 6‟-tetrachloro-1, 1‟, 3, 3‟-tetraethylbenzimidazolylcarbocyanine
iodide
MD Mitochondrial dysfunction
MDS Mitochondrial depletion syndromes
MRC Mitochondrial respiratory chain
mtDNA Mitochondrial DNA
ND1-6 NADH dehydrogenase subunit I-VI (Complex I)
NRF-1 Nuclear respiratory factor I
NRF-2 (GABP) GA binding protein transcription factor, alpha subunit
ρ˚ cells Mitochondrial depleted cells
PD Parkinson disease
PGC1α/PGC1A Homo sapiens peroxisome proliferator-activated receptor gamma,
coactivator 1 alpha (PPARGC1A), mRNA
POLG Polymerase gamma
POLRMT Polymerase (RNA) mitochondrial (DNA directed)
PPP Pentose phosphate pathway
238
ROS Reactive oxygen species
TFAM Mitochondrial transcription factor A
SOD Superoxide dismutase
Autophagy
ATG Autophagy-related genes
LC3 Microtubules-associated protein light chain 3
PAS Pre-autophagosomal structure
TOR Target of rapamycin
Techniques
FACS Fluorescence-activated cell sorting
PCR Polymerase chain reaction
qPCR Quantitative polymerase chain reaction
RISC RNA-induced silencing complex
RNAi RNA interference
SCNT Somatic cell nuclear transfer
shRNA Short hairpin RNA
siRNA Small interfering RNA
SCNT Somatic cell nuclear transfer
TEM Transmission electron microscopy
239
Appendix Table 2. Cell lines used in this study
Cell lines Description Reference
Normal human
dermal fibroblasts
(HFF)
Normal human dermal fibroblasts and cultured
with F-DMEM.
ATCC: ATCC
PCS-201-012
Human
fibroblast-treated
with EtBr
Human fetal fibroblast cells and cultured with
F-DMEM, addition of uridine. In-House
Human
fibroblast-treated
with chloramphenicol
(CAP)
Human fetal fibroblast cells and cultured with
F-DMEM, addition of uridine. In-House
HEK 293T
Called as 293T/17, Human embryonic kidney
cells (Clone 17) expressing T antigen for plasmid
episomal replication and cultured with
F-DMEM.
Invitrogen,
ATCC: CRL-11268
Human foreskin
fibroblasts (hFFi) or
mouse embryonic
fibroblasts (mEFi)
Fibroblasts were collected and irradiated in 0.33
Х 106 cells/cm
2 and cultured. They are
co-cultured with hESC or conditioned medium
collection purpose.
ASCC
Mel, hES3, H9
Human embryonic stem cells and cultured with
KSR on hEFi or culture with conditioned
medium on Matrigel.
ASCC
240
Appendix Table 3-A. Cell Culture Medium Recipes: The various culture medium compositions
and recipes used in this thesis are listed as below, respectively. Where required, medium
formulations were filtered using a 0.22 μm low protein binding syringe filter to ensure sterility.
Materials Abbreviation
Company Cat. No Description
Tissue culture flasks
and plates
B&D Bioscience
NA Tissue culture plastic for cell culture
Tissue culture plates B&D Bioscience
NA Tissue culture plastic for cell culture
High glucose Dulbecco‟s Modified Eagles Medium
HG-DMEM Invitrogen 11965 DMEM containing high glucose (4 g/l) with sodium pyruvate
Low glucose Dulbecco‟s Modified Eagles Medium
LG-DMEM Invitrogen 11865 DMEM containing high glucose (1 g/l) with sodium pyruvate
Phosphate buffered saline PBS Invitrogen E404 Buffered saline with required pH and osmolarity for cells
Fetal Bovine Serum FBS Invitrogen 10099-141
FBS used in this was from Australian cows with no BVDV and demonstrated ability of culture HEK293, and fibroblasts cells.
Knockout Serum Replacement
KSR Invitrogen 10828-028
KSR used to culture human embryonic stem cells and conditioned medium collection.
TrypLE Express TrypLE Invitrogen 12605 Recombinant trypsin solution
Dimethylsulphoxide DMSO Sigma-Aldrich
D2438 Solvent used for efficient cryopreservation of cells to dissolve reagents.
Ethanol Sigma-Aldrich
E7023 Solvent used for efficient to dissolve reagents.
Basic fibroblast growth factor
bFGF Millipore GF-003-AF
Growth factor for maintaining hESCs.
Activin A R&D Systems
338-AC
Growth factor for the differentiation of hESCs into primitive streak cells
Bone morphogenic BMP-4 R&D 314-BP Growth factor for the
241
protein-4 Systems differentiation of hESCs into primitive streak cells
Bovine serum albumin BSA Sigma-Aldrich
A9647 Protein presented at a very high concentrations in serum
Sodium pyruvate Sigma-Aldrich
P5607 Non-glucose substrate for cells
Penicillin/Strptomycin P/S Invitrogen 1750-063
Antibiotic formation for cell in cell culture to reduce contamination
MEM Non-Essential Amino Acids Solution
NEAA Invitrogen 11140-050
Supplement for culture hESCs.
β-mercaptoethanol B-Me Sigma-Aldrich
M6250 Supplement for culture hESCs.
L-Glutamine L-Glu Invitrogen 25030081
Supplement for culture hESCs.
Puromycin dihydrochloride Puromycin Sigma-Aldrich
P7130 Antibiotic formation for selecting the stable hESC lines
Blasticidin Invitrogen R210-01
Antibiotic formation for selecting the stable hESC lines
Geneticine Invitrogen 10131-027
Antibiotic formation for selected the stable hESC lines
Chloramphenicol CAP Sigma-Aldrich
C0378 Reagent for depleting mitochondria in cell culture
Ethidium bromide EtBr Sigma-Aldrich
E7637 Reagent for depleting mitochondria in cell culture
Carbonyl cyanide 3-chlorophenylhydrazone
CCCP Sigma-Aldrich
C2759 Uncoupler of oxidative phosphorylation in mitochondria
Rapamycin Rap Sigma-Aldrich
R8781 Induced autophagosomes in hESCs
Uridine Sigma-Aldrich
U3003 Supplement for mitochondrial-depleted cells
Appendix Table 3-B. Cell Culture Medium Recipes
242
hESCs growth medium:
80% KnockOut Dulbecco‟s modified Eagle‟s medium nutrient mixture
F-12, (KO-DMEM F-12, Invitrogen) 394 ml
20% Knock-Out Serum Replacement (KOSR) (Invitrogen) 100 ml
1% NEAA (Gibco BRL) 5 ml
0.1 mM β-mercaptoethanol (Sigma-Aldrich) 928 μl
4 ng/ml human recombinant basic fibroblast growth factor (bFGF)
(Invitrogen) (fresh addition)
60,000 cells/cm2 of mEFi or hFFi were seeded for conditioned medium
collection for 7 days and filtered before used.
Inactivated human foreskin fibroblast (hFFi) as feeder growth medium
90% Dulbecco‟s modified Eagle‟s medium, (KO-DMEM F-12,
Invitrogen) 440 ml
10% FBS (Invitrogen) 50 ml
1% L-Glu (Gibco BRL) 5 ml
1% NEAA (Gibco BRL) 5 ml
Human fibroblast cells (hFF) growth medium:
90% Dulbecco‟s modified Eagle‟s medium, (DMEM, Invitrogen) 435 ml
10% FBS (Invitrogen) 50 ml
1% L-Glu (Gibco) 5 ml
1% NEAA (Gibco) 5 ml
1mM Sodium pyruvate (Gibco) 5 ml
Human fibroblast cells (hFF) with ethidium bromide or chloramphenicol-treated growth
medium
90% Dulbecco‟s modified Eagle‟s medium, (DMEM, Invitrogen) 430 ml
10% FBS (Invitrogen) 50 ml
1% L-Glu (Gibco) 5 ml
1% NEAA (Gibco) 5 ml
1mM Sodium pyruvate (Gibco) 5 ml
1% of 50 mg/ml uridine (Sigma-Aldrich) 5 ml
243
Ethidium bromide (EtBr) (100 ng/ml) or chloramphenicol (CAP) (100
μg/ml) (Sigma-Aldrich)
HEK 293T (ATCC: CCRL-11268) growth medium:
90% Dulbecco‟s modified Eagle‟s medium, (DMEM, Invitrogen) 445 ml
10% FBS (Invitrogen) 50 ml
500 ng/ml Geneticine (Gibco) 5 ml
Cryopreservation medium for hFF, mEFi, and HEK293T
90% FBS 900 µl
10% DMSO 100 µl
Cryopreservation medium for hESCs
Conditioned media 900 µl
10% DMSO 100 µl
244
Appendix Table 4. Molecular Biology Reagents
Materials Abbreviation Company Cat. No Description
Luria Broth Base LB Sigma-Aldrich
L1900 Bacteria growth
QIAquick Gel Extraction Kit
QIAGEN 28704 Gel extraction
NucleoSpin®
Plasmid QuickPure
Macherey 740615.10 Plasmid extraction for viruses production purpose
NucleoBond®
Xtra Midi /
Maxi Macherey 740410.10 Plasmid
extraction from bacteria
RNeasy Mini Kit QIAGEN 74104 Total RNA extraction
DNeasy Blood & Tissue Kit
QIAGEN 69504 Total genomic DNA extraction
MMLV Reverse transcriptase
MMLV Invitrogen AM2043 Moloney Murine Leukemia Virus Reverse Transcriptase for cDNA synthesis
Oligo d(T)12-18
Primers
Invitrogen
Integrated DNA Technologies (IDT)
12577011
N/A
Primers for cDNA synthesis or PCR reactions.
RNase Sigma-Aldrich
R6513 Apply for DNA and plasmid DNA extraction.
Protease K Roche 03115828001 Dissolve 1 mg/ml of protease K in 1 ml of double-distilled water.
245
Protease inhibitors cocktail
Roche 05 892 791 001 Combination of protease inhibitors in 10 ml of PBS in cell lysate for Western Blot
DNase
Deoxynucleotide solution mix
1 kb and 100 bp DNA ladders
Restriction enzymes
dNTP
New England Biolabs (NEB)
M0303L
N0447
N332, N3231
Molecular biology purpose
SoSoFast SYBR reagent
SYBR green
Bio-Rad
ABI
172-5200
4309155
For quantitative real-time PCR.
Goat Serum
Sigma-Aldrich
G9023 Blocking reagent for immunostaining
Bromophenol Blue
β-mercaptoethanol
BME
Sigma-Aldrich
B0126
M6250
Dye
BME is suitable for reducing protein disulfide bonds prior to polyacrylamide gel electrophoresis and apply in protein lysis buffer.
Tween-20 Sigma-Aldrich
P1379 Buffer detergent
Triton X-100 Sigma-Aldrich
T9284 Cell permeabilisation detergent
Milli-Q water In house NA Ultra-pure water
Tris (hydroxymethyl-amino
TRIS Amerseco 0234 Buffer salt
246
methane
Ethylenediaminetetraacetic acid
EDTA Ajax Finechem
A180 Metal chelator
Hydrogen chloride HCl Ajax Finechem
256-2.5L-GL Buffer salt
Tryphan Blue Sigma-Aldrich
T8254 Satins dead but not live cells
Glycerol Sigma-Aldrich
5516 Used in slide embedding solution
Permount Mounting medium
Invitrogen 00810 Slide mounting medium
Doxycycline Dox Sigma-Aldrich
D9891 Doxycycline is a member of the tetracycline antibiotics group, and applied in tet-on induce system.
Retinoic Acid RA Sigma-Aldrich
R2625 Retinoic Acid applied for hESCs differentiation
LipofectamineTM
LTX & Plus Reagent
Invitrogen 15338-199 For plasmid DNA delivery.
Total Glutathione Detection Kit
Assay Designs
900-160 Total Glutathione Detection in mitochondrial –depleted fibroblast cells.
Paraformaldehyde PFA Sigma-Aldrich
P6148 Fixative
Sodium dodecyl sulphate
SDS Sigma-Aldrich
71728 Detergent for decellularisation process
247
Appendix Table 5. Buffer Receipts Applied in Molecular Biology Techniques
6x DNA loading dye (250 ml)
80% glycerol 93.6 ml
0.5 M EDTA 3 ml
Bromophenol Blue 0.3 g
Xyanol 0.3 g
50x TAE (1L)
Tris base 242 g
Glacial acetic acid 57.1 ml
0.5 M EDTA, pH 8.0 100 ml
0.5 M EDTA, pH 8.0 (M.W. 336.2) (500 ml)
EDTA 84.05 g
5 M NaOH 71 ml
3 M sodium acetate, pH 8.0 (500 ml)
Sodium acetate 204.15 g
Glacial acetic acid 90 ml
248
Appendix Table 6. PCR Primer Sequences
Genes Forward 5‟-3‟
Reverse 5‟ 3‟ Tm Eff Size Acc. No.
Beta-ACTIN GCTGTGCTACGTCGCCCTG GGAGGAGCTGGAAGCAGCC
60 0.983 60 NM_001101
OCT4 TGAAGCTGGAGAAGGAGAAG ATCGGCCTGTGTATATCCC
60 0.985 134
NM_002701, NM_203289, NM_001173531
NANOG GATTTGTGGGCCTGAAGAAA AAGTGGGTTGTTTGCCTTTG
60 0.984 101 NM_024865
SOX2 AACCCCAAGATGCACAACTC CGGGGCCGGTATTTA TAATC
60 0.996 52 NM_003106
MtDNA copy number
D-loop CCCTAACACCAGCCTAACCA GTTAGCAGCGGTGTGTGTGT
60 0.997 165 NC_012920
Mitochondria-encoded genes
MT-ND1 ATGGCCAACCTCCTACTCCT GCGGTGATGTAGAGGGTGAT
52 0.999 214 YP_003024026
MT-ND4 CCTGACTCCTACCCCTCACA
ATCGGGTGATGATAGCCAAG
52 0.990 150 YP_003024035
MT-ND5 ACATCTGTACCCACGCCTTC TCGATGATGTGGTCTTTGGA
60 0.987 189 YP_003024036
MT-COX2 TTCATGATCACGCCCTCATA TAAAGGATGCGTAGGGATGG
52 0.999 186 YP_003024029
MT-COX3 CCCGCTAAATCCCCTAGAAG GGAAGCCTGTGGCTACAAAA
60 0.990 82 YP_003024032
MT-CYTB TATCCGCCATCCCATACATT GGTGATTCCTAGGGGGTTGT
52 0.996 169 YP_003024038
Mitochondrial biogenesis-related genes
TFAM CCGAGGTGGTTTTCATCTGT TCCGCCCTATAAGCATCTTG
52 0.995 203 NM_003201
NRF-1 ACAGAAGAGCAAAAGCAGAG
GCTGATCTTCAAAGGCATAC 60 0.831 111 NM_0010
40110
POLG CAAGGTCCAGAGAGAAACTG 60 0.951 116 NM _0011261
249
GCAATGCTCTCAAGCTTATT 31
PGC1α CCATATTCCAGGTCAAGATCAAG TCACATACAAGGGAGAATTTCG
60 0.997 118 NM_013261
Ectoderm differentiation-markers
PAX6 CAGCACCAGTGTCTACCAACCA CAGATGTGAAGGAGGAAACCG
60 0.995 67 NM_000280
Mesoderm differentiation-markers
BRACHYURY (BRY)
TCAGCAAAGTCAAGCTCACCA
CCCCAACTCTCACTATGTGGATT 60 0.98 102
NM_003181
MIXL1 CCGAGTCCAGGATCCAGGTA
CTCTGACGCCGAGACTTGG 60 0.95 58
NM_031944
Endoderm differentiation-markers
GATA4 TCTCACTATGGGCACAGCAG
GGGACAGCTTCAGAGCAGAC 60 0.98 116
NM_008092
IGF2 TGGCATCGTTGAGGAGTGCTGT ACGGGGTATCTGGGGAAGTTGT
60 0.995 136
NM_000612
NM_001007139
NM_001127598
SOX17 GGCGCAGCAGAATCCAGA CCACGACTTGCCCAGCAT
60 0.995 59 NM_022454
Cloning
GFP CTGGTCGAGCTGGACGGCGACG
CACGAACTCCAGCAGGACCATG 60 N/A 631 N/A
TFAM
CACCATGGCGTTTCT CCGAAG
TTAACACTCCTCAGCACCATA TTTTCGTTG
60 N/A 741 NM_003201
250
Appendix Table 7. Target sequences of shRNAs against human TFAM
shRNA cloned into constitutive knockdown plasmid (the pLenti5-U6-TFAMi, Invitrogen)
Name Sequence (5‟-3‟)
TFAMi-1
(266-286)
F:CCGGGGGAACTTCCTGATTCAAAGACTCGAGTCTTTGAATCAG
GAAGTTCCCTTTTTG
R:AATTCAAAAAGGGAACTTCCTGATTCAAAGACTCGAGTCTTTG
AATCAGGAAGTTCCC
TFAMi-2
(591-611)
F:CCGGGGAATTATATATTCAGCATGCCTCGAGGCATGCTGAATA
TATAATTCCTTTTTG
R:AATTCAAAAAGGAATTATATATTCAGCATGCCTCGAGGCATG
CTGAATATATAATTCC
TFAMi-3
(618-638)
F:CCGGGGACGAAACTCGTTATCATAACTCGAGTTATGATAACGA
GTTTCGTCCTTTTTG
R:AATTCAAAAAGGACGAAACTCGTTATCATAACTCGAGTTATG
ATAACGAGTTTCGTCC
shRNA cloned into inducible knockdown plasmid (the pLKO-Tet-on-TFAMi)
Name Sequence (5‟-3‟)
TFAMi-3
(489-509)
F:CCGGCGTTTATGTAGCTGAAAGATTCTCGAGAATCTTTCAGCT
ACATAAACGTTTTTG
R:AATTCAAAAACGTTTATGTAGCTGAAAGATTCTCGAGAATCTT
TCAGCTACATAAACG
TFAMi-5
(7764-784)
F:CCGGCGTGAGTATATTGATCCAGAACTCGAGTTCTGGATCAAT
ATACTCACGTTTTTG
R:AATTCAAAAACGTGAGTATATTGATCCAGAACTCGAGTTCTGG
ATCAATATACTCACG
TFAMi-6
(Targetless-
shRNA)
F:CCGGCCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTT
AACCTTAGGTTTTTG
R:AATTCAAAAACCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGG
GCGACTTAACCTTAGG (Sarbassov, Guertin, et al. 2005).
F: Forward; and R: Reverse
251
Appendix Table 8. Buffers for Immunostaining and Microscopy
Phosphate buffer saline (PBS) 200 ml
PBS tablet 1 tablet
Make up with ddH2O to 200 ml
4% Paraformaldehyde, pH 7.5 100 ml
Paraformaldehyde 4 g
Make up with 1x PBS to 100 ml and used NaOH to adjust the pH to 7.5 and aliquot
for -20°C storage.
Permeabilization buffer 500 ml
10% Triton 500 μl
Make up with 1x PBS to 500 ml.
Hoechst 33258 or DAP 1 ml
Hoechst 33258 or DAPI 10 mg
Make up with ddH2O to 1 ml and aliquot into small aliquots.
Blocking buffer
5% skim milk power in PBS or 1% bovine serum albumin (BSA)
Make up with 1x PBS
10x Phosphate buffer (PB) 1 L
1.37 M NaCl 80 g
30 mM KCl 2 g
65 mM Na2HPO4H2O 17.8 g
15 mM KH2PO4 2.4 g
Make up with ddH2O to 1 L.
252
Appendix Table 9-A. Working Concentrations for fixed-cell-immunostaining
Antibody Application Company Animal Source
Dilution
Anti-human-TRA-1-60 IC; FC Millipore mouse 1:500 (to 1 mg/ml)
Anti-human-POU5F1 IC; WB Sigma-Aldrich rabbit 1:500
Anti-human-β-ACTIN
(45 kDa) WB
Cell Signaling Biotechnology
rabbit 1:2000
Anti-human-TFAM (24 kDa) WB; FC
Cell Signaling Biotechnology
rabbit 1:2000
Anti-mouse IgG Alexa Fluro 488 IC Invitrogen goat 1:2000
Anti-rabbit IgG Alexa Fluro 568 IC Invitrogen goat 1:2000
Anti-mouse IgG HRP WB Santa Cruz Biotechnology
goat 1:4000 (to 200 μg/0.5 ml)
Anti-rabbit IgG HRP WB Santa Cruz Biotechnology
goat 1:4000 (to 200 μg/0.5 ml)
Note: skim milk was used as blocking in Western Blotting, and 1% BSA was used as blocking in fixed-cell immunostaining and flow cytometry.
IC, Immunocytochemistry; FC, Flow cytometry; WB, Immunoblotting
253
Appendix Table 9-B. Working Concentrations for live-cell-staining
Materials Application Company Cat. No Description
MitoTracker Red IC; IFC Invitrogen M22425
Mitochondria staining dye excitated at 581 nm for imaging purpose: (Flow cytometry use 100 nM final concentration; immunocytochemistry use 100 nM final concentration)
MitoTracker Deep Red FC Invitrogen M22426
Mitochondria staining dye excitated at 644 nm for AMNIS purpose: (Flow cytometry use 100 nM final concentration; immunocytochemistry use 100 nM final concentration)
Mitochondria staining kit (JC-1)
IC; FC Sigma-Aldrich CS0390
For mitochondrial potential changes detection: (Flow cytometry use 0.1 µg/ml final concentration of JC1;immunocytochemistry use 0.1 µg/ml final concentration of JC1)
IC, Immunocytochemistry; IFC, Imaging flow cytometry; FC, Flow cytometry
Reference:
Sarbassov, D. D., D. A. Guertin, S. M. Ali and D. M. Sabatini (2005). “Phosphorylation and
regulation of Akt/PKB by the rictor-mTOR complex.” Science 307 (5712):1098-1101.
255
Supplementary Method
Immunoblotting
Western blot analysis was performed on cytosolic of hESCs with drugs as described in
this thesis. First the cells were lyzed with an extraction buffer (Tris 10 mM, pH 7.0, NaCl 140
mM, PMSF 2 mM, DTT 5 mM, NP-40 0.5%, pepstatin A 0.05 mM and leupeptin 0.2 mM) for
protein extraction, and then centrifuged at 12,500 g for 30 min. To measure the expression
levels of proteins, the total proteins were extracted and Western blotting analyses were
performed as established in the Wolvetang laboratory. An aliquot of 20 μg total protein of
each cell lysates was loaded and separated on 10-15% Tris-Glycine-SDS polyacrylamide
gels and electroblotted onto PVDF membranes (PALL Life, Science, Pensacola, FL). After
the incubation with 5% fat-free skim milk in TBS buffer (10mM Tris, 150mM Nacl, pH7.4) for
1 hour at room temperature, primary antibodies, which consisted of β-ACTIN (1:2000, Cell
Signaling Biotechnology), TFAM (1:2000, Cell Signaling Biotechnology), OCT4 (1:500,
Sigma-Aldrich) were applied to probe the blots (Appendix Table 9). After incubation at 4
degree overnight, the membrane were washed with TBS-T (0.05% Tween-20 in TBS buffer)
three times and then incubated with horseradish peroxidase (HRP)-conjugated secondary
antibodies (1:4000, Santa Cruz Biotechnology). The membranes were then developed
using Western Pico Super ECL reagent (Pierce). Images were developed by the Typhoon
scanner (GE Healthcare), and signal intensity quantification was performed by Image J
software (http://rsb.info.nih.gov/ij/).
256
Supplementary Figures
Figure S1 Characterization of the hESC with Tra-1-60 in undifferentiated and differentiated
hESCs.
Markers for pluripotency (TRA-1-60, green) and mitochondria (MitoTracker Red, red)
were analyzed in fixed hESCs. (A) Undifferentiated hESCs exhibited significantly protein
expression of TRA-1-60 immunoreactivity and low in mitochondrial staining in the fixed
sample, and (B) 14 days differentiated hESCs exhibited significantly reduction in TRA-1-60
immunoreactivity and significantly increased in mitochondrial staining. Nuclei are
counterstained with DAPI (blue). Representative images are shown. Scale bars represent
50 m.
257
258
Figure S2 Constitutive overexpression of TFAM protein levels in hES cell lines (Mel 1, hES3
and H9).
Three samples were hESC without lentivirus-transduction (negative control), hESC
transduced with EF1α-GFP and hESC transduced with EF1α-TFAM, respectively. (A)
Western blot analysis of TFAM expression in the control and lentivirus-transduced hESCs
was performed using antibodies against the proteins indicated on the left. β-actin was
included as a loading control. Untreated hESCs were analyzed as a negative control. (B)
TFAM expression in hESCs was quantified by phosphor-imager analysis of the western
blots. Western blot analysis confirmed that the level of TFAM was higher by overexpressing
TFAM in three different hES cell lines.
259
260
Figure S3 Inducible knockdown of TFAM levels in hES cell lines (Mel 1, hES3 and H9).
Three TFAM shRNA were TFAM-shRNA-3, TFAM-shRNA-5 and TFAM-shRNA-6
(targetless), respectively. (A) Western blot analysis of TFAM expression before and after
doxycycline addition in the control and lentivirus-transduced hESCs was performed using
antibodies against the proteins indicated on the left. β-ACTIN was included as a loading
control. Untreated hESCs were analyzed as a negative control. (B) TFAM expression in
hESCs was quantified by phosphor-imager analysis of the western blots. Western blot
analysis confirmed that the level of TFAM can be knockdown by TFAM-shRNAi-3 and
TFAM-shRNAi-5 in three different hES cell lines.
261
Figure S4 Expression of relative OCT4 expression in the cell lysates detected by using
Western blot assay (A), and quantified analysis represented in histogram (B). Untreated
hESCs were analyzed as a negative control. β-ACTIN was included as a loading control.
Western blot analysis confirmed that the level of POU5F1 protein was not affect in a
short-term treatment, and decreased dramatically upon 3 days of RA-induced differentiation
and spontaneously differentiation.
262
Figure S5 Characterization of
the hES-LC3-GFP reporter
cell line.
(A) Localisation of
TRA-1-60 (green) (B)
Localisation of POU5F1 (red)
within the hES-LC3-GFP cell
line. Nuclei are
counterstained with DAPI
(blue). Representative
images are shown. Scale
bars represent 50 m.
263
264
Figure S6 Characterization of mitochondria in undifferentiated hESCs with 50 μM CCCP in 2
hours or 24 hours.
(A) Undifferentiated hESCs treated with 2 hours of CCCP and stained with JC-1. (B)
Undifferentiated hESCs treated with 24 hours of CCCP and stained with JC-1. The
JC-aggregates form (Red) had shown significantly reduction in 24 hours of CCCP compared
with 2 hours of CCCP in hESCs. This suggested that CCCP significantly depolarised the
mitochondrial membrane potential in hESCs. Low and high membrane potentials are
indicated by green and red staining, respectively. Nuclei are counterstained with DAPI (blue).
Representative images are shown. Scale bars represent 50 m inverted confocal
laser-scanning microscope (Zeiss LSM 710).
265
Figure S7 Scheme of different treatments in hES3-LC3-GFP cells videos by imaging
fluorescence microscopy.
Data were displayed as in images. Abbreviations: control, hES3-LC3-GFP cells treated
with DMSO; Rap, hES3-LC3-GFP cells treated with rapamycin; CCCP, hES3-LC3-GFP
cells treated with carbonyl cyanide 3-chlorophenylhydrazone; EtBr, hES3-LC3-GFP cells
treated with ethidium bromide; Spd. Diff-7, hES3-LC3-GFP cells culture without basic
fibroblast growth factor for 7 days.
266
267
268
269
Figure S8 A more detailed dosage of treatment toxin and differentiation-induced mitophagy
in hES-LC3-GFP cells.
Images represented from left to right. Images are combination of LC3-GFP signal
(green) and MitoTracker Red signal (red) channels. Treatment conditions are listed at left:
(A) control (DMSO), (B and C) rapamycin (positive control, 200nM and 500 nM) in 0, 5 and
10 minutes, respectively, (D-F) CCCP (mitophagy induction, 10, 5, and 100 µM) in 0, 7 and
15 minutes, (G) EtBr (mitophagy induction, 100 ng/ml) treatment, (H) 7 days of bFGF
withdrawal (spontaneous differentiation) in 0, 10 and 20 minutes, (I) control in 0, 8, 16 hours,
(J-L) EtBr (10, 50, and 100 ng/ml) treatment in 0, 8 and 16 hours, (M) EtBr (100 ng/ml for 1
week) treatment in 0, 8 and 16 hours, respectively. Representative images from
supplementary videos are shown and mitophagy events are circled by red circles. Scale
bars represent 50 m. Abbreviations: control, hES-LC3-GFP cells treated with DMSO; Rap,
hES-LC3-GFP cells treated with Rapamycin; CCCP, hES-LC3-GFP cells treated with
carbonyl cyanide 3-chlorophenylhydrazone; EtBr, hES-LC3-GFP cells treated with ethidium
bromide; Spd. Diff-7, hES-LC3-GFP cells culture without basic fibroblast growth factor for 7
days.
270
Figure S9 Schematic structure of TFAM. TFAM is composed of seven exons (indicated as
“E” inside blue boxes). The first HMG-motif contains Ex1 to Ex 4 and second HMG-motif
contains end of Ex4 to Ex 6. There had a small linker region connected between two
HMG-motifs and a small carboxy-terminal tail at the end of the second HMG-motif.
There are two sets of shRNA that are constructed in either a constitutive repression
vector or an inducible-repression vector. The shRNA construct in the constitutive
expression vector are 1 (266-286), 2 (591-611), and 3 (618-638) as indicated by the green
arrows. The shRNA construct in the inducible expression vector are 3 (489-509), and 5
(764-784), as indicated by the red arrows.
N-terminus C-terminus
E1 E3 E2 E5 E4 E7 E6
Constitutive
knockdown
shRNA1
Constitutive
knockdown
shRNA
2 & 3
Inducible
knockdown
shRNA 3
Inducible
knockdown
shRNA 5