What are Embryonic and Adult stem cells?

Stem Cell and Organ Regeneration

Alice BasinDaiji Kano

Experimental Cell and Developmental Biology 03-345Section B

04/13/2010

Embryonic Stem (ES) Cells Undifferentiated cells derived

from the inner cell mass (ICM) of the blastocyst stage embryos.

Are capable of indefinite self renewal in culture: (applies to Mouse ES cells) supplied with LIF (Leukemia Inhibitory Factor) belongs to IL-6 cytokine family. It binds to its gp130 receptor and leads to activation of differentiating and anti-differentiating signals at the same time.

Maintain normal karyotpe, pluripotent and have the ability to differentiate into multiple cell types of the three germ layers.

Google Images: ://theblackcordelias.files.wordpress.com/2009/03/stem-cells.jpg

Embryonic Stem Cells; human vs. mouse

• LIF can replace the requirement for feeder cells and serum entirely for mouse ES cells (mESCs)but not for human ES cells (hESCs). hESCs have the ability to form trophoblast cells in response to bone morphogenetic proteins, mESCs do not.

• hESCs and mESCs differ in the expression of several cell surface antigens.

• Have the ability to treat degenerative diseases and major traumatic injuries, which may result in a significant improvement in the quality and length of life for affected patients. 

• Can be used as a model for early human embryonic development

• Figure on the right shows that when ES cells were injected into immuno compromised mice, teratomas of muliple cell types formed.

Teratomas formed by the human ES cell lines in SCID-beige mice. Human ES cells after 4 to 5 months of culture from about 50% confluent six-well plates were injected into the rear leg muscles of 4-week-old male SCID-beige mice. Seven to eight weeks after injection, the resulting teratomas were examined histologically

http://www.sciencemag.org/cgi/content/full/282/5391/1145/F4

Adult Stem Cells

• Tissue specific, only able to give rise to progeny cells corresponding to their tissue of origin.

• In contrast to ES cells, immature versions of the cells normally found in the tissue from which they were removed and so are usually only able to differentiate into mature cells of the same or similar type.

• Similar to ES cells, adult stem cells also have the ability to divide or self-renew indefinitely, and generate all the cell types of the organ from which they originate.

• Unlike ES cells, the use of adult stem cells in research and therapy is considered to be less of an ethical concern as they are derived from adult tissue samples rather than destroyed human embryos.

Stem Cells and Organ Regeneration

Stem cells and organ regeneration

• Liver is the only organ in body capable of regeneration

• Organ transplantation:– Need for a donor: availability is

decreasing– Demands that specific conditions

are met such as blood type– Complicated process (surgically and

emotionally)• Major surgeries often come with

great risks• Are people comfortable with being n

% pork?

• What else can we do?• Stem cell mediated organ

regeneration

http://www.ptei.org/assets/METHOD_ARM.jpg

Stem cells and organ regeneration

• How do stem cells contribute to organ regeneration?

– Adult cardiac stem cells are multipotent and support myocardial regeneration

– Mesenchymal stem cells (MSC) differentiate into bone, cartilage, and fat cells

– Hematopoietic stem cells (HSC) contribute to the regeneration of renal tubules after renal ischemia-reperfusion injuries

Stem cells and organ regeneration• Can take the form of:

– Scaffold transplantation: Stem cells are grown on a 3D scaffold. The cells start secreting growth factors and form a living tissue, which is then transplanted in the patient. The scaffold biodegrades as the stem cells differentiate to repair the organ. 2D->3D = better mimics actual organ and therefore more efficient

– Hematopoetic Stem Cell (HSC) Mobilization: mobilization of the multipotent stem cell that give rise to all the blood cell types to the site of action, followed by differentiation at the target location

– HSC injection: similar to HSC mobilization– MSC (Mesenchymal Stem Cell): often

used in adjunction with stem cell transplantation. Can differentiate into osteoblasts, (bone cells), chondrocytes (cartilage cells) and adipocytes (fat cells)

http://www.biomaterials.org/week/bio25_clip_image003.jpg

http://www.sigmaaldrich.com/etc/medialib/life-science/stem-cell-biology/mesenchymal-stem-cell.Par.0001.Image.457.gif

• Advantages over the conventional organ transplantation:– Availability and feasibility– Autografting and isografting

• Still a controversial topic– long-term or permanent presence of foreign cells in the recipient, i.e.,

cells that cannot be retrieved• tumor formation• fibrosis

Stem cells and organ regeneration

Final Project

Our project

• Project Goal: to examine the effect of caffeine treatment on myoblast differentiation.

• Hypothesis: If caffeine is added to myoblasts and myotube stages of C2C12 cells, then this will cause an increase in intracellular calcium from intracellular stores which will accelerate myogenesis.

Our project

• Details of what we are going to test:

1. We are going to test whether or not caffeine causes the increase of intracellular calcium and if that effects cell differentiation.

2. We are going to have cells in NES, containing EGTA (calcium chelator) in order to confirm whether or not the presence of [Ca2+

] ex is necessary for myogenesis.

3. [Ca2+]in will be monitored by Fura-2 tagging of [Ca2+]in.

– Fura-2 is a calcium ion chelator that fluoresces in the UV range• Used as an indicator of [Ca2+] fluctuation

Protocol for treatment of cells

We will be treating 7 dishes of cells in total:• Day 0 (Monday)

– Cells to be proliferated only (no differentiation)1. Control plate with no treatment2. Plate with 20mM Caffeine treatment

– Cells to be differentiated on Day 2 (Thursday)3. Control plate with no treatment4. Plate with 2mM Caffeine treatment5. Plate with 20mM Caffeine treatment

• Day 2 (Thursday)– Cells whose [Ca2+]in will be observed by Fura-2

treatment6. Control plate with no treatment7. Plate with 20mM Caffeine treatment

– Might be a different concentration depending on observations from Lab Day 2

Current Research related to Final Project:“Caffeine and Nicotine decrease acetylcholine receptor clustering

C2C12 myotube culture”

• Experimental Goals: assess the effects of short-term and long-term exposure to caffeine and nicotine on the frequency of AChR clustering on myotubes and possibly on myogenesis.

• Hypothesis: Physiologically significant concentrations of caffeine would decrease AChR clustering, and that combinations of caffeine and nicotine would decrease AChR clustering beyond the effect of either treatment alone.

Methods:• Short term treatment: C2C12 cell cultures were maintained as untreated

controls or exposed to caffeine, nicotine, or caffeine and nicotine during the last 48 h of 72 h in Differentiation Media (DM) . Caffeine concentrations from1mM-1 nM were tested for the ability to decrease agrin-induced and spontaneous AChR clustering, with 10 μM caffeine being demonstrated as sufficient to do so.

• Long term treatment :C2C12 cell cultures were maintained initially in10-cm plates as untreated controls or exposed to 10 μM caffeine, 1 μM nicotine, or 10 μM caffeine and 1 μM nicotine in Growth media (GM) for several generations over 2 weeks. Treatments continued when cultures were transferred to cover-slips and then for 72 h in DM.

• AChR clustering assay: C2C12 cell cultures were maintained in six-well plates with experimental manipulations as described above. AChRs were labeled by the binding of α-bungarotoxin conjugated to tetramethyl rhodamine. Cultures were incubated in the toxin-containing medium for 30 min at 37°C to label AChRs.

Data/Results

Fig. 1 Caffeine decreases the frequency of agrin-induced AChRclustering with a dose response. AChR clusters were determined forC2C12 myotube cultures treated for 48 h in DM with various levels ofcaffeine and for the final 16 h with 10 ng/ml agrin. Each columnrepresents the average for 25 fields of view (error bars SEM).*Statistically decreased in comparison with control, at P<0.01 withStudent’s t-test

• “When exposed to physiologically significant amount of caffeine, C2C12 myotubes cluster AChRs in response to agrin at a decreased frequency when compared with untreated culture”

Fig. 3 C2C12 myotubes were viewed and photographed by fluorescence microscopy after 72 h in DM, with exposure to caffeine and/or nicotine for the last 48 h in DM and to agrin for the last 16 h in DM. a With 10 ng/ml agrin. b With 10 μM caffeine and 10 ng/ml agrin.

Image source: Kordorsky-Herrrera et al

Discussion/Conclusion

• Caffeine (as well as nicotine) decreases AchR clustering• Intracellular calcium is known to be involved in agrin-

dependent Ach aggregation• Caffeine may impact calcium in a similar way calcium

chelators like BAPTA does—by depleting intracellular calcium stores—and therefore interfere with calcium-dependent agrin-induced synapse formation

References1. Wobus, Anna M.: Google Books: Stem Cells: Handbook of experimental pharmoclolgy, Springer

2. Thompson, James A. et al. “Embryonic Stem Cells derived from human blastocysts:http://www.sciencemag.org/cgi/content/full/282/5391/1145 Science AAAS

3. Stemagen: Stem Cell Research and development:Stem Cell Overview: http://www.stemagen.com/overview.htm

4. Van Hoof et al. Molecular and Cellular Proteomics: “A quest fo human and mouse embryonic stem cell-specific proteins

5. Kordorsky-Herrrera et al. Cell Tissue Res. “Caffeine and Nicotene decrease acetylcholine receptor clustering in C2C12 mouse myotube culture” (2009) Springer-Verlag

6. Levenberg, S. et al. Differentiation of human embryonic stem cells on three-dimensional polymer scaffolds. Proc. Natl. Acad. Sci. USA 100, 12741–12746 (2003).