identification of molecular and functional heterogeneity
TRANSCRIPT
Identification of Molecular and Functional Heterogeneity of Epithelial Progenitor Cells in the
Upper Airway
by
Monica Allison Clifford
A thesis submitted in conformity with the requirements
for the degree of Master of Science
Medical Biophysics
University of Toronto
© Copyright by Monica Clifford 2013
ii
Identification of Molecular and Functional Heterogeneity of Epithelial Progenitor Cells in the
Upper Airway
Monica Allison Clifford
Master of Science
Medical Biophysics University of Toronto
2013
Abstract
Upper airways are lined with a pseudostratified mucociliary epithelium
maintained by basal cells. To investigate functional and phenotypic heterogeneity
within the human basal cell compartment, we used a combination of limiting dilution
assays and surface marker profiling on primary cultures of basal cells with verified
progenitor activity. The limiting dilution assay suggested functional heterogeneity in the
ability of basal cells to repopulate a filter and maintain a barrier at ALI. The frequency of
cells with this activity varied between patient strains and ranged from 0.08%-1% of basal
cells. Validation of large-scale comprehensive surface marker profiling on basal cells led
to identification of 74 antigens demarking consistent subpopulations. Preliminary
functional analyses suggest differences in differentiation potential of some
subpopulations. This work supports the idea that the basal cell compartment may be
functionally heterogeneous, and provides new molecular tools for interrogation of
human basal cells.
iii
Acknowledgments
It is a great pleasure to thank the many people who have made this thesis possible.
It is difficult to overstate my gratitude to my supervisor Dr. Nadeem Moghal. With his
enthusiasm and passion for science he inspired me and with his patience and knowledge he
taught me. Throughout my degree and thesis preparation he provided encouragement, sound
advice and lots of good ideas. I would have been lost without him.
I wish to thank my lab mates, Boram Kim and Emily van de Laar, and my many colleagues for
their support, guidance, assistance and companionship.
I am grateful to my committee Dr. Sean Egan and Dr. Norman Iscove for their wisdom and
guidance throughout my degree.
Finally, I wish to thank my family, friends and the many people who provided me with the
support I needed to succeed and who made this experience memorable.
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Contents
Abstract ............................................................................................................................................ ii
Acknowledgments ........................................................................................................................... iii
List of Tables .................................................................................................................................... iv
List of Figures .................................................................................................................................. vii
List of Appendices.......................................................................................................................... viii
List of Abbreviations ........................................................................................................................ ix
1. Introduction ................................................................................................................................. 1
1.1 Structure of Normal Adult Airway ......................................................................................... 1
1.2 Classical and Non-Classical Stem Cell Theory ........................................................................ 3
1.3 Candidates for Lung Stem Cells ............................................................................................. 4
1.3.1 Progenitor Populations in the Distal Airways and Respiratory Region .......................... 5
1.3.2 Progenitor Populations in the Proximal Airway ............................................................. 7
1.4 Evidence for Heterogeneity within the Basal Cell Compartment ....................................... 10
2. Experimental Design and Methods ........................................................................................... 13
2.1 Isolation of Human Tracheal Epithelial Cells ....................................................................... 13
2.2 Growth and purification of HTECs by culturing ................................................................... 14
2.3 In vitro differentiation using ALI culture ............................................................................. 15
2.5 Antigen retrieval .................................................................................................................. 17
2.6 Antibody Staining ................................................................................................................ 17
2.7 Enface staining..................................................................................................................... 18
2.8 High- Throughput Antibody Screen ..................................................................................... 19
2.9 RNA Extraction and cDNA Synthesis .................................................................................... 19
2.10 Real-Time PCR ................................................................................................................... 19
2.11 Statistical Analysis of Limiting Dilution Analysis Results ................................................... 20
3. Results ....................................................................................................................................... 20
3.1 Early passage HTECs are p63-positive and retain regenerative properties ........................ 20
3.2 Ciliogenesis can be used to evaluate culture performance at ALI. ..................................... 24
3.3 Kinetic analysis of mucociliary differentiation of P0-plastic cells seeded at ALI. ................ 27
3.4 ALI cultures may maintain a progenitor population during differentiation. ....................... 33
3.5 Functional heterogeneity is suggested by limiting dilution analysis ................................... 36
v
3.6 Culture-isolated basal cells have heterogeneous surface marker expression. ................... 40
3.7 Differences in isolated cell populations can be determined using ALI culture. .................. 42
4. Discussion .................................................................................................................................. 46
4.1 Establishment of culture systems and Assays ..................................................................... 48
4.2 Evidence for Functional Heterogeneity ............................................................................... 52
4.3 Evidence for Molecular Heterogeneity ............................................................................... 54
7. References ................................................................................................................................. 60
Appendices .................................................................................................................................... 64
Appendix A ................................................................................................................................ 64
Appendix B................................................................................................................................. 67
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List of Tables
Table 1: LDA results are unable to support that differentiation at ALI is dependent on a
single factor.
Table 2: LDA results show frequency of a cell capable of repopulating an ALI culture.
Table 3: Frequency of a cell capable of repopulating an ALI culture is significantly
different between patient strains.
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List of Figures
Figure 1. P0plastic HTECs are a purified population of basal cells
Figure 2. P0p cells retain the ability to differentiate in xenograft and ALI cultures (Str. 37
and 38)
Figure 3. P0p cells retain the ability to differentiate in ALI cultures (Str. 23)
Figure 4. Accurate evaluation of ciliogenesis is possible by enface staining and FOXJ1
expression but not by 2D ALI sections.
Figure 5. Qualitative assessment of differentiation in ALI cultures at multiple time points
by immunofluorescence.
Figure 6. Significant changes in gene expression occur during differentiation at ALI and
vary between patient samples.
Figure 7. Differentiation of basal cells at ALI can maintain a progenitor cell population.
Figure 8. Cell surface markers identify subpopulation of P0p basal cells.
Figure 9. P0p basal cells sorted for expression of CD54 resulted in morphologically
distinct ALI cultures.
Figure 10. P0p basal cells sorted for expression of podoplanin gave rise to
indistinguishable ALI cultures.
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List of Appendices
Appendix A: List of subpopulations identified by HTS
Appendix B: Primer sequences
ix
List of Abbreviations
ALI air liquid interface
BASC bronchioalveolar stem cell
BrdU 5-bromo-2-deoxyuridine
BSA bovine serum albumin
βTUBIV beta tubulin IV
C Celsius
CCA Clara Cell-specific marker
CD cluster of differentiation
cDNA complementary DNA
cm centimeter
CO2 Carbon dioxide
DAPI 4',6-diamidino-2-phenylindole
DASCs Distal airways stem cells
DMSO dimethyl sulfoxide
DNA Deoxyribonucleic acid
Np63 delta-N p63 isoform
EDTA Ethylenediaminetetraacetic acid
EGF epidermal growth factor
ELDA extreme limiting dilution analysis
EtOH ethanol
FACS Fluorescent activated cell sorting
FBS fetal bovine serum
H&E hematoxylin and eosin
x
HCl Hydrochloric acid
HTEC Human Tracheal Epithelial Cells
HTS high-throughput screen
IF immunofluorescence
K14 keratin 14
K5 keratin 5
LHC-9 Laboratory of Human Carcinogenesis-9
MEM minimal essential media
ml milliliter
mm millimeter
MUC16 Mucin 16
MUC5ac Mucin 5ac
N normal
ng nanogram
nM nanomolar
NO2 nitric dioxide
NOD/SCID
non-obese diabetic/severe combined
immunodeficiency
P0p passage 0, plastic
P0pA passage 0 plastic to ALI
P0plastic passage 0, plastic
P1 passage 1
P2 passage 2
P2pA passage 2 plastic to ALI
P3 passage 3
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p63 tumour protein 63
PAP plastic, ALI, plastic
PAPA plastic, ALI, plastic, ALI
PBS phosphate buffered saline
PSG penicillin streptomycin L-Glutamine
qPCR quantitative polymerase chain reaction
RA retinoic acid
RNA ribonucleic acid
SMG submucousal gland
SO2 Sulfur dioxide
SP-C prosurfactant apoprotein-C
str. strain
TAC transit amplifying cell
TAp63 transactivating p63 isoform
TASCs tracheal airway stem cells
TF Tissue factor
TNS trypsin neutralizing solution
µg microgram
UHN University Health Network
µm micrometer
1
1. Introduction
1.1 Structure of Normal Adult Airway
The human lung is a complex organ that delivers oxygen to the circulatory
system. It is composed of a series of airways that lead ultimately to the respiratory
region where gas exchange occurs. To facilitate gas exchange the structure of the lung
provides an extensive interface between the environment and organism. This extensive
interaction highlights the airway epithelium’s essential barrier function and role in
defense against infectious and harmful inhaled agents [1-4]. Air entering the lungs
initially passes through proximal and conducting airways where it is conditioned prior to
entering the respiratory region. The numerous cell types found in lung epithelium
contribute to the function and maintenance of this barrier [1-4].
The proximal airways, comprised of trachea and main stem bronchi, are lined
with a pseudostratified columnar epithelium [5]. This epithelium contains three main
cell types: basal, secretory, and ciliated [2]. Ciliated cells are most prevalent, accounting
for more than 50% of the total cell number [3,6]. They are involved in tight junction
formation, and function to transport mucus from the lung to the throat by means of
ciliary beating [2,3]. Goblet cells are the predominant secretory cell in proximal airways
and account for the majority of mucin production [3]. They are also present at the
luminal surface and are involved in barrier maintenance through tight junction
formation [2]. Other secretory cells include serous cells and clara cells, though these are
present at a very low frequency [2]. A major difference between human and murine
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airways is the frequency and distribution of clara cells. In mice, clara cells are the
predominant secretory cell and are found throughout proximal and distal airways [7]. In
humans, clara cells are largely restricted to bronchioles [8]. Basal cells account for up to
30% of the total cell number in human trachea but decrease in number in bronchi and
bronchioles [2]. They are present at the basement membrane and serve as an
anchoring point for the epithelium by formation of hemidesmosomes [9,10]. Basal cells
are cuboidal non-columnar cells, distinguished by their expression of tumour protein 63
(p63), cytokeratins 5 and 14 (K5/K14), as well as the cell surface marker CD44 [10-12].
P63 exists in different isoforms in the cell. Emerging data has suggested that the
isoform lacking an N-terminal transactivating domain (Np63) regulates expression of
K5 and K14 in keratinocytes. It may therefore be essential for maintaining basal cell
identity [13]. The isoform possessing the transactivating domain (TAp63) is thought to
play a role in maturation and differentiation of keratinocytes and esophageal cells. It
may therefore also have a role in maturation of lung basal cells into other, differentiated
cell types [14]. Basal cells represent a progenitor cell compartment for proximal
airways, giving rise to secretory and ciliated cell lineages [4,15-18]. Neuroendocrine
cells and submucosal glands (SMG) are also found in this region of the airway [19,20].
Epithelial composition of the human distal airway is distinct from the
pseudostratified columnar epithelium of the proximal airway [21]. The bronchioles are
lined with a simple cuboidal epithelium composed mostly of ciliated and clara cells; they
are devoid of cartilaginous rings and SMGs [5]. Moving distally, bronchioles connect to
terminal bronchioles, which have a simple cuboidal epithelial structure composed
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mostly of clara cells with a large decline in the number of ciliated cells [5]. This is the
final region of conducting airways and leads into alveoli, where gas exchange occurs.
Alveoli are lined with two cell types: type I and type II alveolar cells [21]. Type I alveolar
cells are thin squamous cells through which gas exchange occurs [21]. Type II alveolar
cells are thought to be progenitor cells of alveoli with the ability to give rise to type I
alveolar cells [21]. Additionally, they secrete pulmonary surfactant proteins essential for
alveoli to overcome the surface tension of water and fill with air upon inhalation [21].
1.2 Classical and Non-Classical Stem Cell Theory
Highly proliferative tissues such as the intestinal epithelium or the hematopoietic
compartment of the bone marrow rely on a classical stem cell hierarchy to maintain
homeostasis [22,23]. In these tissues a stem cell undergoes asymmetric division,
resulting in self-renewal (i.e. production of another stem cell) and creation of a daughter
cell. The daughter cell is still relatively undifferentiated but proliferates much more
rapidly than the stem cell; these are referred to as progenitor cells, or transit amplifying
cells (TACs). TACs have a finite capacity for self-renewal and are responsible for the
generation of the differentiated cell types that compose the mature tissue, such as
columnar cells in the intestinal epithelium, or circulating blood [22,23]. In classical stem
cell hierarchies the stem cells cycle rarely and possess the capacity for life-long self-
renewal.
Lung epithelium is a relatively quiescent tissue compared to blood, or intestinal
epithelium [24]. The low turnover rate required for homeostasis has made it difficult for
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researchers to identify stem/ progenitor cells involved in long-term maintenance.
Therefore, most research has focused on characterizing proliferation that occurs in
response to injury. Several cell types have been identified as facultative progenitors
including: type II alveolar cells and clara cells [25,26]. The transit amplifying properties
of these relatively differentiated cells suggests that lung epithelium may have a non-
classical stem cell hierarchy similar to that found in other endoderm derived tissues
such as the liver [27]. Mature hepatocytes appear to be responsible for repair following
injury [27]. However, the liver also contains relatively undifferentiated oval cells, which
have been shown to act as facultative stem cells under some conditions [27]. Basal cells
in the proximal airway epithelium may play an analogous role, as they possess capacities
for regeneration as well as for differentiation into columnar cells. Such ‘non-classical’
stem cell hierarchies may represent an alternative means for homeostasis and repair, or
may be reflective of the difficulty of identifying rare stem cells in a relatively quiescent
tissue. The presence of facultative stem cells in lung epithelium does not preclude the
possibility of a classical stem cell hierarchy. Recent findings suggest that more primitive
progenitors, and possibly stem cells, do exist in the lung [28].
1.3 Candidates for Lung Stem Cells
Exposure of lung epithelium to damaging and infectious agents in the
environment necessitates repair. In a recent study, Kajstura et al. proposed the
existence of a primitive CD117+, KLF4+, NANOG+, OCT3/4+ and SOX2+ stem cell that
facilitates extensive repair, supporting a classical stem cell hierarchy in the lung [28].
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They showed that cells isolated from human lung tissue specimens, expanded in F12
media supplemented with 10% serum and sorted for CD117 were self-renewing,
clonogenic, and multipotent in vitro [28]. A cryogenic injury model was used to ablate
cell populations from regions of the mouse lung, after which, CD117+ cells isolated from
human tissue and expanded in vitro were transplanted into the region. These
transplanted cells regenerated human bronchioles, alveoli and pulmonary vessels in the
mouse lung. These cells showed multipotency and were serially transplantable, two key
features of stem cells [28]. These cells are found throughout the human lung, but are
present in higher numbers in the distal airway. The proposed CD117+ stem cell would
represent a novel population upstream of previously described regional progenitors.
The majority of current evidence suggests that repair processes call on regional
progenitor cells present in the airway epithelium. The study of airway maintenance and
repair, as well as the population of progenitor cells responsible for these processes,
relies on ex vivo cultures of human epithelial cells and in vivo mouse models. Studies in
these systems have led to an understanding of some of the hierarchical relationships
that exist; basal cells give rise to secretory as well as ciliated cells in the proximal airway;
clara cells give rise to ciliated cells in bronchioles; and type II alveolar cells give rise to
type I alveolar cells in respiratory regions [16,25,26].
1.3.1 Progenitor Populations in the Distal Airways and Respiratory Region
The mouse airway epithelium can be used to study lung injury repair. Several
mouse injury models based on inhalation of cytotoxic agents have been established. In
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the respiratory region, type I alveolar cells are selectively destroyed by inhalation of
hyperoxic agents such as ozone and nitrogen dioxide (NO2) or the chemotherapeutic
agent blyeomycin [29,30]. In a study of alveolar repair, Evans et al. labeled dividing
cells with tritiated thymidine following NO2 treatment [25]. They found that type II
alveolar cells took up label, but over a 14 day chase period there was an increase in the
proportion of labeled type I cells [25]. This suggests that type II alveolar cells serve as a
regional progenitor for type I cells. Although clara cells and type II alveolar cells
demonstrate some capacity for regeneration, they also have a more differentiated
phenotype, suggesting they may be more mature, or facultative progenitors.
A naphthalene induced injury model is also widely used. Administration of
naphthalene selectively ablates clara cells based on their expression of cytochrome
P450F2 [31]. Once inside the cell, naphthalene is converted to a cytotoxic epoxide by
P450, leading to cell death [31]. Studies of mouse bronchioles have revealed a rare
population of clara cells which don’t express cytochrome P450F2, and are thus resistant
to naphthalene injury [32,33]. Following treatment with naphthalene, these rare clara
cells may act as a progenitor population to regenerate bronchiolar epithelium [33].
However, in proximal airways, the contribution of these resistant clara cells is minimal.
The basal cells of the trachea and bronchi regenerate the clara cell compartment
following naphthalene injury [16].
A putative stem cell residing at the bronchioalveolar junction has been described
in distal airways of the mouse lung [34]. A cell that was double positive for the type II
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alveolar cell marker, prosurfactant apoprotein-C (SP-C) and Clara Cell-specific marker
(CCA) was found in a murine adenocarcinoma model as a population present only in
tumours [34,35]. Kim et al. defined this double-positive cell population as the
bronchioalveolar stem cell (BASC) [34,35]. BASCs are characterized as Sca1-positive,
CD34-positive, CD45-negative, Pecam-negative cells. When FACS-purified on the basis
of these markers, and expanded on a mouse fibroblast feeder layer, BASCs gave rise to
cells expressing clara cell, and type I alveolar cell markers in Matrigel [34]. These
findings suggest that BASCs have multipotential differentiation capacity and may
represent a rare population of stem/progenitor cells in distal airways. However, the in
vivo importance of BASCs in lung homeostasis has not supported by lineage-tracing
assay [26]. Indeed, by following the fate of lineage-labeled Scgb1a1 (CCA+) cells, they
found no evidence for a contribution of CCA-positive cells, including BASCs, to alveolar
homeostasis or repair [26]. Also, BASCs have not been found in humans.
1.3.2 Progenitor Populations in the Proximal Airway
The study of human tracheal epithelial cells has relied primarily on two ex vivo
systems; the rat tracheal xenograft model and the Air-Liquid Interface (ALI) culture
system [36-38]. In the xenograft assay, a rat trachea is decellularized by multiple rounds
of freeze-thaw. The trachea is then assembled into an open-ended cassette and
inserted subcutaneously on the back of an immunocompromised mouse [37]. Human
epithelial cells are injected into the lumen of the rat trachea and over the course of 5-6
weeks, these cells regenerate a fully differentiated epithelium which includes SMGs
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[37]. Alternatively, ALI culture is an in vitro system where cells are seeded onto the
surface of a porous Transwell membrane and grown under submerged conditions until
confluence is reached [36,39]. Once the cells have formed a confluent monolayer,
media from the upper chamber is removed and cells are fed basolaterally, leaving the
apical surface exposed to air [36,39]. The ALI system allows for differentiation into
ciliated cells, and secretory cells, and maintains cells in a basal position.
Using the xenograft model Englhardt et al. interrogated cultured primary human
tracheal/bronchial epithelial cells [40]. The cells were infected with retrovirus carrying
either ß-galactosidase or alkaline phosphatase at an efficiency of 5-10% [40]. Xenografts
where then established with between 0.5-1x106 cells. After several weeks, the resulting
epithelium contained labeled colonies of different sizes and of distinct composition [40].
Over 40% of labeled colonies contained basal, intermediate, ciliated and goblet cells.
However, the majority of colonies were composed of a subset of these cell types. For
example nearly 30% of colonies were composed of basal, intermediate and ciliated cells,
but lacked goblet cells, while less than 3% of colonies were composed of basal cells
alone. Interestingly, no colonies were found that lacked both basal and intermediate
cells. These data demonstrate that human tracheal epithelial cells, expanded transiently
in vitro as P0plastic cells, possess the potential to give rise to a new, fully differentiated
epithelium in vivo. Additionally, the varying clonal output from individual labeled cells
suggest heterogeneous lineage potential of cells in the initial population. However, it is
also possible that the initial population was homogeneous, with individual cells giving
rise to distinct clonal outcomes as a varying stochastic response to environmental cues.
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Basal cells of the trachea were speculated to be progenitors as early as 1963,
based on lack of discernible functional features and their location in the epithelium [11].
In 2007, Hajj et al. proved this by using CD151 and Tissue Factor (TF) surface markers to
purify human basal cells [41]. The CD151/TF double positive population was isolated
from a suspension of primary human airway epithelial cells by Fluorescence Activated
Cell Sorting (FACS). The ability of double positive cells to generate a well-differentiated,
functional epithelium relative to the marker negative fraction was assessed both by
xenograft assay and ALI culture. Only the CD151/TF-positive basal cell fraction
possessed the ability to proliferate and differentiate in these assays [41]. These results
show that basal cells can act as progenitors for the proximal airway.
Complimentary in vivo murine experiments using injury repair models also
support basal cells as being the main regenerative cell for proximal airways [16]. Using
SO2 to induce epithelial damage, Rock et al. studied repair by following the fate of
lineage-labeled basal cells [16]. A transgenic mouse was developed to follow the fate of
K5+ basal cells by combining K5-CreER and a Rosa26-lacZ reporter. Following tamoxifen
injections to activate Cre, the fate of labeled cells was tracked during homeostasis or
during repair from SO2 injury. K5+ basal cells gave rise to label containing ciliated and
clara cells, showing that basal cells function as progenitors for columnar cells during
homeostasis and repair [16].
Together these results show that the proximal airway epithelium has capacity for
extensive repair and regeneration, that this capacity is found within the basal cell
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compartment, and that basal cells regenerate non-basal cell types. These data further
suggest that there is heterogeneity of lineage potential of within the basal cell
compratment. However, while it is accepted that cells present in the basal
compartment act as progenitors, the degree of heterogeneity or hierarchical
organization within this compartment is unknown.
1.4 Evidence for Heterogeneity within the Basal Cell Compartment
Based on the understanding that basal cells house regenerative potential, the
distribution of this potential within the basal compartment has been the focus of a
number of studies. Heterogeneity within the basal cell population could be described
as: i) functional differences between basal cells, in terms of lineage potential or
proliferative capacity, ii) molecular differences between populations of basal cells, or iii)
anatomical differences in location, such as the residence of stem cells in specific niches.
Using murine adapted influenza H1N1 infection as a novel injury model, Kumar
et al. provided evidence for functionally distinct classes of p63+ basal cells [42]. Airway
cells were isolated from nasal, tracheal and distal airways of healthy human subjects.
Cells with in vitro clonogenic potential were found at frequencies ranging from 1:500 to
1:2000. Clonogenic cells were p63+ and K5+ basal cells, but given the frequency of p63+
K5+ basal cells is far greater than 1:500 in some airway segments, these data suggest
not all basal cells have the same ability to give rise to colonies in vitro [42]. Lineage
potential of these cells was also investigated using in vitro differentiation assays.
Clonogenic cells isolated from the tracheal airway, termed tracheal airway stem cells
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(TASCs), formed pseudostratified epithelia, and differentiated into goblet and ciliated
cells in ALI culture [42]. Clonogenic distal airway stem cells (DASCs) formed monolayer
epithelia and showed only limited induction of mucin expression and ciliated cell
formation in ALI culture [42]. These findings suggest that intrinsic differences exist
between regionally distinct p63+ progenitor cells, and that only rare cells in the basal
cell compartment (represents approximately 30% of all tracheal epithelial cells) possess
clonogenic potential. The presence of functional heterogeneity within the basal cell
compartment is further supported by distinct lineage composition of colonies derived
from single labeled basal cells during tracheal xenograft regeneration [43].
Potential molecular heterogeneity within the basal cell compartment has been
suggested based on differential cytokeratin expression. While expression of K5 and K14
are associated with basal cells in the airway epithelium [10], it has been reported that
these keratins may not be uniformly expressed in all basal cells [16,44-46]. A number of
studies in murine models have shown enrichment for K5 or K14 promoter activity in a
subset of basal cells in the upper trachea, suggesting there are subpopulations of basal
cells which have higher levels of cytokeratin expression [44-46]. An in vitro study by
Schoch et al. suggests basal cells expressing different levels of K5 could be functionally
distinct [44]. In this study, by using a transgenic mouse in which the K5 promoter drives
expression of enhanced green fluorescent protein (EGFP), they isolated high K5/EGFP-
expressing tracheal basal cells by FACS and evaluated their colony forming potential
under ALI conditions. Interestingly, K5 high expressing cells (EGFP bright) showed a 4.5-
12
fold greater efficiency of colony formation and a 12-fold greater ability to form large
colonies relative to the K5 low-expressing basal cells [44].
The stem cell compartment in a number of tissues, including the intestinal
epithelium, exists in a specific anatomical niche [23]. There is some evidence that
stem/progenitor activity might be enriched in SMGs in the upper airway of mice [44-46].
Label retaining cells at the opening of SMGs have been described by Borthwick et al. in a
pulse chase experiment [46]. Using sulfur dioxide inhalation to injure the lung
epithelium, followed by intraperitoneal injections of BrdU to label cycling cells, they
observed an enrichment of label retaining cells (LRCs) in SMGs. LRCs are thought to
possess stem- or progenitor-like characteristics as they undergo a limited number of
divisions in response to injury, and then enter a quiescent state. However, the
relationship of these LRCs to basal cells is not known [46]. To investigate the
contributions of SMG basal cells to regeneration of damaged epithelia, Hegab et al.
developed a method to separate surface epithelial basal cells from SMG basal cells [45].
In vitro, the cells isolated from surface epithelium gave rise to a distinct colony
morphology in 3D cultures compared to SMG cells, suggesting that these two
populations possess inherent biological differences [45]. Furthermore, they performed
a K14 lineage trace showing that K14+ basal cells, which are enriched in SMGs, can
generate SMGs and surface epithelium during repair following severe ischemic injury
[45]. Taken together their results suggest that functionally distinct, K14+ SMG cells may
be multipotent progenitors capable of regenerating SMGs and surface epithelia [45].
13
Current evidence supports p63+ basal cells being an important progenitor
population of the proximal, and possibly, distal airway epithelium. These cells have
critical roles in repair, and their study could lead to novel stem cell based therapies for
proximal airway diseases like cystic fibrosis. Furthermore, basal cells are thought to be
the cell of origin for squamous cell carcinomas (SCCs) of the lung as well as a subset of
adenocarcinomas. Given the important role of basal cells in health and disease states,
we seek to study molecular and functional heterogeneity within the basal cell
compartment of the healthy adult human airway epithelium. Such studies may help
uncover hierarchical organization within the basal cell compartment or may help to
define states associated with distinct basal cell behaviours. To perform this work we
use functionally verified populations of cultured, primary, unpassaged healthy human
airway basal cells (P0plastic cells). We predict that P0plastic cells contain identifiable
molecular and functional heterogeneity which is reflective of the in vivo basal cell
compartment.
2. Experimental Design and Methods
2.1 Isolation of Human Tracheal Epithelial Cells
Epithelial cells are isolated from healthy organ donor tracheas obtained as
surgical waste from lung transplants with informed patient consent from the Trillium
Gift of Life Network and Research Ethics Board approval (UHN). Tissue is stored in a
solution of Minimal Essential Media (MEM) with antibiotics and placed at 4°C until
retrieved for epithelial cell isolation. All steps of the dissociation are carried out on ice,
14
or at 4°C. Initially, fatty tissue is removed and the trachea is rinsed in a solution of
MEM with antibiotics. It is then cut into 1-2 cm2 pieces and digested in a solution of
MEM with Protease type XIV (Sigma P5147), DNase (5 mg) containing L-Glutamine Pen-
Strep (PSG), Fungizone (Amphotericin B) and Gentamycin. Digestion is carried out at 4°C
for 24 hours; after 24 hours, tissue pieces are moved to fresh protease solution. Upon
collection, isolated human tracheal epithelial cells (HTECs) are counted using a
hemocytometer and either plated directly for use in experiments or suspended at 1x106
cells/ml in freezing media composed of LHC-9 with 10% fetal bovine serum (FBS) and
10% Dimethylsulfoxide (DMSO). Suspensions are frozen slowly overnight at -80°C then
transferred to a liquid nitrogen freezer for long-term storage.
2.2 Growth and purification of HTECs by culturing
Cell culture and propagation methods used for primary HTECs were first
established in 1981 by Lechner et al. [47]. Prior to initiation of culture, dishes are
prepared by coating with type 1 collagen (PureCol, Advanced BioMatrix). PureCol is
diluted to 48ug/ml in 0.01N hydrochloric acid (HCl); 10ml of this solution is added to a
10 cm dish and incubated at 37°C with 5% carbon dioxide for 2 hours. After incubation,
the solution is removed and plates are ready for use in HTEC culture. Initially, frozen
vials of HTECs are thawed into a collagen coated culture dish in 10 ml of LHC-9
supplemented with Amphotericin B; LHC-9 media is formulated for isolation and
expansion of p63-positive basal cells [47]. Media is changed 16 hours after thawing to
remove DMSO and FBS. Media is changed every other day and cells are maintained in
15
culture for approximately 10 days or until 70% confluence is reached, at which point
cells are referred to as P0plastic cells and are used for subsequent experiments.
Subculturing of cells is performed by treating cultures with 0.025% trypsin/EDTA, trypsin
is then neutralized with Trypsin Neutralizing Solution (TNS) (ReagentPack, Subculturing
reagent kit, Lonza, CC-5034). Cells are typically replated at a density of 1500-3000
cells/cm and maintained in LHC-9 without Amphotericin.
2.3 In vitro differentiation using ALI culture
Growth and differentiation of P0plastic cells on porous transwell membranes is
performed as previously described [36]. Matsui et al. initially described ‘Matsui’ media,
used for expansion and differentiation of P0plastic cells at ALI [39]. Transwell
membranes are coated with 48ug/ml PureCol in 0.01N HCl for 2 hours at 37C. PureCol
is removed and 1.5 ml Matsui expansion media (0.33nM Retinoic acid (RA) and 5ng/ml
epidermal growth factor (EGF)) is added to the bottom chamber. Cells are suspended
at required concentration in Matsui expansion media to seed the desired number in a
0.5 ml aliquot in the upper chamber. Cells are grown at 37C and 5% CO2, media is
changed every other day. Once confluence is reached, media is removed from the
upper chamber and media in the lower chamber is switched to Matsui differentiation
media with high RA (50nM) and low EGF (0.5ng/ml). Cells are maintained in culture at
an air-liquid interface for a minimum of 3 weeks at which point they are evaluated for
differentiation.
2.4 Histology of ALI Cultures
16
An agar embedding method described by the UHN Pathology Research Program
was adopted to facilitate cutting sections from paraffin embedded samples. At the
point of harvest, cultures are washed twice with PBS and 10% buffered formalin is
added above and below the membrane to a total volume of 1 ml. Fixation is carried out
at room temperature for 1 hour, formalin is removed, and the cultures are rinsed an
additional three times with PBS. Membranes supporting the fixed ALI culture are
removed from the Transwell insert using a scalpel to cut carefully around the edges
while keeping the membrane pressed flat against the cutting surface. The membrane is
placed tissue side down in an embedding mold containing molten agar (3% low gelling
temperature agar in distilled water) ensuring the membrane is well away from all edges
and completely covered with agar. The agar is allowed to set at room temperature. The
agar block containing a fixed culture is removed from the mold and excess agar is
trimmed so the block will fit into an embedding cassette, leaving at least 1 mm of agar
on all sides. Agar blocks can be stored in 70% ethanol until processed.
Once prepared, agar blocks can be processed like a regular block of tissue.
Embedding cassettes are loaded into a Shandon Excelsior tissue processor and
processed overnight. Briefly, this machine takes the blocks through stages of
dehydration in an alcohol series, alcohol is displaced with xylene, and xylene is
subsequently replaced with increasing concentration of paraffin in xylene until the
blocks contain only paraffin.
17
Using a microtome, 6 µm sections are cut from paraffin embedded samples
perpendicular to the direction of the membrane in the block. Sections are floated on a
42C water bath to flatten and soften the paraffin, and then scooped onto pre-cleaned
charged glass slides. The slides with cut sections are placed in a 58C oven until dry and
the paraffin is melted onto the slide. This can be carried out overnight.
Prior to H&E or antibody staining, the paraffin must be cleared from the sample.
Paraffin is initially removed by soaking slides in xylene for 10 minutes; this step is
repeated with fresh xylene. The slides are then taken through an ethanol series of: 2x
100% for 10 minutes, 90% for 5 minutes, 70% for 3 minutes, 50% for 3 minutes, 35% for
3 minutes and PBS for 3 or more minutes.
2.5 Antigen retrieval
Antigen retrieval must be performed on formalin fixed samples prior to antibody
staining [48,49]. The optimal retrieval process depends on the antigen. We find heat
retrieval in citrate buffer is sufficient for our antigens of interest [48,49]. Deparaffinized
samples are placed in containers of 10mM citrate buffer pH 6 in an antigen retriever
(2100 Retriever). The assembly is brought to over 120C under pressure for at least 3
minutes. The cycle takes approximately 20 minutes to complete; the whole apparatus is
allowed to cool for 100 minutes before removing samples for a total run time of 2 hours.
2.6 Antibody Staining
Following antigen retrieval, cells are permeablized in PBS with 0.1% Triton X-100
for 5 minutes. Samples are then blocked in 3% bovine serum albumin (BSA)/ 0.1%
18
Triton X-100 in PBS for 30 minutes and incubated with primary antibody diluted in BSA/
0.1% Triton X-100 in PBS for 1 hour at room temperature. Primary antibodies used
were: CD44 (IM7) cat# 17-0441-81, MUC16 (oc125) cat# sc-33344, Muc5ac (45M1) cat#
MS145-p1, p63 (BC4A4) cat# sc-56188, and βTUBIV (ONS.1A6) cat# T7941. After
incubation with primary, samples were washed 3 times with 0.1% Triton X-100 in PBS
and incubated with secondary antibody in BSA/ 0.1% Triton X-100 in PBS for 2 hours at
room temperature or overnight at 4°C. The secondary antibodies were alexafluor
(Invitrogen) 488 goat anti-rabbit (1:500), 555 Goat anti-rabbit (1:500) and 568 goat anti-
mouse (1:500). Samples are washed 3 times with 0.1% Triton X-100 in PBS. Tissue
sections and cytospun preparations were mounted in vectashield mounting media with
DAPI.
2.7 Enface staining
For a quantitative assessment of differentiation, enface staining technique was
established. ALI membranes are formalin fixed as described above. ALI membranes are
stained as described above; antigen retrieval is not required for βTUBIV staining. Upon
completion of staining membranes are washed three times in 0.1% Triton X-100 in PBS
then dehydrated through an alcohol series from 70% for approximately 1 minute, to
90% ETOH briefly and rinsed in 100% ETOH to remove any residual water from the
surface of the ALI. The membrane is carefully cut from the well insert with a sharp
scalpel while keeping the membrane pressed against the cutting surface. In a fume
hood, the membrane is placed in a drop of 30% Permount in xylene on a pre-cleaned
glass microscope slide, epithelial side up. More Permount-xylene solution is added on
19
top of the membrane and a cover glass is carefully placed to avoid bubbles and flatten
the membrane, the slide is dried in a fume hood overnight. Imaging of enface staining
was performed using Metamorph® tiling utility.
2.8 High- Throughput Antibody Screen
Cells were expanded under submerged conditions to 70% confluence as
P0plastic. Cells were trypsinized with 0.0125% trypsin+EDTA (Clonetics), neutralized
with TNS, pelleted and resuspended in Hanks buffered salt solution with 1% FBS. Cells
were submitted to the antibody core facility. Cells were then stained with the panel of
antibodies in a 96-well plate format; marker expression was analyzed by flow cytometry.
2.9 RNA Extraction and cDNA Synthesis
Total RNA was extracted using Ambion Micro RNA kit, RNA isolate was treated
with DNase I to remove contaminating genomic DNA. A total of 1ug of RNA was
converted into cDNA in a 20ul reaction volume using the High Capacity cDNA Reverse
Transcriptase kit (ABS). An automated thermocycler was used to provide the following
conditions: 10 minutes at 25°C, 120 minutes at 37°C, 5 seconds at 85°C and 4°C until
transferred to -20°C for long-term storage.
2.10 Real-Time PCR
cDNA stock solution was prepared by diluting cDNA to a concentration of 2 ng/µl
in autoclaved miliQ water. The SYBR Green system (Biorad) was used for qPCR
reactions. 4 µl of diluted cDNA was added to a reaction mixture containing 5 µl of SYBR
green super mix (Biorad) and 1 µl of primer mix containing 3µM of forward and reverse
20
primers (Appendix B). Reaction conditions for detection of amplification were 95°C for 3
minutes, 95°C for 10 seconds, and 60°C for 30 seconds for 40 cycles.
2.11 Statistical Analysis of Limiting Dilution Analysis Results
Stem/progenitor cell occurrence follows a Poisson distribution. Limiting dilution
analysis results are analyzed by a webtool: Extreme limiting dilution analysis [50]. The
program enables inclusion of densities that result in all failures or all successes. Results
are based on the assumption of a linear relationship between seeding density and
outcome.
3. Results
3.1 Early passage HTECs are p63-positive and retain regenerative properties
To begin to address the degree of molecular heterogeneity within the human basal cell
compartment, we used previously described methods to extract large airway progenitor
cells from adult human tracheal tissue [51-53]. Although our goal was to analyze basal
cells in as close to an in vivo state as possible, we found that the most effective means
of isolating these cells also cleaved many cell surface markers. To bypass this problem
and increase the yield of cells for high-throughput assays we expanded enzymatically
digested, P0 tracheal suspensions without passaging, in a serum free medium previously
described to maintain large airway progenitor activity [47]. After expansion, we
evaluated the purity of this P0-plastic population by immunofluorescent (IF) antibody
staining for basal and columnar cell markers. We used α-p63, α-βTUBIV, α-MUC16 and
21
α-Muc5ac to assess the presence of basal cells, ciliated cells, mucinous cells and goblet
cells respectively. Staining showed approximately 90% of cells were positive for p63
(Fig. 1a). No cells had polarized apical βTUBIV staining, a hallmark feature of ciliated
cells, though a small percentage stained weakly for βTUBIV in cytoplasmic foci (Fig.
1b) [54,55]. No cells expressed MUC16 (fig. 1c) or Muc5ac (data not shown). To
assess if p63-negative nuclei represented contaminating cell types, we examined
another basal cell marker, CD44 by FACS analysis, showing that P0plastic cells are
~100% positive for CD44 (fig. 1e) [12]. Based on CD44 staining data, we re-
examined p63 expression by IF and found a number of nuclei initially scored
negative actually had low levels of p63. Together these data indicate that P0plastic
cells represent a close to pure population of basal cells, with a low amount of
heterogeneity in p63 expression.
To verify that proliferating P0plastic basal cells retained progenitor activity,
we seeded cells derived from different donor tracheas into rat tracheal xenograft
(Strain 37)[37] and ALI culture (Strain 38 and 23)[38] systems, which support the
differentiation of basal cells into columnar cells. These cells formed well-
differentiated epithelia in xenograft and ALI cultures showing they retained
functional in vivo progenitor properties (fig. 2a, b; fig 3a). The presence of ciliated,
secretory and basal cells was confirmed in ALI-culture sections stained for βTUBIV
(fig. 2c; fig. 3b), MUC16 (fig. 2d; fig. 3c), and p63/CD44 (fig. 2e; fig. 3d). Although
other studies have reported differentiation of basal cells into Muc5ac-positive goblet
cells under ALI culture conditions, we did not detect this lineage (fig. 2f; fig. 3e)[39].
22
Instead, we found robust differentiation into MUC16-positive mucinous cells,
another abundant in vivo mucinous large airway lineage [56].
Figure 1. P0plastic HTECs are a purified population of basal cells. Cytospun
unpassaged, HTECs expanded in submerged condition on collagen coated plastic (P0p)
stain positively for (A) basal cell marker p63 (86.3 +1.2%), but do not express (B) tubIV
(0%) or (C) Muc16 (0%). (D) Percentage of cells expressing each marker. (E)
Expression of basal cell marker, CD44, was evaluated by FACS on P0p cells.
23
Figure 2. P0p cells retain the ability to differentiate in xenograft and ALI cultures.
(A) P0p cells were seeded into denuded rat tracheas and implanted into NOD/SCID
mice. After 33 days, xenografts were fixed, sectioned and stained by H&E (Str. 37). (B-
F) P0p cells were grown at ALI cultures for 21 days, after which filters were fixed,
sectioned and stained with H&E and indicated antibodies (Str. 38). All scale bars
represent 50 m.
24
Figure 3. P0p cells retain the ability to differentiate in ALI cultures (Str.23). (A-E)
P0p cells were maintained at ALI for 21 days, after which filters were fixed, sectioned
and stained with H&E and indicated antibodies (Str. 38). All scale bars represent 50 m.
3.2 Ciliogenesis can be used to evaluate culture performance at ALI.
To quantify p63-positive progenitor activity in ex vivo cultures we focused on the
ciliated cell lineage, which is a major lineage derived from p63-positive progenitors [16].
Three methods to evaluate the extent of ciliogenesis were explored: 2D sections, enface
staining and gene expression analysis.
Intracellular localization of βTUBIV at the apical surface is a definitive marker for
mature ciliated cells [54,55]. However, the distribution of βTUBIV-positive cells is not
uniform in differentiated ALI cultures (fig. 4a). This can be seen in sections from the
25
same membrane where in some sections ciliated cells are sparsely distributed (fig. 4a),
while in others they are densely distributed (fig. 4b). In contrast, basal cells, as marked
by p63, appear uniformly distributed as assessed by random sections of the same
membrane (fig. 4c and d). The impact of uniformity of cellular distribution on
estimating the mean number of cells is demonstrated by comparing standard deviations
in the percentages of ciliated cells and basal cells (fig. 4e). The percentage of total cells
having undergone ciliogenesis was 10.4 ±5.8%, compared to 25.4 ±2.7% for evenly
distributed basal cells. Thus, without extensive sectioning, random sections do not
readily allow for accurate extrapolation of the extent of differentiation for an entire
membrane, making it difficult to quantify differences in ciliogenesis between samples.
To circumvent this problem, we sought to establish a quantitative method to assess the
entire membrane for ciliated cells.
An enface staining technique was adopted as a non-biased method of
determining the extent of ciliogenesis. The membrane is stained for βTUBIV and images
of the entire membrane are captured and stitched together using Metamorph® tiling
software (fig. 4f). Software analysis of the enface image provides either: (1) a count of
ciliated cells, which is useful under conditions where limited differentiation occurs; or
(2) a percentage of reconstituted epithelium that is covered by cilia. In theory, this
approach accurately evaluates the extent of ciliated cell differentiation across the entire
culture, and should allow for quantitative comparison between cultures.
26
Figure 4: Accurate evaluation of ciliogenesis is possible by enface staining and
FoxJ1 expression, but not by 2D ALI sections. (A,B) 2D sections stained for tubIV show irregular distribution of ciliated cells, in contrast, (C,D) p63-positive cells are evenly distributed. (E)The variability in percentage and frequency of positive cells as calculated
based on staining of 2D sections gives low confidence in values for tubIV when compared to normally distributed p63. (F) Enface staining allows quantitative determination of total ciliogenesis. (G) Analysis of gene expression provides a quantitative method to compare induction of FoxJ1 between cultures, physiologic controls (tracheal cells) and baseline controls (P0plastic basal cells). Scale bars in IF
images: 50 m; enface image: 1 mm.
27
In addition to βTUBIV staining, we also evaluated FOXJ1 gene expression in
differentiated cultures (fig. 4g). FOXJ1 is a transcription factor involved in ciliogenesis in
various tissues; in the airway epithelium, its expression is restricted to ciliated cells [57].
Gene expression analysis by qPCR has the advantage of being very sensitive and, like
enface staining, uses the entire population of cells from the culture. qPCR analysis does
not permit determination of ciliated cell number, but we have found that induction of
FOXJ1 gene expression tracks with emergence of ciliated cells (fig 6g-l, and 7c). Gene
expression analysis allows for direct quantitative comparison between samples, as well
as comparison to physiological (tracheal cells) and base line control cells (P0plastic,
starting population). Our qPCR analysis of FOXJ1 in ALI cultures revealed that P0plastic
basal cells retained a robust ability to induce FOXJ1 to in vivo, tracheal levels (fig. 4g).
3.3 Kinetic analysis of mucociliary differentiation of P0-plastic cells seeded at
ALI.
In order to characterize the differentiation potential of putative subpopulations
of basal cells, we analyzed the kinetics of mucociliary differentiation from bulk P0-plastic
cultures at ALI. P0plastic progenitors isolated from three different donor tracheas were
grown at ALI, and cultures evaluated for differentiation markers at six stages of
repopulation and differentiation. The stages examined were: proliferating subconfluent
cultures, covering 50-70% of filter, cultures just achieving confluence, which is the point
when media was removed from the upper chamber and cells were exposed to air, 3
days post-confluence, 7 days post-confluence, 14 days post-confluence, and 21 days
28
post-confluence. Cultures were evaluated by IF staining on sections, gene expression
analysis, and enface staining.
As p63 progenitors differentiate, we expect to see a shift from a population of
basal cells to a heterogeneous culture containing mixed lineages. Thus, we first
examined changes in the proportion of basal cells by immunostaining sections for p63.
We found that at subconfluence and at confluence nearly all nuclei are p63-positive (fig
5a, b). As early as 3 days post-confluence, a p63-negative layer of nuclei was seen
above the basally located p63-positive nuclei, consistent with pseudostratification (fig.
5c). This staining pattern of basally located p63-positive nuclei and apical p63-negative
nuclei persisted until the cultures reached maturity at 21 days post-confluence (fig. 5d,
e, and f). To investigate the contributions of TAp63 and Np63 to immunostaining and
to quantify p63 expression, we looked at gene expression levels of both p63 isoforms.
We saw the expected downward trend in expression of Np63; however, in one patient
strain there was an initial increase leading up to confluence, prior to its eventual decline
(fig. 6a). In contrast, TAp63 initially increased in all patient strains, and declined
following confluence in 2 of 3 patient strains (fig. 6b). All progenitor strains showed the
expected decline in Np63 expression that would occur during differentiation.
However, our data also suggest there may be some strain-dependent differences in
regulation of p63 isoforms during differentiation at ALI. The significance of these
differences is not known.
29
30
Figure 5: Qualitative assessment of differentiation in ALI culture at multiple time
points by immunofluorescence. (A-F) p63, (G-L) tubIV, and (M-R) Muc16 expression is assessed at multiple time points during repopulation and differentiation of ALI cultures by P0p cells. Expression of p63 in: (A) subconfluent cultures, (B) at confluence, and (C-F) in post confluent cultures, p63 staining is seen only in basally located cells. (G) Some
cells in subconfluent cultures have cytoplasmic tubIV foci, (H-I) these are not observed at confluence or 3 days post-confluence, (J) appearance of pre-ciliated cells is seen at 7 days post-confluence, and (K-L) mature ciliated cells are present at 14, and 21 days post-confluence. (M-O) Muc16 is not seen until 3 days post confluence, (P-R) and is
maintained until 21 days post-confluence. Scale bars: 25 m.
Polymerization of βTUBIV into apically projecting microtubules of cilia is a
definitive marker of the airway ciliated cell lineage [54,55,58]. In sections of
subconfluent, confluent, and 3 day post-confluence ALI cultures, we observed some
cytoplasmic βTUBIV staining, but no apical polymerization (fig. 5g, h, and i). At 7 days
post-confluence, intracellular accumulation of high levels of βTUBIV was seen in some
cells, presumably those undergoing ciliogenesis (fig. 5j). However, only at 14- days and
21-days post-confluence did apical localization of βTUBIV appear, indicating mature
ciliated cells (fig. 5k, l).
Similar kinetics for ciliogenesis were observed by following FOXJ1 expression by
qPCR. FOXJ1 expression was first detectable at 3-7 days post-confluence (fig. 6c), and
reached over 700% of physiological levels in two of three patient samples by 21 days
post-confluence (Fig 6c). Although the extent of induction varied substantially between
patient strains, the kinetics of ciliogenesis were similar. Enface staining for βTUBIV also
showed kinetics similar to the emergence of FOXJ1 which is consistent with FOXJ1’s late
role in ciliogenesis [59]. By enface staining, 3-day post-confluent cultures also showed
intracellular βTUBIV staining, but no mature ciliated cells (data not shown). At 7 days
31
post-confluence, a limited number of mature ciliated cells were apparent, and by 14
days and 21 days post-confluence, cultures had large numbers of ciliated cells (fig. 7e-g).
Monitoring culture progression by light microscopy, we consistently observed
mucous-like secretions on the surface of differentiating cultures prior to detecting any
sign of ciliogenesis. To examine for emergence of mucinous lineages, we followed
expression of MUC16, which in vivo, marks abundant surface and glandular tracheal
cells [56]. In subconfluent cultures, and when confluence was first achieved, we saw no
reactivity with an α-MUC16 antibody (fig. 5m, n). However, as early as 3 days post-
confluence, we observed cell-specific staining at the apical surface of the epithelium (fig.
5o), which became more intense and more wide-spread by 1 week post confluence (fig.
5p). After this period it became difficult to determine which cells were unambiguously
MUC16 positive, possibly due to the antigen being clipped and spread across the
epithelial surface (fig. 5q, r). By qPCR, MUC16 gene expression was first detectable at
confluence, but at less than 1% of physiological levels (fig. 6d). At 3 days post-
confluence, expression in two of three patient samples reached over 10% of physiologic
levels, while in the third strain expression had reached less than 2% of these levels. In
subsequent weeks there was strain to strain variability in gene expression. In Str.
30277, MUC16 expression peaked at 7 days, reaching ~80% of physiologic levels but
then declined (fig. 6d). In Str. 78297, MUC16 levels rose to over 200% of physiological
expression by day 21 (fig. 6d). In Str. 39, MUC16 expression plateaued on day 14, at
25% of physiologic levels (fig. 6d). Our data indicate that for quantification of mucinous
cell numbers by antibody staining, cultures between 3 and 7 days post confluence are
32
optimal. However, for greater sensitivity, qPCR on bulk populations of cultures older
than 7 days post confluence is better suited.
33
Figure 6: Significant changes in gene expression occur during differentiation at
ALI and vary between patient samples. Gene expression changes in (A) Np63, (B)
TAp63, (C) FOXJ1 and (D) MUC16 were monitored during repopulation and
differentiation of P0p cells in ALI cultures. Enface staining for TUBIV showing (E) a
limited number of ciliated cells at 7 days post-confluence, (F, G) but substantial ciliated
cell coverage at both 14 and 21 days post-confluence.
3.4 ALI cultures may maintain a progenitor population during differentiation.
Serial passaging of HTECs on 2D plastic, under submerged culture conditions has
been reported to result in attenuation of basal cell progenitor capacity as assessed in ALI
culture [38]. Gray et al. demonstrated that NHTBE cells grown to a P3 plastic stage
retain the ability to functionally differentiate at ALI into an epithelium possessing many
properties of the in vivo epithelium including mucociliary differentiation and tight
junction formation [38]. For large scale or long-term studies it would be of great
interest to preserve progenitor activity ex vivo. The ALI culture system recapitulates an
in vivo environment by creating an air-liquid interface where cells are fed basolaterally
and exposed to air at the apical surface. This environment, combined with factors in the
media, leads to mucociliary differentiation of p63-positive basal cells, and formation of a
pseudostratified epithelial layer. It is unknown if this in vivo-like environment is better
able to maintain stem/progenitor cells than 2D-culture. Alternatively, differentiation
signals could push the stem/ progenitor cell population toward a terminally
differentiated fate, where basal cells seen in ALI cultures serve only a structural role in
epithelial anchoring.
34
35
Figure 7. Differentiation of basal cells at ALI can maintain a progenitor
population. (A) P0plastic cells were passaged on either ALI or plastic, re-equilibrated
on plastic and their ability to differentiate at ALI was re-assessed; arrows indicated
points where samples were collected. Differentiated cultures grown from either plastic-
passaged (P2pA) or ALI-passaged (PAPA) cells were assessed for expression of: (B)
FoxJ1, (C) Muc16, (D) Np63 and (E) TAp63. Enface staining for tubIV show extent of
ciliogenesis in (F) P2pA, plastic-passaged and (G) PAPA, ALI-passaged cultures.
To address this question, we compared progenitor activity of ALI-passaged and
plastic-passaged cells relative to the P0plastic progenitor population. Initially, after
expansion, P0p cells were seeded in replicate at ALI or passaged on plastic as P1 cells
(fig. 7a). After a well differentiated epithelium was generated at ALI (3 weeks), or P1
plastic cells reached 70% confluence, CD44+ basal cells were FACS purified from the
cultures (fig. 7a). To control for potential changes in progenitor activity due to culturing
in the different media associated with ALI vs. plastic culture we re-equilibrated both ALI
and plastic sorted cells in LHC-9 media on 2D plastic before seeding the P3 cells into ALI
for final analysis (fig. 7a). Both PAP and P2p cells proliferated in ALI culture wells at
comparable rates to form a confluent monolayer. After establishment of the ALI, both
populations maintained a barrier.
We next examined ciliated cell lineage potential of PAP and P2p progenitor cells.
In general, passaging under both conditions reduced ciliogenic differentiation. FOXJ1
induction was significantly reduced under both conditions in all three stains relative to
P0pA (fig. 7b). However, in Str. 30277, P2p basal cells had significantly larger induction
of FOXJ1 gene expression at ALI. These data indicate the ability to give rise to ciliated
cells is significantly impacted by prolonged culture under both conditions (fig. 7b). A
36
dramatic decline in extent of ciliogenesis from P0plastic to P2plastic cells was seen by
enface staining (fig. 7f and g); given the deterioration of P2pA cultures in 2 of 3 patient
strains, we were unable to attribute failure of PAPA cultures in these 2 strains to their
previous differentiation at ALI. In Str. 39 we observed ciliary differentiation of PAPA
cultures at levels comparable to P2pA cultures (fig. 7g, h). MUC16 was induced at levels
similar to P0pA under both treatment conditions in all strains, suggesting that MUC16
lineage potential is maintained during the culture period examined (fig. 7c). Evaluation
ofNp63 and TAp63 showed no consistent difference following treatment conditions
(fig. 7d and e). These results demonstrate that both plastic and ALI culture can maintain
MUC16 competent lineage potential. However ALI is not better than plastic for
maintaining ciliogenic potential, and may be worse.
3.5 Functional heterogeneity is suggested by limiting dilution analysis
While some work supports the concept of functional heterogeneity within the
basal cell compartment, definitive evidence for a hierarchical organization is still lacking
[28,40,45]. To examine potential functional heterogeneity in human basal cells, we
attempted to quantify the frequency of ALI repopulating cells in limiting dilution assays.
We hypothesized that cells which could: proliferate, form tight junctions, and
differentiate on ALI, would have properties equivalent to in vivo progenitors. P0plastic
cells were seeded on ALI filters at seeding densities ranging from 10 000 to less than 100
cells per membrane. Cultures were assessed on their ability to: form a barrier, to
37
maintain a barrier once exposed to air, and to differentiate as assessed by the presence
of beating cilia. We employed the ‘Extreme Limiting Dilution Analysis’ (ELDA) webtool
(http://bioinf.wehi.edu.au/software/elda/) to determine the frequency of a cell that
met these criteria [50].
In order for ELDA to provide an accurate estimate of frequency, it requires a
mixed outcome at a single seeding density on replicate filters. Some filters must show
success, while others show failure [50]. Furthermore, ELDA analysis assumes a single-hit
model, in which a single cell, the stem cell, is the only factor that affects success of the
culture; therefore the single hit hypothesis must be met before frequency of a
stem/progenitor cell can be estimated [50]. If additional factors or parameters that
affect the success of a culture can also be diluted, ELDA cannot be used to quantify the
frequency of a single progenitor population [50].
We initially used presence of functional cilia, directly assessed by light
microscopy, as the output for success and the indicator of presence of ALI-repopulating
cells. However, in experiments using basal cells derived from 11 donor tracheas, none
provided sufficient data to test the single-hit criteria (table 1) and allow estimation of
the frequency of ciliogenic progenitors. In no case did we observe a mixed response of
cultures successfully differentiating and failing to differentiate at a single seeding
density (Appendix C). Although there was strain to strain variability, we did determine
that in all cases, a seeding density of 6 000 cells/ filter was sufficient for success at ALI
(table 1). When success was re-defined to include those cultures that only reached
38
confluence and formed tight junctions, but did not undergo differentiation, 5 patient
strains gave sufficient data to test the single hit hypothesis. In all of these patient
strains, the single hit criterion was met. The estimated frequency of a repopulating cell
ranged from as high as 1 in 96 in one patient strain, to as low as 1 in 1297 in another
(Table 2), suggesting human basal cells are functionally heterogeneous in their ability to
repopulate an ALI filter (Table 3).
Table 1: LDA results are unable to support that differentiation at ALI is
dependent on a single factor. P0plastic cells were seeded in replicate filters at
multiple dilutions and grown to confluence while being fed apically and
basolaterally. If confluence was reached, cultures were exposed to air apically
and fed basolaterally until differentiation was observed or culture failed to
differentiate. Those cultures which underwent mucociliary differentiation were
scored as successes and ELDA [50] was used to determine frequency of cell that
leads to success. No experiment satisfied the criteria of the statistical test and
frequency could not be estimated. Experimental data is available in Appendix C.
Lowest density at which differentiation occurred is presented.
Patient Sample
Reject single-hit hypothesis (p<0.05)
Lowest successful seeding density
18 Insufficient data 10 94214 Insufficient data 100 78297 Insufficient data 250 79168 Insufficient data 500 39 Insufficient data 500 23 Insufficient data 1000 11111 Insufficient data 1000 38 Insufficient data 1500 29945 Insufficient data 2000 32 Insufficient data 6000 55 Insufficient data 6000
39
Table 2: LDA results show frequency of a cell capable of repopulating an
ALI culture. P0plastic cells were seeded in replicate filters at multiple dilutions
and grown to confluence while being fed apically and basolaterally. If confluence
was reached, cultures were exposed to air apically and fed basolaterally until
differentiation was observed or culture failed to differentiate. Those cultures
which reached confluence and/or underwent differentiation were scored as
successes and ELDA [50] was used to determine frequency of cell that leads to
success. Lower and Upper bounds represent 95% confidence intervals.
Patient Sample
Lower Estimated frequency
Upper Reject single-hit hypothesis (p<0.05)
78297 511 164 53 No (0.483) 79168 203 96 45 No (0.906) 29945 1030 519 261 No (0.267) 32 398 182 83 No (0.823) 55 3441 1297 489 No (0.297)
Table 3: Frequency of a cell capable of repopulating an ALI culture is
significantly different between patient strains. Pairwise comparison of
estimated repopulating cell frequency between patient samples was performed
by Pearson Chi square test using ELDA tool [50].
Patient Sample 1
Patient Sample 2
P-value Significance
29945 32 0.0502 29945 55 0.152 29945 78297 0.0966 29945 79168 0.00249 * 32 55 0.00228 * 32 78297 0.884 32 79168 0.25 55 78297 0.00837 * 55 79168 8.56e-05 * 78297 79168 0.461 *
40
3.6 Culture-isolated basal cells have heterogeneous surface marker
expression.
The functional heterogeneity in basal cells observed here and in other work,
suggests there might be identifiable molecular heterogeneity within basal cells
[28,40,44-46]. To investigate the degree of molecular heterogeneity in human basal
cells, we stained tracheal basal cells with a panel of 339 antibodies raised against cell
surface markers. We chose a P0plastic population for this analysis for several reasons.
First, efficient isolation of lung epithelial cells required enzymatic digestion that clipped
off many surface proteins, potentially compromising our ability to detect some
subpopulations. Second, from the amount of donor tissue typically received, insufficient
cell numbers were obtained for such a screen without expansion. Third, one round of
expansion in LHC-9 media had the additional benefit of helping to remove columnar
cells as well as contaminating blood and stromal cells. To minimize potential artifactual
surface marker expression due to prolonged cell culture, we limited our analysis to
unpassaged P0plastic cell populations with verified progenitor activity (fig. 2 and 3).
High-throughput screens (HTSs) were carried out on two different patient
strains. Any distinctly shifted population that was reproduced across both patient
strains was considered a hit (fig. 8a-c). If the regenerative potential of lung epithelium
follows a classical stem cell hierarchy, as observed in blood, rare cells may represent the
stem or progenitor cell. These P0p populations could be identified as rare marker-
positive or rare-marker negative cells. Alternatively, if a non-classical stem cell model is
followed, in which a large number of facultative stem/progenitor cells are
41
Figure 8: Cell surface markers identify subpopulations of P0plastic basal cells.
(A) Markers represent percentage of marker-positive cells in Str. 38, plotted against
percentage marker-positive in Str. 23 for a given cell-surface protein. Insets show (B)
small subpopulation, present on less than 2% or (C) those on greater than 98% of cells.
(D) A summary of all markers, categorized based on highest level of expression in any of
the three screens into: 0-1.9%, 2-9.9%, 10-89.9%, 90-97.9%, 98-100% (Total), the
number selected for further screening based on HTS (HTS), and the number confirmed
by single tube staining (validated) are shown (for list of markers selected based on HTS
see Appendix A).
42
present in the tissue, we might expect to see more prevalent populations of cells
representing progenitors. These cells may also exist as marker-positive or marker-
negative subpopulations.
From the HTS, 112 markers were heterogeneously detected in both patient
strains of basal cells (fig. 8a-c, appendix A). These markers were examined in a third
patient strain of basal cells by FACs staining in a single tube format. Single tube staining
confirmed subpopulations denoted by 74 of these 112 markers; 35 markers were not
detectable, and 3 markers were detected on 100% of cells. Of the 74 markers of
heterogeneity that validated, 28 marked rare subpopulations consistently representing
fewer than 2% of the bulk population (fig. 8b, c). An additional 20 markers were present
on 2- 10% of the bulk population (fig. 8d). The remaining 26 markers were detected on
a relatively high percentage of cells, demarking more than 10% of the bulk population
(fig. 8d). Although the hits were similar between the two strains examined by HTS,
there was large variability in the percentage of cells expressing these markers between
patient samples (Appendix A). Single tube FACs analysis consistently identified smaller
populations (Appendix A), which might be due to technical differences in staining
between HTS and single tube format.
3.7 Differences in isolated cell populations can be determined using ALI
culture.
While comprehensive functional evaluation of all reproducible subpopulations
would be beyond the scope of this work, we chose a few candidate subpopulations for
functional studies. Bulk population cells were FACS sorted based on expression of CD54
43
(fig. 9a, 5.4%) or podoplanin (fig. 10a, 85%), and seeded in replicate at ALI (10 000 cells
per well).
Monitoring progress of CD54-positive and -negative cultures revealed a marked
difference in epithelial morphology arising immediately after confluence and persisting
until 3 weeks post confluence when cultures had fully differentiated (fig. 9b-d). CD54-
positive cultures resembled control cultures, with many gland-like structures. In
contrast, CD54-negative cells formed very smooth cultures, with few gland-like
structures. During the three week period following establishment of ALI, CD54-positive
cultures appeared to slough a significant amount of debris or cells. This process was not
observed in CD54-negative cultures. These differences did not impact the extent of
ciliogenesis by enface staining (fig. 9e-g). Although gene expression analysis for
induction of FOXJ1 showed high variability between replicate cultures of each
subpopulation, no significant difference between CD54-positive and CD54-negative
cultures and unsorted controls were detected (fig. 9h). Evaluation of MUC16 induction
also showed no reproducible difference between populations (fig. 9i).
Cells sorted for the expression of podoplanin (fig. 10a) and grown at ALI showed
no differences during culture maturation at ALI. Enface staining revealed similar levels
of ciliogenesis in podoplanin-positive, podoplanin-negative and unsorted cultures (fig.
10b-d). Similar to our analysis with CD54 subpopulations, there were large differences
in FOXJ1 expression between replicate cultures, but overall, no reproducible difference
44
Figure 9: P0p basal cells sorted for expression of CD54 resulted in morphologically distinct cultures. (A) FACS plot showing gating used to sort P0plastic cells based on podoplanin expression. Phase contrast microscopy showing morphology of: (B) CD54-positive, (C) CD54-negative, and (D) unsorted ALI cultures at 3
weeks post-confluence (scale bars: 50 m). Enface staining of: (E) CD54-positive, (F) CD54-negative, and (G) unsorted ALI cultures at 21 days post-confluence (scale bars: 1 mm). QPCR analysis showing (H) FoxJ1 and (I) Muc16 gene expression between CD54-positive and CD54-negative cultures.
45
Figure 10: P0p basal cells sorted for expression of podoplanin gave rise to
indistinguishable cultures. (A) FACS plot showing gating used to sort P0plastic cells
based on podoplanin expression. Enface staining of: (B) podoplanin-positive, (C)
podoplanin-negative, and (D) unsorted ALI cultures at 21 days post-confluence (scale
bars: 1 mm). QPCR analysis showing (E) FoxJ1 and (F) Muc16 gene expression
between podoplanin-positive and podoplanin-negative cultures.
46
between podoplanin-positive and –negative subpopulations (fig. 10e). Similarly, no
significant differences were observed in MUC16 induction between the podoplanin-
positive and podoplanin-negative subpopulations (fig. 10f).
Rare populations of cells are of great interest, as they may represent stem or
progenitor cells. However, these populations pose a challenge for study due to their
limiting number. To bypass this issue, we adopted the strategy of depleting the bulk
population of such rare cell populations. If the depleted cells represent an essential
population it is expected that cultures would perform poorly even at high seeding
densities. Depletion of populations expressing: CD116, CD117, CD127 or CD337 showed
no noticeable decline in performance (data not shown). These data suggest that such
subpopulations do not represent essential populations for success at ALI, or that the
culture conditions employed here do not require such populations.
4. Discussion
Under steady state conditions the airway epithelium is relatively quiescent.
However, it is regularly exposed to harmful environmental agents and pathogens, and is
the site of a number of prevalent diseases such as lung cancer, and cystic fibrosis.
Hence, a mechanism is required for normal, long term maintenance that can also
respond rapidly to injury and disease conditions. Presumably, the regenerative process
responsible for maintaining epithelial integrity involves a local stem cell population. This
population could follow a classical stem cell hierarchy as seen in several highly
proliferative tissues including the intestinal epithelium and blood, where a rare stem cell
47
gives rise to progeny cells [22]. Alternatively, the lung epithelium could follow a non-
classical stem cell hierarchy as proposed in the liver [27,60]; in this model a large
number of equipotent, facultative stem cells would act in steady state and following
injury to repair or maintain the epithelium. Arguments can be made for either model in
the lung epithelium; a classical stem cell model might provide for long term
regenerative potential to maintain a tissue over the lifespan of the organism. A non-
classical model could be sufficient for long term maintenance of a tissue with a low
turnover rate, while providing a large number of progenitor cells capable of responding
quickly to acute injury. No consensus has been reached as current evidence supports
either model existing in the lung.
Basal cells are known to possess the regenerative capacity of the tracheal
epithelium [16,40,41], but also serve to anchor the entire epithelium to the basement
membrane through their unique formation of hemidesmosomes [9,10]. In light of the
multiple known functions for basal cells, in either a classical or non-classical model, we
might expect to find heterogeneity amongst basal cells at varying stages of maturity,
lineage commitment, or functional commitment. In any case, basal cell subpopulations
could be molecularly and functionally distinct.
In this work I explored cellular heterogeneity of the human trachea progenitor
cell compartment. Some evidence from murine studies has supported the idea of
functional difference in subsets of basal cells based on expression of cytokeratins, K5
and K14 [16,44-46]. Other work suggests that the anatomical location of basal cells is a
48
predictor of progenitor capacity [40,45,46]. In humans, the existence of cellular
heterogeneity has been supported by several lines of evidence [28,40,42]. In vitro
studies suggest there are differences in the differentiation potential of human p63+
progenitor cells that reside at different locations within airways. P63+ cells isolated
from the trachea were shown to differentiate into ciliated and goblet cells at ALI, while
those from the distal airway formed monolayers with very little mucociliary
differentiation [42]. Differences in clonogenic lineage potential of tracheal basal cells
have been shown in tracheal xenografts [40]. Furthermore, 1.6% of cells isolated from
murine trachea have some clonogenic potential, and 0.05- 0.1% of human and murine
tracheal cells can give rise to large colonies in vitro [42,44]. Since basal cells represent
approximately one third of all epithelial cells in the proximal airway [2], these numbers
suggest that rare basal cells possess clonogenic potential, while even fewer possess a
large capacity for proliferation. These results support the existence of subpopulations
within the basal lineage with distinct functional properties. Based on this evidence, we
set out to discover the frequency of such a population of cells in the human trachea, and
to find molecular markers which could be used to identify and separate functionally
distinct subpopulations.
4.1 Establishment of culture systems and assays
To begin to investigate heterogeneity in the basal cell compartment we first had
to obtain functional progenitor populations and establish techniques to evaluate them.
Using previously described methods, human tracheal epithelial cells (HTECs) were
dissociated from tracheal tissue and cultured to selectively expand basal cells while not
49
supporting growth or survival of fibroblasts or columnar cells [47]. These culture-
purified basal cells, grown on plastic without passaging (P0plastic), possess the capacity
to differentiate into other functional tracheal lineages [38]. Using these methods, we
derived basal cells from a number of donor tracheas and confirmed their purity and
progenitor function.
The basal cell purity of P0plastic cells was initially assessed by FACS analysis
showing over 99% expressed CD44, an in vivo basal cell specific marker (fig. 1). Purity
was also evaluated by IF staining; nearly 90% of P0plastic cells stained positively for p63,
another basal cell-specific marker, and no cells expressed columnar cell markers MUC16
or polarized apical βTUBIV, markers for mucinous and ciliated cells respectively (fig. 1).
Given these data and our observation that with longer exposure times we could detect
low levels of p63 in “p63 negative” nuclei, we conclude that virtually all P0plastic cells
are basal cells. The reduced levels of p63 immunoreactivity observed in some cells
could be a staining artifact, or could represent cells undergoing a cellular process which
corresponds with decreased expression of p63; these changes may occur during cell
division or migration. Such processes may also account for the observed βTUBIV
cytoplasmic foci, which could represent cells in a particular stage of the cell cycle, or
cells undergoing specific morphological changes [58].
To verify progenitor activity of the P0plastic basal cells and to establish methods
to functionally interrogate basal cell subpopulations, we characterized the behaviour of
basal cells in ALI cultures. Basal cells were initially seeded subconfluently at ALI, grown
50
to confluence while being fed both basolaterally and apically, and then fed only
basolaterally for 3 weeks. Assessment of p63 immunostaining and qPCR showed that
basal cells grown at ALI retain their basal identity until cultures reach confluence. By
three days post-confluence there were many p63 negative nuclei, and secretory cells
were detected by MUC16 protein and gene expression suggesting that progenitors were
transitioning into other lineages. Emergence of ciliated cells was apparent by 7 days
post confluence as assessed by IF staining. At 14 days post confluence, the presence of
beating cilia was visible by light microscopy, which corresponds with large increases in
FOXJ1 expression. These data demonstrate that P0plastic basal cells are a pure
population of progenitors that have retained mucociliary potential.
Having purified a population of functional progenitors we sought to develop a
method to functionally evaluate progenitor cells that is: consistent between samples,
easily monitored and compared, and gives good differentiation. Using the ALI culture
system to differentiate progenitor cells, we looked at ciliogenesis as a progenitor
output. Ciliogenesis is an ideal lineage readout as: it is robust; it can be observed
microscopically in living culture by light microscopy; and it can be measured
quantitatively at the level of gene and protein expression.
Histological sections have historically been used to examine ciliogenesis. They
reveal epithelial morphology, and IF staining provides a qualitative view of cell types
present and may allow some cell types to be quantified [41,61-63]. However, we found
histological sections to have limited value for quantitative analysis when limited
51
differentiation is achieved. This is largely due to uneven distribution of ciliated cells. To
resolve this issue, we investigated two alternative methods to quantitatively assess
differentiation: qPCR and enface staining. We show that FOXJ1 gene induction tracks
well with emergence of ciliated cells as determined by IF staining for βTUBIV, (fig. 5 and
6). Furthermore, using IF staining, ciliated cells are easily delineated from non-ciliated
cells because βTUBIV is contained within membrane bound cilia projections. The enface
staining and tiling method described here allows the extent of differentiation to be
measured across the entire ALI culture, removing any variability due to uneven
distribution of ciliated cells.
We observed macroscopic secretions on the apical surface of the ALI culture
preceding appearance of functional cilia, suggesting the early emergence of secretory
cells. Surprisingly, antibody and gene expression analysis suggest these secretions are
due to MUC16 secretory cells, not due Muc5ac+ goblet cells (fig. 2, 3). Although we
found that P0plastic cells can differentiate into goblet cells in xenograft cultures, we
have not observed the emergence of goblet cells in ALI cultures, suggesting our ALI
conditions lack required signaling for this fate. Instead, our ALI conditions drive
physiologic levels of MUC16 secretory cell differentiation, another abundant secretory
lineage in the upper airway [56] (fig. 2, 3). We observed that Muc16 gene and protein
expression show similar kinetics at ALI, and are induced in all samples. However, trends
in induction and expression are not as robust or consistent between patient strains as
those observed with ciliated cell markers (fig. 5 and 6).
52
Previous work has indicated that serial passage of human basal cells on plastic
leads to significant decline in progenitor potential [38]. We asked whether serial
passage of cells on ALI culture might provide better preservation of progenitor activity.
This could be important for maintaining progenitor cells for long term experiments or
expanding limited primary cells to acquire large numbers required for high-throughput
assays. We compared the progenitor activity of plastic-passaged cells to ALI-passaged
cells by comparing their ability to differentiate at ALI (fig. 7). Consistent with previous
reports, we found that the progenitor potential of plastic passaged cells fell drastically
over 2 passages. A similar decline was observed in the activity of ALI-passaged cells.
The extent of ciliogenesis was substantially lower in P2pA and PAPA cultures than in
P0pA from all patient strains examined. However, there was little impact on MUC16
lineage potential following P2pA and PAPA cultures, which showed similar MUC16
expression to P0pA cultures. These data show that ALI passaging is not better than
submerged LHC-9 culture at preserving mucociliary progenitor activity. The differential
impact of ciliary and mucinous potential could suggest these lineages are derived from
different progenitor cell populations, which are differentially maintained during culture.
Alternatively, the ciliogenic potential may be maintained but the signaling environment
of culture may preferentially drive a mucinous fate. Further development of both ALI
and submerged culture conditions could permit better maintenance or detection of
progenitor potential.
4.2 Evidence for functional heterogeneity
53
To investigate functional heterogeneity within the basal cell compartment, we
employed a limiting dilution assay (LDA). In theory, if rare stem cells exist in our
P0plastic basal cell population, they would be diluted out as cultures were seeded at
lower densities. The distribution of these cells would follow a Poisson model, meaning
at some critical seeding density we would have a high probability of seeding one or zero
stem cells. At this density we would see some cultures succeed, while those cultures
not containing a stem cell would fail. Alternatively, we could see a similar result if the
cells were equipotent, but produced a paracrine factor required for growth, since a
minimum number of cells might be required for sufficient concentrations of that factor
to accumulate.
Using this technique, we were able to obtain sufficient data from 5 patient
strains to confirm that a single factor may be responsible for success of the cultures, as
defined by the cultures reaching confluence and maintaining a barrier (table 2). We
found that basal cells isolated from different patients performed significantly differently
in ALI cultures (table 3). The estimated frequency of a basal cell capable of
repopulating an ALI culture ranged from 1 in 96 to 1 in 1297 (0.08%-1%) (fig. 3). These
values are similar to those previously reported in human and mouse for the frequency
of cell capable of giving rise to large colonies (0.05- 0.1%) [42,44]. The slightly lower
frequency of clonogenic cell observed in these studies may be due to failure to sort
basal cells from columnar cells, differences between species, damage from the sorting
process [44], or culture conditions [42] used in these studies. Alternatively, this could
54
reflect natural variation between patients based on biological differences such as age, or
gender.
The data presented here supports the existence of subsets of ALI repopulating
cells rather than the existence of a dilutable paracrine factor essential for growth. First,
the complete failure of one replicate culture at the same seeding density that results in
successful repopulation in another replicate culture is difficult to reconcile with dilution
of a paracrine factor unless an extreme error in seeding density occurred. Second, the
sometimes large differences in induction of differentiation genes between replicate
cultures (fig. 6, 7, 10 and 11), seeded at high initial densities, are consistent with the
notion that there are rare cells that give rise to differentiated progeny, and that these
cells are variably diluted during seeding. This observation is also difficult to reconcile
with the model that equipotent cells simply produce a limiting paracrine factor,
especially when cells are seeded at high densities. Our data support a model where rare
cells, representing less than 1% of P0plastic basal cells, have the ability to undergo
massive expansion and repopulate an ALI filter. However, the mucociliary
differentiation of these cultures may be dependent on additional factors or cell types.
None of the LDA experiments yielded sufficient results to test whether a single factor
was responsible for successful mucociliary differentiation. However, the difference in
our ability to test the single hit hypothesis for repopulation but not differentiation
suggests a different factor, or number of factors, is required for each process.
4.3 Evidence for molecular heterogeneity
55
Large scale comprehensive surface marker profiling has not been reported for
lung basal cells in any species. To comprehensively investigate molecular heterogeneity
within the basal cell population we employed a high-throughput screen consisting of
339 antibodies. Here we consider a subpopulation to be any subset of P0plastic cells
that have differential expression of the examined surface marker. We initially identified
115 cell surface markers that denoted subpopulations of basal cells in 2 independent
patient strains. Of these markers, 74 were confirmed in a non-high throughput test on
cells from different patient strains, indicating reproducible surface marker
heterogeneity among human basal cells. The observed heterogeneity could be due to
the presence of hierarchical relationships between basal cells with different proliferative
and lineage potential, or result from equipotent basal cells that are in different states.
For example, these states could reflect different stages of maturation or differentiation
or stress response to in vitro culture. The cell surface marker signature of any single
subpopulation of basal cells could be comprised of single or multiple markers within the
group of 74. At present, it is unclear how many distinct populations are represented by
the 74 markers.
While the extent to which this surface marker heterogeneity reflects in vivo
populations has not been addressed in this work, our results provide tools for future
directed in vivo analyses. A previous study by Atsuta et al. looking at integrin expression
on human bronchial epithelial cells supports some of our findings. They also found
evidence for expression of 12 surface markers identified in our screen on P0plastic
bronchial epithelial cells [64]. However, they did not examine potential heterogeneity in
56
expression of these markers. Our data suggest that 9 of these 12 markers are actually
heterogeneously expressed among basal cells.
Little work has been done to identify normal lung stem cell markers. However,
one recent report by Kajstura et al. proposed the existence of rare human CD117+,
KLF4+, NANOG+, OCT3/4+ and SOX2+ stem cells in the lung. While most of these cells
are p63 negative and lack epithelial markers, some evidence was provided that
indicated a fraction of these cells could acquire a p63 positive fate [28]. In our work, we
found inconsistent expression of CD117 across different patient samples. Furthermore,
we were unable to expand viable primary unpassaged HTECs using the culture
conditions that were reported to promote expansion of the CD117+ stem cells [28].
While it is possible that our failure to consistently detect such a cell could be due to its
relative enrichment in the distal rather than primary airways, the existence of CD117+
stem cells has yet to be confirmed by other labs.
It is possible that distinct progenitor cells throughout the airways share some
surface antigens. Studies of lung progenitor cells in distal regions of the airway have led
to the identification of several surface markers. Kim et al. described a bronchioalveolar
stem cell as Sca1+ and CD34+. Sca-1 is a murine specific antigen and we did not detect
CD34 immunoreactivity in our basal cells.
In some cases, cancer stem cells share phenotypic properties with normal stem
cells [65]. Several surface markers have been identified for lung cancer stem cells, also
known as tumour initiating cells (TIC). Tirino et al. proposed that a CD133+ cell fraction
57
of non-small cell lung cancers (NSCLC) includes TICs [66]. They found a CD133+ fraction
in 72% of NSCLC, and showed that culturing these cells as non-adherent spheres
increased the proportion of CD133+ cells, which resulted in a nearly 4-fold increase in
tumour initiating capacity of these cultures in NOD/SCID mice [66]. However, CD133+
cells were not detected in our screen, which suggests that such cells may only arise in
disease states, or may reflect non-basal cell progenitors. More recent work by Zhang et
al. did not support CD133 as a marker for NSCLC TICs, but provided evidence for the
CD166+ fraction as marking the majority of TICs [67]. In our screen, all basal cells
expressed CD166, which could be consistent with some TICs arising from the p63+ basal
cell compartment.
Putative stem cell populations have been characterized in a number of other
epithelia. Notably, mammary glands are composed of a stratified epithelium where
p63+ basal cells are thought to contain the progenitor activity. CD49f, CD24 and CD29
have been identified as potential markers of mammary epithelial stem cells [68,69], and
CD49f has also been used to enrich progenitor activity in p63+ population of
keratinocytes [70]. All three of these markers were identified in our screen as being
heterogeneously expressed in lung basal cells and are promising candidates for further
study.
To investigate the existence of potential functional differences between marker-
positive and marker-negative subpopulations of P0plastic basal cells we looked at CD54
and podoplanin, markers of relatively abundant subpopulations which showed
58
promising results in preliminary experiments. Cells were sorted based on expression of
CD54 or podoplanin, and fractions were assessed at ALI for: proliferative potential,
extent of differentiation, and emergence of mature cell types. For both CD54 and
podoplanin, the positive and negative fractions had indistinguishable lineage potential
as assessed by qPCR and enface staining of 21 day post confluent cultures. However,
monitoring of CD54 subpopulations revealed marked, reproducible differences in
culture morphology during epithelial regeneration (fig. 10). CD54 expression on airway
epithelial cells has been linked to local molecular events in inflammation in response to
allergen exposure, playing a role in eosinophil recruitment and infiltration [71]. Thus,
these morphological differences between CD54+ and CD54- generated ALI epithelia
could reflect functional differences in the ability to promote specific inflammatory
processes (fig. 10) [71,72]. The failure to detect a difference between podoplanin
subpopulations could reflect stochastic changes in ex vivo expression without functional
significance, or could reflect limitations of our assays in detecting functional differences
between subpopulations of basal cells. Notably, we only examined two lineage markers
and our assays do not extensively evaluate the self-renewal potential of basal cells.
The markers we have identified can now be used to help distinguish a classical
from non-classical stem cell model for epithelial regeneration in the trachea. Candidate
subpopulations can be sorted and re-cultured, and the proliferative ability of these cells,
as well as their ability to regenerate marker positive and marker negative states can be
assessed. Sorted cells can also be interrogated in ALI models employing serial
passaging, as well as tracheal xenograft models, which may help uncover functional
59
differences between basal cell subpopulations. Additionally, antibody staining of
tracheal tissue could be used to distinguish relevant in vivo subpopulations from those
generated artifactually by ex vivo culture. Finally, multi-parametric FACS analysis of
P0plastic basal cells will be useful to determine how many subpopulations are
represented by the markers identified here.
Here I present data demonstrating human tracheal P0plastic cells are a purified
population of basal cells that retain progenitor activity (Fig. 1, 2 and 3). I provided
detailed analysis of the differentiation process of P0plastic basal cells on ALI, showing
robust induction of markers for both ciliogenic and secretory differentiation programs,
as well as optimized methods for assessing progenitor output and differentiation (Fig. 4,
5 and 6). I demonstrated that ALI culture conditions are able to maintain a progenitor
population at an extent similar to LHC-9 conditions (Fig. 7). Performing a limiting
dilution assay on basal cells with in vitro and in vivo validated progenitor activity, I
determined the frequency of clonogenic cells capable of repopulating ALI ranges from
0.08%-1%, and varies significantly between patients. Importantly, I contributed a list of
74 validated cell surface markers that are heterogeneously expressed amongst P0plastic
basal cells. Subpopulations varied in frequency from <1% to ~50%. Preliminary
functional analyses suggest there may be differences in the differentiation potential of
some of the subpopulations. This work supports the notion that the basal cell
compartment may be functionally heterogeneous, and provides a new arsenal of
molecular tools for the directed investigation of heterogeneity among human basal
cells.
60
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Appendices
Appendix A
Antibody Str. 38 Str. 23
Single
tube
CD50 0.0367 2.55E-03 0
CD123 0.18 0.139 0.054
CD180 0.182 0.0448 0
CD195 0.283 1.36 0
CD249 0.332 0.829 0
CD33 0.399 0.64 0.017
CD22 0.441 0.548 0.055
CD122 0.502 0.231 0.46
CD62L 0.511 1.11 0
CD312 0.519 1.25 0
CD181 0.533 0.555 0.48
CDW93 0.567 0.241 0
CD62P 0.609 0.4 0
CD275 0.808 0.173 0.49
CD116 0.948 0.618 0.89
CD266 0.978 0.991 0.071
CD213a2 1.05 0.589 0
CD159c 1.07 1.5 0
CD182 1.07 0.312 0.12
VBTA8 1.07 1.49 0.88
CD32 1.09 0.364 0
CA9 1.11 1.64 0.036
CD16 1.13 0.461 0.085
CD82 1.22 1.35 0.045
CD6 1.26 1.31 0
CD132 1.42 0.383 0.87
CD7 1.65 1.15 0.11
HLA DM 1.69 0.953 0
CD193 1.83 1.95 0.37
CD247 0.207 2.22 0
CD138 0.697 5.36 0
CD85g 1.41 2.35 0
CD56 1.71 2.32 0
CD164 2.01 1.34 0
CD158e2 2.07 1.28 0.65
CD99 2.09 3.36 0
CD227 2.09 2.43 1.48
CD38 2.61 5.97 0
CD117 2.66 0.338 6.35
CD2 2.68 3.38 0
CD87 3.09 0.757 0
65
CD97 3.09 3.37 0
SSEA-4 3.11 0.632 0.58
CD175s 3.17 8.88 0
FOXP3 3.9 0.0561 0
VDTA2 4.16 2.38 1.31
CD267 4.42 1.17 1.01
CD125 4.52 1 0
CD157 4.89 13 0.015
CD268 4.91 1.31 1.96
CD13 5.37 9.3 31.6
CD90 5.54 1.93 4.42
CD49D 5.55 8.4 1.85
CD51 5.76 1.34 0
GMA DTA 5.86 0.805 0.76
CD36L1 6.18 5.53 1.22
CD91 7.46 6.05 0
CD127 8.14 0.707 0.61
CD61 3.17 17.7 0
CD170 4.57 18.8 0
CD295 6 13.9 0.43
CD314 14.8 2.55 1.86
CD337 17.8 4.26 0.72
CDH3 18.1 49.3 0
CD158A 19.2 0.821 0.015
CD253 19.3 2.37 0.057
CDW218a 20 24.9 0.48
SSEA-3 20.6 3.16 0.017
CD245 21 46.2 22.8
CD252 23.2 2.16 0.31
CD10 27.7 9.02 2.79
CD102 29.3 21.9 3.18
CD263 30.8 2.81 0.63
CD49A 38.7 37.4 0
6D12 39.9 20.7 30.5
CD66C 45 17.5 17.5
CD271 21.9 87.7 2.28
CD205 41.2 61.3 0
CD201 32.8 55.7 17.8
CD148 46.3 56.4 1.51
CD151 58 1.82 7.28
CD130 61.5 14.2 1.41
CD54 67 43.4 31.2
CD108 72.4 33.3 41.9
CD105 76.9 68 0
CD71 82.9 28.7 0.91
CD264 85.8 36.9 42.4
66
CD65s 86.9 76.1 15.9
CD63 87 77.8 6.84
Podoplanin 89.7 71.3 58.6
CD261 82 92.1 0.36
CD262 82.1 90.3 21.7
CD223 57.4 94.1 44.6
CD44 66.4 94.2 N/A
EGF-R 91.3 96.6 5.42
CD66 95.7 76.3 39
CD9 96.9 96.7 73.1
CD340 97 99.1 75.6
CD221 98.2 99 1.45
CD142 98.2 96.2 95.1
CD47 98.5 99.9 3.49
CD326 98.5 98.7 65.7
CD81 98.5 93.9 75.9
CD24 98.7 74.9 62.3
CD172a 98.8 50.8 47.4
CD29 98.8 99.8 100
CD119 99.1 97.7 1.34
CD58 99.1 99.5 99.42
CD276 99.4 99.7 100
CD49F 99.5 99.9 99.1
CD147 99.7 99.5 100
TNFR RLP 85.1 99.3 1.17
67
Appendix B
Sequence of primers
Gene Forward Primer Reverse Primer MUC16
Np63 TAp63 FOXJ1 TBP
TGC GGT GTC CTG GTG ACC ACC CGC GGA AAC AAT GCC CAG ACT CAA TGT ATC CGC ATG CAG GAC T CAC CTG AGC CGA GCC GGG ACT TAG CGG TGT GCA CAG GAG CCA AGA GT
CAC CGG CAA GTT CCA GTC ATT GC TGC GCG TGG TCT GTG TTA CTG TGT TAT AGG GAC TGG TGG AC CTC CCG TTA CAC GGC CTC CCG ATT TTC TTG CTG CCA GTC TGG
68
Appendix C Experimental data from LDA experiments
Str: 29945 Str: 38 Str: 23 Str: 78297 Str: 32 Str: 18 Str: 79168 Str: 39
Cell No: 2000 6000 6000 1500 31000 31000 100 6000
Confluence 6/ 6 6 /6 6/ 6 6/ 6 2/ 2 4/4 4/6 8/ 8
Barrier 6 6 6 6 2 4 0 8
Cilia 6 6 6 6 2 4 0 8
Cell No: 500 3500 3000 1000 6000 6000 50 500
Confluence 4/6 6/6 6/6 6/6 2/ 2 4/4 2/6 8/8
Barrier 2 6 6 6 2 4 0 8
Cilia 0 6 6 6 2 4 0 8
Cell No: 250 2500 2000 500 1000 1000 10 100
Confluence 2/6 6/6 6/6 6/6 4/ 4 4/4 1/ 6 8/8
Barrier 0 6 6 6 4 4 0 5
Cilia 0 6 6 6 0 4 0 0
Cell No: 50 1500 1000 250 500 500 1
Confluence 0/6 4/6 6/6 5/6 4/4 4/4 0/ 6
Barrier 0 4 6 5 0 4 0
Cilia 0 4 6 5 0 4 0
Cell No: 12000 250 250
10000, 5000, 2500, 1500, & 500
Confluence 2/ 2 2/ 4 4/4 1/1
Barrier 2 0 4 1
Cilia 2 0 4 1
Cell No: 500
50 50
Confluence 0/2
2/ 4 4/4
Barrier 0
0 4
Cilia 0
0 4
Cell No:
10 10
Confluence
0 of 4 4
Barrier
0 4
Cilia 0 4
69
Str: 31 Str: 27 Str: 55 Str: 94214
Cell No: 6000 6000 6000 10 000
Confluence 7/ 7 5 / 5 8/ 8 16/16
Barrier 7 5 8 16
Cilia 7 0 8 16
Cell No: 100 500 500 100
Confluence 6/ 7 5/5 2/ 6 16/ 16
Barrier 2 5 0 16
Cilia 0 0 0 16
Cell No: 100 100
Confluence 3/ 5 0/ 6
Barrier 1 0
Cilia 0 0