1 Department of Cell and Molecular Pathology, St John's Institute of Dermatology
2 Richard Dimbleby Department of Cancer Research/Cancer Research UK Laboratory, Guy's, King's and St Thomas' School of Medicine, St Thomas' Hospital, London, UK
3 FACS Laboratory, Cancer Research UK, London Research Institute, 44 Lincoln's Inn Fields, London, UK
* Author for correspondence (e-mail: hong.wan{at}kcl.ac.uk)
Accepted 5 June 2003
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Summary |
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Key words: Desmoplakin, Desmoglein 3, Desmosomes, Stem cells, Keratinocytes
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Introduction |
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Skin homeostasis is governed by a small subpopulation of keratinocyte stem cells (Potten and Morris, 1988; Lavker and Sun, 2000
; Watt, 2001
). It has been known for some years that basal keratinocytes are not homogeneous and possess diverse cell populations including stem cells, transit amplifying cells and post-mitotic cells undergoing differentiation (Potten and Morris, 1988
; Jones and Watt, 1993
; Jones et al., 1995
; Dunnwald et al., 2001
). Owing to the slow cycling nature of epidermal stem cells in vivo, conventional approaches to determine stem cell location have relied on cell kinetic analyses using either bromodeoxyuridine (BrdU) or [3H]thymidine to follow up the label-retaining cells within the tissue. Thus, the location of stem cells in a variety of epithelia has been determined in vivo (Lavker and Sun, 1983
; Cotsarelis et al., 1989
; Taylor et al., 2000
). For instance, stem cells in monkey palm were identified at the tips of deep rete ridges by pulse-labelling with [3H]thymidine (Lavker and Sun, 1983
).
Cultured human keratinocytes, however, may not maintain all their in vivo characteristics after removal from their `niche' or microenvironment but are clonogenic (Potten and Morris, 1988; Jones and Watt, 1993
; Jones et al., 1995
) and able to generate three distinct clonal forms owing to the varied self-renewal capacities of individual keratinocytes (Barrandon and Green, 1987
). Holoclones, founded mainly by stem cells, appear large with a smooth circular perimeter and have the highest reproductive capacity. Paraclones, founded by cells undergoing terminal differentiation, appear small and are known as abortive clones. Meroclones are intermediate between holoclones and paraclones in their morphology and behaviour. It has been suggested that analysis of colony formation and frequency of clone types after a period of growth in vitro predicts the intrinsic nature and capacity of the proliferative potential of the original keratinocyte populations (Barrandon and Green, 1987
).
Growing interest has focused on the role that stem cells play in homeostasis, wound repair and tumorigenesis (Tu et al., 2002) as well as on their identification and use as a therapeutic option in tissue bioengineering, gene therapy and for the treatment of a variety of diseases (Rama et al., 2001
). Considering the potential ethical issue of using embryonic stem cells, identifying multiple markers to isolate pure keratinocyte stem cell populations from adults is of value. It is important both for understanding the basis of stem cell science and also for stem-cell-mediated clinical replacement therapy. However, studies of stem cell properties have been hampered by the lack of appropriate surface markers that could facilitate their identification and isolation. Many attempts have been made to identify putative markers of epidermal stem cells (Jones and Watt, 1993
; Jones et al., 1995
; Li et al., 1998
; Dunnwald et al., 2001
), one of which is the ß1 integrin (Jones and Watt, 1993
; Jones et al., 1995
), the positive cell surface marker. It has been shown that the basal keratinocytes with a high colony forming efficiency (CFE) have the highest ß1 integrin staining characteristics (Jones and Watt, 1993
; Jones et al., 1995
). Other studies suggest that a keratinocyte population containing stem cells can be isolated according to high levels of integrin
6 expression and low levels of transferrin receptor (Li et al., 1998
; Tani et al., 2000
). However, negative markers alone are also considered useful for isolating the putative stem cell populations. For example, gap junction protein connexin 43 has been proposed to be a negative marker for epidermal stem cells (Matic et al., 1997
; Matic et al., 2002
). Several molecular signal pathways such as NF-
B, Wnt/ß-catenin, Sonic hedgehog/patched and delta/notch have been described in the literature as being involved in the control of the stem cell compartment (Watt, 2001
).
Based on the evidence that desmosomes become more numerous as keratinocytes differentiate and mature, it seemed reasonable to hypothesise that desmosomes may appear sparse in the epidermal stem/progenitor cells. In this study we report, for the first time, that this indeed is the case. We found that the epidermal basal keratinocytes, either in vitro or in vivo, show heterogeneity in desmosome expression. Clusters of cells with low Dp expression can be identified at the tips of deep rete ridges in palm skin as well as within the colonies of cultured keratinocytes. Fractionated populations, enriched for ß1-bright cells exhibit inversely low desmosomal protein expression. We show that sorting keratinocytes that express low levels of Dsg3 results in enrichment for a cell population that exhibits features in common with the epidermal stem cells, i.e. high clonogenecity, CFE and proliferative potential. Moreover, we show that sorting for ß1 integrin-bright/Dsg3-dull keratinocytes enhances even further the enrichment of epidermal stem cells.
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Materials and Methods |
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Immunofluorescence for adult palm skin section and cultured keratinocytes
Normal palm and non-palm (mainly breast) skins were obtained under approval from St Thomas' Hospital's ethics committee. Cryosections of skin and the cultured keratinocytes were processed and labelled as described previously (Wan et al., 2003).
Human primary keratinocyte culture
The adult human keratinocyte cultures were generated following a standard procedure (Navsaria, 1994) and as described previously (Wan et al., 2003
).
Laser scanning confocal microscopy
Dual immunofluorescence images were acquired following the procedure described previously (Wan et al., 2003).
In situ hybridisation
In situ hybridisations for Dp mRNA on cryosections of palm skin and cultured keratinocytes were performed following the protocol provided by the manufacturer (BIOGNOSTICK, Germany) as detailed elsewhere (Wan et al., 2003).
Immunoblotting
Protein extracts and western blotting for the major desmosomal proteins were carried out as described previously (Wan et al., 2003).
Adhesion to type IV collagen
This procedure was carried out as previously described (Jones and Watt, 1993; Jones et al., 1995
). Briefly, the plastic dishes were coated overnight at 4°C with 100 µg/ml collagen (Sigma), and then incubated with 0.5 mg/ml heat-denatured bovine serum albumin (BSA) (Sigma) at 37°C for 1 hour and washed in serum-free medium before use. The suspended cells were harvested using trypsin-EDTA and allowed to attach to the substrate for 20 minutes (rapid adhesion). The unattached cells were then transferred to a fresh dish and allowed to attach for 24 hours (slow adhesion). After 24 hours, the unattached cells were removed and fresh medium was added. Cells plated into the dish without adhesion separation were used as the control. Cells were cultured for a further 8 hours before protein extraction or fixation for immunofluorescent labelling.
Immunofluorescent labelling and flow cytometry
Immunofluorescent labelling and flow cytometry were carried out as described previously (Jones and Watt, 1993). Briefly, for fluorescence-activated cell sorting (FACS) analysis, passage 1 keratinocytes were suspended in 0.25% trypsin containing 1 mM calcium chloride in PBS and filtered through a 70 µm nylon mesh (Falcon). For single labelling, cells were incubated on ice with a saturating concentration of Dsg3 Ab 5H10 or CD29:FITC for 5-10 minutes before washing in wash buffer [Dulbecco's modified Eagle's medium (Gibco) plus 0.1% bovine serum albumin (BSA; Sigma)]. Primary antibody-labelled cells were incubated with Alexa Fluor 488-conjugated IgG for 5 minutes on ice. After washing thoroughly, cells were kept either in PBSABC plus 0.1% BSA and 0.1% NaN3 for flow cytometry or in keratinocyte-culture medium containing 1% foetal bovine serum (FBS) for FACS. Propidium iodide (5 µg/ml) was added for viability gating. For dual labelling, cells were either labelled with 5H10-RPE or 5H10:Zenon Alexa Fluor 647 followed by CD29:FITC. Non-viable cells were gated out using DAPI.
Cells were sorted using a FACS Vantage (BD Biosciences, San Jose, CA) or a Moflo (DakoCytomation, Fort Collins, CO) sorter. The sorted cells were collected into tubes containing keratinocyte culture medium supplemented with 10% FBS before being plated at different cell densities on J2 feeder cells, which were treated with mitomycin C (Sigma) prior to use.
Colony analysis
Cells were grown on feeders for different time periods. The colonies were assessed by phase-contrast microscopy (Zeiss TELAVAL 31) and photographed (KYOCELA, Japan). At the end of each experimental time point, colonies in wells (n=3) were fixed with equal volumes of ice-cold methanol and acetone for 15 minutes, washed in PBS and stained either with 1% Rhodamine B (Sigma) and 1% Nile Blue (Sigma) (Jones and Watt, 1993) or with LP34 and DH1 then DAKO EnVision Doublestain System. The stained colonies were scanned and the images were analysed in Adobe Photoshop. The feeder background was masked with white (255 in greyscale) to avoid its influence before analysis of the colonies in software OPTILAB PRO 2.6.1 (GRAFTEK, France). Parameters of colony density per dish and colony number and area (size) were acquired. The colony density was calculated as the percentage of each dish covered by colonies. Results are presented as mean±s.e.m. of triplicate samples. CFE was the ratio of colony number to plating cell number (Jones and Watt, 1993
). The mean colony area was calculated from all the individual colonies and presented as the mean±s.e.m. Statistical analysis was conducted using one-way analysis of variance (ANOVA). Significant differences were determined at P<0.05.
Colony assessment and total cell number determination
To determine the frequency of abortive colonies, colonies were selected randomly and counted blindly using a phase-contrast microscope. Colonies were scored on their morphological feature (Jones and Watt, 1993; Jones et al., 1995
). Total colony numbers after 2 weeks in culture were counted and total cell number was determined using a haemocytometer. Differences of clone type between groups were analysed by Chi-Square and differences were deemed statistically significant if P<0.05.
Viability assay
The FACS-sorted and control cells, and non-FACS-sorted control cells were plated at a series of titration densities in 96-well plates coated with 100 µg/ml collagen. The viability assay was performed following the protocol provided with CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega).
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Results |
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High ß1 integrin-expressing keratinocytes have low level of Dp expression
To test our hypothesis that the putative adult epidermal stem cells express fewer desmosomes we used an established protocol to enrich for keratinocytes that contain stem cells from human skin (Jones and Watt, 1993; Jones et al., 1995
). We separated trypsin-dissociated adult palm keratinocytes into two populations by rapid (<20 minutes) and slow (>20 minutes to 24 hours) adhesion to type IV collagen, a selection procedure based on levels of functional ß1 expression. Unfractionated cells were used as the control. Western blotting analyses (n=8) from a minimum of five experiments revealed that rapidly adhering cells appeared to have low Dp expression. In contrast, the slowly adhering cells showed about a fourfold increase in Dp expression, while an intermediate level of expression of this protein was seen in the control cells, as expected (Fig. 2). Other desmosome proteins, including Dsgs, Dscs, Pg and Pkp1 also showed qualitatively similar expression profiles when the rapidly and slowly adhering cell populations were compared (Fig. 2). To confirm that the rapidly adhering cells contained higher levels of the surface activated ß1 integrin (Jones and Watt, 1993
; Jones et al., 1995
) we fixed the cells prior to direct immunofluorescent staining with an antibody recognising functional cell surface ß1 integrin [12G10 (Mould et al., 1995
) directly conjugated to Cy5]. We found that there was a significantly higher level of activated ß1 integrin expression in the rapidly, rather than the slowly, adhering cells (data not shown). We also compared ß1 integrin expression in these two populations by flow cytometry and found consistently higher surface ß1 integrin levels in the rapidly, rather than slowly, adhering cells (data not shown), as previous reported (Jones and Watt, 1993
). Immunofluorescent staining for Dp in these two cell populations showed relatively less punctate staining in the rapidly- as compared to the slowly- adhering cells although diversity of Dp expression was still seen within the rapidly adhering cells (data not shown), suggesting existence of keratinocytes heterogeneity. Taken together, these findings show that selecting palm keratinocytes, by rapid adhesion to a ß1 integrin ligand (type IV collagen), enriches for a subpopulation that has a low level of desmosomal protein expression including Dp and Dsgs.
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Adult palm keratinocytes with a low Dsg3 level are capable of forming large, actively expanding colonies
The different expression level of desmosomal proteins between rapidly and slowly adhering cell populations suggested that desmosomes might serve as a negative marker for putative epidermal stem cell enrichment. In order to sort for the viable cells with low desmosome expression using FACS, antibodies directed against the extracellular domains of desmosomal cadherins in basal keratinocytes were required. We therefore tested two available antibodies, Dsc3-U114 (mouse antibody directed to the extracellualr anchor domain of human Dsc3) and 5H10 (mouse antibody directed against the amino-terminal EC1 and part of EC2 in the extracellular domain of Dsg3). We found that only 5H10 worked in flow cytometry and thus we developed an assay for detecting the cell surface desmosomal protein Dsg3. Palm keratinocytes at passage 1 were dissociated with 0.25% trypsin in the presence of 1 mM Ca2+ (Takeichi, 1977) and labelled with 5H10. Bound antibody was detected with goat anti-mouse IgG conjugated to Alexa Fluor 488 prior to FACS analysis (Fig. 3A). The viable keratinocytes were selected in the square box on the basis of their light-scattering characteristics. Dead cells were gated out with propidium iodide (less than 10% of the cells). The selected cells were further fractionated into two categories on the basis of Dsg3 expression. We sorted for 20% of cells with the lowest level of Dsg3 (Dsg3dim) and for the 40% of cells with the highest level of Dsg3 (Dsg3bri). Cells were then plated onto the feeder cells (J2 strain) at three different densities, and grown for 5, 7, 9, 11 (Fig. 3B) or 14 days. At each time point, colonies were fixed prior to staining for both keratins and involucrin, and visualised with EnVision Doublestain System (DAKO). Parameters of colony density (Fig. 3C), CFE (Fig. 3D) and colony size (Fig. 3E) were scored (see Materials and Methods). Overall, the Dsg3dim population developed higher colony densities and larger colony sizes than Dsg3bri cells at almost every time point measured. By day 11 both colony density and size were increased significantly compared with the Dsg3bri population (Fig. 3C,E). A slightly higher CFE was also observed in Dsg3dimrather than Dsg3bri population at 11d (Fig. 3D). These results suggested that cells in the Dsg3dim population had greater growth ability and were capable of generating large, actively expanding colonies in short-term (2 weeks) culture.
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Dsg3dim keratinocyte populations shows greater overall proliferative capacity and survive better than Dsg3bri cells in long-term culture
In order to investigate the longer-term culture of the adult keratinocytes sorted for either low- or high-Dsg3 levels, cells were monitored for multiple further passages until all the cells were completely exhausted. At each passage, cells were counted and plated at the same density. Cells formed heterogeneous colonies in the cultures, and we found that there were three major and distinct colonies in our study of the adult human keratinocytes. One resembled holoclones as described by Barrandon and Green (Barrandon and Green, 1987), and cells in this type of the colony were all very small and capable of generating large, actively growing colonies after 2 weeks. Another type of colony resembled the paraclones or abortive clones (Barrandon and Green, 1987
; Jones and Watt, 1993
; Jones et al., 1995
) and was very small in size. Cells in this colony resembled the differentiating squamous cells and were almost all positive for involucrin (data not shown). The third type of colony showed intermediate features. In our study we therefore assessed the frequency of the abortive clones. We found that in older cell generations (4-14 weeks), differences in colony appearance between the two sorted populations were more apparent. Fig. 4 shows examples of the different colonies observed in Dsg3dim and Dsg3bri sorted cell cultures at an early passage number. Colonies in Dsg3bri cultures were very small and mostly abortive within the first 4 weeks (passage 2). By passage 3 (5-6 weeks), very few or no colonies were found in the Dsg3bri population. In contrast, in the Dsg3dim population, there were still many large colonies observed from passage 2 (Fig. 4A,B), and the total cell output in this population was sixfold that of Dsg3bri cells (30x105 Dsg3dim versus 5x105 Dsg3bri). Moreover, the Dsg3dim cell population was capable of sustaining longer-term generation for a further 9-10 weeks and thus had remarkably longer culture duration than Dsg3bri cells. Double staining the colonies for Dp and involucrin expression showed that a higher proportion of cells and colonies in the Dsg3bri population were involucrin-positive compared with cells of the Dsg3dim population, suggesting that the majority of cells in the Dsg3bri population were terminally differentiating (data not shown). To assess the clone type quantitatively, we selected colonies randomly in each population in passage 2 and scored the abortive colonies under `blind' conditions (Fig. 4B). The total number of colonies assessed in Fig. 4B reflected the frequency and population density of colonies in flasks. Of 96 colonies assessed for the Dsg3dim population, only 59 (61.5%) were abortive colonies. In contrast, 32 out of 33 colonies (97.0%) in Dsg3bri cells were abortive (
2: P<0.05). Furthermore, these data suggested that cells with low Dsg3 expression had a greater overall proliferative potential and were capable of expanding more and for a longer duration in culture.
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To explore whether we were able to achieve similar results in keratinocytes derived from a site other than thick skin, we repeated the same studies on thin skin keratinocytes, in two separate experiments. Again, we found that keratinocytes from thin (mainly breast) skin sorted into low and high Dsg3 populations also showed significantly larger, more actively growing colonies but lower frequency of abortive colonies in the population with a low expression level of Dsg3, suggesting high clonogenecity and CFE in this population (data not shown). These data demonstrated that sorting keratinocytes from thin skin for low desmosome expression also enabled us to enrich for a cell population with some common features of the putative stem/progenitor cells. There is, therefore, no difference between thick and thin skin for these characteristics although they are known to differ in terms of desmosome composition and distribution (Wan et al., 2003).
To verify whether the FACS procedure affects the cell viability in each fraction, we also performed a cell proliferation assay (data not shown). We found that at each time point examined, for up to 5 days, there was no difference between the viability of fractionated cells compared with the control undergoing the FACS.
Combining the two surface markers of ß1 integrin and Dsg3 to sort keratinocytes for a ß1bri/Dsg3dim population enriches considerably the putative keratinocyte stem cell population
The ß1 integrin has been proposed to be a useful cell surface marker for the enrichment of epidermal keratinocytes containing stem cells (Jones and Watt, 1993; Jones et al., 1995
). However, the ß1 integrin-bright population may contain both stem cells and transit amplifying cells (Potten and Morris, 1988
; Jones and Watt, 1993
; Jones et al., 1995
; Li et al., 1998
; Birkenbach and Dunnwald, 2002
), implying that this method of sorting stem cells, although powerful, is not sufficient to identify the true stem cell compartment. We further enriched the colony-forming cell population by combining two surface markers, ß1 integrin and Dsg3, to obtain a ß1bri/Dsg3dim population (from either thick or thin skin). The dissociated keratinocytes (of passage 1) were dual labelled with 5H10 in conjunction with Zenon Alexa Fluor 647 and CD29:FITC. Dead cells were gated out with DAPI. We sorted for two cell populations, i.e. ß1bri/Dsg3dim: the cells with the highest ß1 and least Dsg3 constituting of 11.5% of the population, and ß1bri/Dsg3bri: cells with the highest ß1 and Dsg3 constituting 5.39% of the population (Fig. 5A). The ß1-bright cells in this study included 15
25% of the total sorted cells (Jones and Watt, 1993
). As colony-forming cells represent a more primitive population and showed low forward light scatter (FSC) and side light scatter (SSC) profiles (Jones and Watt, 1993
), we investigated light characteristics of these two cell populations using an FSC vs SSC plot. The majority of cells (85%) in the ß1bri/Dsg3dim population had lower SSC characteristics (Fig. 5Ab) than the ß1bri/Dsg3bri population (40% only) (Fig. 5Ac), possibly suggesting that cells in ß1bri/Dsg3dim population may be less mature. Colony forming assays in short-term culture showed more colonies in the ß1bri/Dsg3dim population than the ß1bri/Dsg3bri population (Fig. 5B,C). There was at least a fourfold increase of colony density and CFE in thick skin keratinocytes, and at least a threefold increase in colony density and CFE in thin skin keratinocytes in the ß1bri/Dsg3dim populations (Fig. 5C). Similar results were consistently observed in three independent experiments and among two to three cell strains (cells from different individuals) in each experiment (n=7). Compared with the results of the colony density and the CFE data with a single Dsg3 marker (Fig. 3C,D), there was a remarkable increase of both colony density and CFE from combining the two markers together. Since kinetic studies suggest stem cells represent 10% of the basal keratinocyte population (Potten and Morris, 1988
; Lavker and Sun, 2000
; Watt, 2001
), whereas sorting for a ß1bri population enriches for 40-50% of the cells (Dunnwald et al., 2001
; Birkenbach and Dunnwald, 2002
), our study clearly has identified a subpopulation in the ß1bri cells (i.e. those that are also Dsg3bri) that did not exhibit stem cell characteristics and showed poor CFE (Fig. 5). Colonies with low Dp staining which were involucrin-negative were seen more frequently in ß1bri/Dsg3dim than ß1bri/Dsg3bri populations (data not shown). Furthermore, we found sustained and significantly increasing differences between these two populations especially in long-term cultures (up to 14 weeks). Cultivation of the ß1bri/Dsg3dim cells showed much better cell survival and longer culture duration than cells in the ß1bri/Dsg3bripopulation (average 14.3 weeks vs 4.2 weeks, Fig. 6). The total cell outputs in the ß1bri/Dsg3dim population were approx. threefold higher at passage 1 (average 10.1x105 ß1bri/Dsg3dim vs 2.8x105 ß1bri/Dsg3bri) with greater differences in growth capacity from passage 2 onwards (14 weeks) since few or none of the cells from ß1bri/Dsg3bri population were able to survive beyond passage 2 (4 weeks). Collectively, these data clearly demonstrate that labelling keratinocytes for ß1 and Dsg3 together and sorting for a ß1bri/Dsg3dim population further enriches the putative epidermal stem cell compartment.
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Discussion |
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The distribution of stem cells, as identified by label-retaining or ß1 integrin-bright cells, is not random but varies in location according to the specific phenotype of epithelial tissue (Jones and Watt, 1993; Jones et al., 1995
). They may occur at the centre of the cluster of basal cells associated with each epidermal proliferative unit (Potten and Morris, 1988
). They have been identified in the bulge of the outer root sheath in the hair follicle (Lenoir et al., 1988
; Cotsarelis et al., 1990
), in the limbus of the cornea (Cotsarelis et al., 1989
), in the flat interpapillary basal layer of oesophageal epithelium (Seery, 2002
), and at the tips of deep rete ridges in the palm (Lavker and Sun, 1983
). These cells exist as clusters in tissue, rather than as sporadic single cells (Jones et al., 1995
; Watt, 2002
). In this study we demonstrate, by immunofluorescence and in situ hybridisation analysis, that there are clusters of cells at the tips of deep rete ridges in human adult palm having low levels of Dp and Dsg expression (Fig. 1A-C), suggesting a overlap of the desmosome-sparse cells and epidermal stem cells in this region. Studies on the existence of the desmosome-sparse cells on other stem cell locations in a variety of epithelial tissues are considered to be necessary.
We demonstrate that cell populations with high levels of ß1 integrin (fractionated by rapid adhesion to type IV collagen (Jones and Watt, 1993), tend to have a low level of Dp and the other major desmosomal proteins (Fig. 2). Studies by others have suggested that the expression differences of cellular proteins between these two populations varied by up to only twofold (Watt, 2001
). In this study we detected considerably greater fold differences in Dp expression although our flow cytometry data still indicated less than twofold differences in ß1 integrin expression between these two populations (data not shown). Whether this greater expression of Dp was overestimated, possibly because of the presence of other cell types in culture requires further studies. Heterogeneous Dp expression in the rapidly adhering cells (ß1-bright) may suggest the presence of some transit amplifying cells in this population, particularly using such a rapid adhesion assay, and also the progressive changes of cell type occurred with cell differentiation and maturity in this population (data not shown) (Jones et al., 1995
; Li et al., 1998
). It is therefore important to further identify and purify the truly stem cells within the ß1-bright cell population.
The ß1 integrins were initially proposed to be a useful cell surface marker for the enrichment of epidermal keratinocytes (Jones and Watt, 1993; Jones et al., 1995
). More recently, questions about the purity of ß1-bright stem cells have arisen, since these cells contain about 40% of the basal keratinocytes (Jones and Watt, 1993
; Jones et al., 1995
), far more than the 10% of basal cells predicted to be stem cells by cell kinetic studies (Potten and Morris, 1988
; Jones et al., 1995
; Li et al., 1998
; Dunnwald et al., 2001
; Birkenbach and Dunnwald, 2002
). In this extended study based on ß1 integrin (Jones and Watt, 1993
; Jones et al., 1995
), we demonstrate that a low level of the second cell surface marker, Dsg3, from the ß1-bright cell population yields pronounced enrichment of cells with a high clonogenecity, CFE and, most importantly, a greater overall proliferative potential (Figs 5 and 6). This last feature is considered to be a very important characteristic for distinguishing the stem cells from transit amplifying cells, since both cell types are mitotic cells at early passages but only the stem cells are able to sustain in long-term generation of keratinocytes (Li et al., 1998
). We find that using such stringent sorting criteria for the ß1bri/Dsg3dim cells enables us to enrich approximately 10% of the basal keratinocyte population. The culture duration of the ß1bri/Dsg3dim cells was
14.3 weeks, in contrast to
4.2 weeks in the ß1bri/Dsg3bri cells (Fig. 6). Our finding clearly indicates a subgroup in the ß1-bright cell population, i.e. the ß1briDsg3bri cells that did not exhibit the stem cell characteristics and were unable to survive in long-term culture. As the ß1bri/Dsg3bri cells only exhibit a short-term survival (
4.2 weeks), it is speculated that they may represent the transit amplifying cells in the ß1bri population. Collectively, these data clearly suggest that combining two markers together offers considerable advantages for the isolation of the putative keratinocyte stem cells, which provides another powerful tool to enrich the putative epidermal stem cells in addition to the combined markers of
6 and 10G7dim (CD71dim) (Li et al., 1998
; Tani et al., 2000
). Surprisingly, both these approaches [our extended study based on ß1 integrin and Li et al. (Li et al., 1998
)] share some features in common. First, both studies use one of the integrin subunits (ß1 or
6) as the positive marker to enrich the stem and some transit amplifying cells with the high cell-matrix adhesive property as well as great growth capacity (Jones et al., 1995
; Li et al., 1998
). Second, both approaches apply the second negative marker, Dsg3 or differentiation-related CD71, to facilitate isolation of the putative stem cells from the transit amplifying cells, which yields approximately 10% of the basal keratinocyte population with properties of not only the high growth capacity but also the pronounced proliferative potential (high CFE and long-term duration of culture). Third, although different human skins and donor age (neonatal foreskin vs adult palm and mainly breast skins) are used in these studies, the duration of the cultures is similar [
12.7 weeks (Li et al., 1998
) vs 14.3 weeks (in this study)]. Further characterisation of the stem cell properties of Dsg3dim and ß1bri/Dsg3dim cell populations, such as label-retaining and full skin regeneration capacities, is needed. Indeed, it would be of interest to further enhance a stem cell population combining two negative markers, connexin 43 (Matic et al., 1997
; Matic et al., 2002
) and Dsg3dim cells.
Unlike most other studies in the literature, where neonatal foreskin was the major source of the primary keratinocytes (Jones and Watt, 1993; Jones et al., 1995
; Li et al., 1998
), the keratinocytes generated in this study, particularly the palm cells, were from the small skin biopsies (
2-3 mm2) of adults up to 50 years of age. Each strain varied according to body site, age and size of specimen. However, regardless of all these factors, we consistently obtained high clonogenecity, CFE and proliferative potential in the Dsg3dim or the ß1bri/Dsg3dim populations. Our findings provide a novel strategy for the improved purification of adult putative epidermal stem/progenitor cells that could pave the way for further studies in stem cell and desmosome biology, and also have implications for the development of new keratinocyte stem cell-mediated clinical treatments.
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Acknowledgments |
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References |
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