CD98, a novel marker of transient amplifying human keratinocytes

Identification of plasma membrane markers of basal keratinocytes is essential for sorting basal cells and, subsequently, adult epidermal stem cells. In this study, we isolated caveolin-1-enriched microdomains from human HaCaT keratinocytes and identified proteins representing potential cell surface markers of the epidermis by a proteomic approach. The purification of this caveolae domain allowed us to characterize 53 proteins of which 26% were transmembrane and 32% associated-membrane proteins. One of them, CD98, was found to be co-localized with beta1 integrin at the plasma membrane of the basal keratinocytes of healthy human epidermis. We then isolated CD98-positive keratinocytes from fresh skin biopsies. Using clonogenic assays, we demonstrate that CD98 may be considered as a marker of transient amplifying human keratinocytes.     

In vivo dedifferentiation of human epidermal cells

Consistent with our previous study, we herein offer further evidence to demonstrate the dedifferentiation of differentiating epidermal cells into stem cells or stem cells -like in vivo. The epidermal sheets eliminated of basal cells were labeled with 6-diamidino-2-phenylindole (DAPI), and then were transplanted onto the full-thickness skin wounds nude mice. Immunohistochemical examination of the survival sheets showed that some cells were positive for both DAPI and either cytokeratins (CK19, CK14) or beta1 integrin in spinous and granular layers at day 7 after transplantation. Furthermore, there was a significant increase in the percentages of both alpha6briCDdim and alpha6briCD71bri populations in survival epidermal sheet grafts 7 d after transplantation compared with those before xenotransplantation (P<0.05), as determined by flow cytometry. The results collectively indicated that some of the differentiated cells in engrafted epidermal sheets dedifferentiated into stem cells or stem cells-like in vivo, which offer us new evidence and insights into the dedifferentiation.     

The spatial relationship between stem cells and their progeny in the basal layer of human epidermis: a new view based on whole-mount labelling and lineage analysis

In order to examine the spatial organisation of stem cells and their progeny in human epidermis, we developed a method for whole-mount epidermal immunofluorescence labelling using high surface beta1 integrin expression as a stem cell marker. We confirmed that there are clusters of high beta1 integrin-expressing cells at the tips of the dermal papillae in epidermis from several body sites, whereas alpha6 integrin expression is more uniform. The majority of actively cycling cells detected by Ki67 or bromodeoxyuridine labelling were found in the beta1 integrin-dull, transit amplifying population and integrin-negative, keratin 10-positive cells left the basal layer exclusively from this compartment. When we examined p53-positive clones in sun-exposed epidermis, we found two types of clone that differed in size and position in a way that was consistent with the founder cell being a stem or transit amplifying cell. The patterning of the basal layer implies that transit amplifying cells migrate over the basement membrane away from the stem cell clusters. In support of this, isolated beta1 integrin-dull keratinocytes were more motile on type IV collagen than beta1 integrin-bright keratinocytes and EGFP-labelled stem cell clones in confluent cultured sheets were compact, whereas transit amplifying clones were dispersed. The combination of whole-mount labelling and lineage marking thus reveals features of epidermal organisation that were previously unrecognised.     

Putative epidermal stem cell convert into corneal epithelium-like cell under corneal tissue <Emphasis Type="Italic">in vitro</Emphasis>

Rhesus putative epidermal stem cells are being investigated for their potential use in regenerative corneal epithelium-like cells, which may provide a practical source of autologous seed cells for the construction of bioengineered corneas. The goal of this study was to investigate the potential of epidermal stem cells for trans-differentiation into corneal epithelium-like cells. Rhesus putative epidermal stem cells were isolated by type IV collagen attachment method. Flow cytometry analysis, immunohistology and RT-PCR were conducted to identify the expression of specific markers (β1m α6 integrin, K15, K1/K10, K3/K12 and CD71) on the isolated rapid attaching cells. The isolated cells were cocultured with human corneal limbal stroma and corneal epithelial cells. After coculture, the expression of the same specific markers was evaluated in order to identify expression difference caused by the coculture conditions. K3/K12 expression was analyzed in coculture cells on day 2, 4, 6, 8 and 10. Putative epidermal stem cells in conditioned culture media were used as control. Putative epidermal stem cells were predominant in rapid attaching cells by type IV collagen attachment isolation. Before being cocultured, the rhesus putative epidermal stem cells expressed K15, α6 and β1 integrin, but no CD71, K1/K10 and K3/K12. After coculture, these cells expressed K3/K12 (a marker of corneal epithelial cells), K15 and β1 integrin, but no K1/K10. Cells being not coculture converted into terminally differentiated cells expressing K1/K10. These results indicate that rhesus putative epidermal stem cells can trans-differentiate into corneal epithelium-like cells and, therefore, may have potential therapeutic application as autologous seed cells for the construction of bioengineered corneas.     

Enrichment of corneal epithelial stem/progenitor cells using cell surface markers, integrin alpha6 and CD71

Corneal epithelial stem cells are believed to reside in the basal layer of the limbal epithelium, but no definitive cell surface markers have been identified. For keratinocytes, stem/progenitor cells are known to be enriched by cell surface markers, integrin α₆ and CD71, as a minor subpopulation which shows high integrin α₆ and low CD71 expressions (α₆^bri/CD71^dim). In the present study, we investigated the possibility that corneal epithelial stem cells can be enriched by integrin α₆ and CD71. The α₆^bri/CD71^dim cells were separated by fluorescence-activated cell sorting, as a minor subpopulation of the limbal epithelial cells. They were enriched for relatively small cells, showing a higher clonogenic capacity and expression of stem cell markers, but a lower expression of differentiation markers, compared to other cell populations. The cells were localized immunohistochemically in the basal region of the limbal epithelium. These results indicate that the α₆^bri/CD71^dim subpopulation enriched corneal epithelial stem cells.     

Putative epidermal stem cell convert into corneal epithelium-like cell under corneal tissue in vitro

Rhesus putative epidermal stem cells are being investigated for their potential use in regenerative corneal epithelium-like cells, which may provide a practical source of autologous seed cells for the construction of bioengineered corneas. The goal of this study was to investigate the potential of epidermal stem cells for trans-differentiation into corneal epithelium-like cells. Rhesus putative epidermal stem cells were isolated by type IV collagen attachment method. Flow cytometry analysis, immunohistology and RT-PCR were conducted to identify the expression of specific markers (β1m α6 integrin, K15, K1/K10, K3/K12 and CD71) on the isolated rapid attaching cells. The isolated cells were cocultured with human corneal limbal stroma and corneal epithelial cells. After coculture, the expression of the same specific markers was evaluated in order to identify expression difference caused by the coculture conditions. K3/K12 expression was analyzed in coculture cells on day 2, 4, 6, 8 and 10. Putative epidermal stem cells in conditioned culture media were used as control. Putative epidermal stem cells were predominant in rapid attaching cells by type IV collagen attachment isolation. Before being cocultured, the rhesus putative epidermal stem cells expressed K15, α6 and β1 integrin, but no CD71, K1/K10 and K3/K12. After coculture, these cells expressed K3/K12 (a marker of corneal epithelial cells), K15 and β1 integrin, but no K1/K10. Cells being not coculture converted into terminally differentiated cells expressing K1/K10. These results indicate that rhesus putative epidermal stem cells can trans-differentiate into corneal epithelium-like cells and, therefore, may have potential therapeutic application as autologous seed cells for the construction of bioengineered corneas. Supported in part by Hi-tech Research and Development Program of China (Grant No. 2003AA205005), the Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP, No.20030558074), the Key Technologies Research and Development Programme of the Tenth Five-Year Plan (Grant No. 2004BA720A15), Scientific and Technological Program (Grant Nos. A3020101 and 2003A3020401) of Guangdong Province     

The role of androgen in determining differentiation and regulation of androgen receptor expression in the human prostatic epithelium transient amplifying population

Abnormal differentiation in epithelial stem cells or their immediate proliferative progeny, the transiently amplifying population (TAP), may explain malignant pathogenesis in the human prostate. These models are of particular importance as differing sensitivities to androgen among epithelial cell subpopulations during differentiation are recognised and may account for progression to androgen independent prostate cancer. Androgens are crucial in driving terminal differentiation and their indirect effects via growth factors from adjacent androgen responsive stroma are becoming better characterised. However, direct effects of androgen on immature cells in the context of a prostate stem cell model have not been investigated in detail and are studied in this work. In alpha2beta1hi stem cell enriched basal cells, androgen analogue R1881 directly promoted differentiation by the induction of differentiation-specific markers CK18, androgen receptor (AR), PSA and PAP. Furthermore, treatment with androgen down-regulated alpha2beta1 integrin expression, which is implicated in the maintenance of the immature basal cell phenotype. The alpha2beta1hi cells were previously demonstrated to lack AR expression and the direct effects of androgen were confirmed by inhibition using the anti-androgen bicalutamide. AR protein expression in alpha2beta1hi cells became detectable when its degradation was repressed by the proteosomal inhibitor MG132. Stratifying the alpha2beta1hi cells into stem (CD133(+)) and transient amplifying population (TAP) (CD133(-)) subpopulations, AR mRNA expression was found to be restricted to the CD133(-) (TAP) cells. The presence of a functional AR in the TAP, an androgen independent subpopulation for survival, may have particular clinical significance in hormone resistant prostate cancer, where both the selection of immature cells and functioning AR regulated pathways are involved.     

PA-FABP, a novel marker of human epidermal transit amplifying cells revealed by 2D protein gel electrophoresis and cDNA array hybridisation

Human epidermal stem cells express higher levels of beta1 integrins than their more differentiated daughters, transit amplifying cells. In a search for additional stem and transit cell markers we used proteomics and differential cDNA hybridisation to compare keratinocytes fractionated on the basis of beta1 integrin expression. There were remarkably few differences between the two populations and none of the RNAs differed in abundance by more than 2-fold. Nevertheless, proteomics revealed upregulated expression of epidermal fatty acid binding protein (PA-FABP, also known as E-FABP), Annexin II and two keratin related proteins in the transit population. An unknown high molecular mass protein was upregulated in the stem cell population. The upregulation of PA-FABP was confirmed by Northern blotting and conventional and whole mount labelling of human epidermis. We conclude that PA-FABP is a novel marker of epidermal transit amplifying cells.     

Role of melanoma chondroitin sulphate proteoglycan in patterning stem cells in human interfollicular epidermis

Human interfollicular epidermis is renewed by stem cells that are clustered in the basal layer in a patterned, non-random distribution. Stem cells can be distinguished from other keratinocytes by high expression of beta1 integrins and lack of expression of terminal differentiation markers; they divide infrequently in vivo but form actively growing colonies in culture. In a search for additional stem cell markers, we observed heterogeneous epidermal expression of melanoma chondroitin sulphate proteoglycan (MCSP). MCSP was expressed by those keratinocytes with the highest beta1 integrin levels. In interfollicular epidermis, expression was confined to non-cycling cells and, in culture, to self-renewing clones. However, fluorescence-activated cell sorting on the basis of MCSP and beta1 integrin expression gave no more enrichment for clonogenic keratinocytes than sorting for beta1 integrins alone. To interfere with endogenous MCSP, we retrovirally infected keratinocytes with a chimera of the CD8 extracellular domain and the MCSP cytoplasmic domain. CD8/MCSP did not affect keratinocyte proliferation or differentiation but the cohesiveness of keratinocytes in isolated clones or reconstituted epidermal sheets was greatly reduced. CD8/MCSP caused stem cell progeny to scatter without differentiating. CD8/MCSP did not alter keratinocyte motility but disturbed cadherin-mediated cell-cell adhesion and the cortical actin cytoskeleton, effects that could be mimicked by inhibiting Rho. We conclude that MCSP is a novel marker for epidermal stem cells that contributes to their patterned distribution by promoting stem cell clustering.