Human Interfollicular Epidermis Research Articles

Deciphering "Immaturity-Stemness" in Human Epidermal Stem Cells at the Levels of Protein-Coding and Non-Coding Genomes: A Prospective Computational Approach.

The epidermis hosts populations of epithelial stem cells endowed with well-documented renewal and regenerative functions. This tissue thus constitutes a model for exploring the molecular characteristics of stem cells, which remain to date partially characterized at the molecular level in human skin. Our group has investigated the regulatory functions of the KLF4/TGFB1 and the MAD4/MAX/MYC signaling pathways in the control of the immaturity-stemness versus differentiation fate of keratinocyte stem and precursor cells from human interfollicular epidermis. We described that down-modulation of either KLF4 or MXD4/MAD4 using RNA interference tools promoted an augmented stemness cellular status; an effect which was associated with significant transcriptional changes, as assessed by RNA-sequencing. Here, we have implemented a computational approach aimed at integrating the level of the coding genome, comprising the transcripts encoding conventional proteins, and the non-coding genome, with a focus on long non-coding RNAs (lncRNAs). In addition, datasets of micro-RNAs (miRNAs) with validated functions were interrogated in view of identifying miRNAs that could make the link between protein-coding and non-coding transcripts. Putative regulons comprising both coding and long non-coding transcripts were built, which are expected to contain original pro-stemness candidate effectors available for functional validation approaches. In summary, interpretation of our basic functional data together with in silico biomodeling gave rise to a prospective picture of the complex constellation of transcripts regulating the keratinocyte stemness status.

Human Basal and Suprabasal Keratinocytes Are Both Able to Generate and Maintain Dermo-Epidermal Skin Substitutes in Long-Term In Vivo Experiments.

The basal layer of human interfollicular epidermis has been described to harbour both quiescent keratinocyte stem cells and a transit amplifying cell population that maintains the suprabasal epidermal layers. We performed immunofluorescence analyses and revealed that the main proliferative keratinocyte pool in vivo resides suprabasally. We isolated from the human epidermis two distinct cell populations, the basal and the suprabasal keratinocytes, according to the expression of integrin β4 (iβ4). We compared basal iβ4+ or suprabasal iβ4− keratinocytes with respect to their proliferation and colony-forming ability and their Raman spectral properties. In addition, we generated dermo–epidermal substitutes using freshly isolated and sorted basal iβ4+ or suprabasal iβ4− keratinocytes and transplanted them on immuno-compromised rats. We show that suprabasal iβ4− keratinocytes acquire a similar proliferative capacity as basal iβ4+ keratinocytes after two weeks of culture in vitro, with expression of high levels of iβ4 and downregulation of K10 expression. In addition, both basal iβ4+ and suprabasal iβ4− keratinocytes acquire authentic self-renewing properties during the in vitro 3D-culture phase and are able to generate and maintain a fully stratified epidermis for 16 weeks in vivo. Therefore, against the leading dogma, we propose that human suprabasal keratinocytes can retro-differentiate into true basal stem cells in a wound situation and/or when in contact with the basement membrane.

Characterization of a melanocyte progenitor population in human interfollicular epidermis.

It is still unknown whether the human interfollicular epidermis harbors a reservoir of melanocyte precursor cells. Here, we clearly distinguish between three distinct types of melanocytes in human interfollicular epidermis: (1) cKit+CD90-, (2) cKit+CD90+, and (3) cKit-CD90+. Importantly, we identify the Kit tyrosine kinase receptor (cKit) as a marker expressed specifically in mature, melanin-producing melanocytes. Thus, both cKit+CD90- and cKit+CD90+ cells represent polydendritic, pigmented mature melanocytes, whereas cKit-CD90+ cells display bipolar, non-dendritic morphology with reduced melanin content. Additionally, using tissue-engineered pigmented dermo-epidermal skin substitutes (melDESSs), we reveal that the cKit expression also plays an important role during melanogenesis in melDESS invivo. Interestingly, cKit-CD90+ cells lack the expression of markers such as HMB45, TYR, and TRP1 invitro and invivo. However, they co-express neural-crest progenitor markers and demonstrate multilineage differentiation potential invitro. Hence, we propose that cKit-CD90+ cells constitute the precursor melanocyte reservoir in human interfollicular epidermis.

Single cell transcriptomics of human epidermis identifies basal stem cell transition states

How stem cells give rise to epidermis is unclear despite the crucial role the epidermis plays in barrier and appendage formation. Here we use single cell-RNA sequencing to interrogate basal stem cell heterogeneity of human interfollicular epidermis and find four spatially distinct stem cell populations at the top and bottom of rete ridges and transitional positions between the basal and suprabasal epidermal layers. Cell-cell communication modeling suggests that basal cell populations serve as crucial signaling hubs to maintain epidermal communication. Combining pseudotime, RNA velocity, and cellular entropy analyses point to a hierarchical differentiation lineage supporting multi-stem cell interfollicular epidermal homeostasis models and suggest that transitional basal stem cells are stable states essential for proper stratification. Finally, alterations in differentially expressed transitional basal stem cell genes result in severe thinning of human skin equivalents, validating their essential role in epidermal homeostasis and reinforcing the critical nature of basal stem cell heterogeneity.

Mitotic Diversity in Homeostatic Human Interfollicular Epidermis.

Despite decades of skin research, regulation of proliferation and homeostasis in human epidermis is still insufficiently understood. To address the role of mitoses in tissue regulation, we utilized human long-term skin equivalents and systematically assessed mitoses during early epidermal development and long-term epidermal regeneration. We now demonstrate four different orientations: (1) horizontal, i.e., parallel to the basement membrane (BM) and suggestive of symmetric divisions; (2) oblique with an angle of 45°–70°; or (3) perpendicular, suggestive of asymmetric division. In addition, we demonstrate a fourth substantial fraction of suprabasal mitoses, many of which are committed to differentiation (Keratin K10-positive). As verified also for normal human skin, this spatial mitotic organization is part of the regulatory program of human epidermal tissue homeostasis. As a potential marker for asymmetric division, we investigated for Numb and found that it was evenly spread in almost all undifferentiated keratinocytes, but indeed asymmetrically distributed in some mitoses and particularly frequent under differentiation-repressing low-calcium conditions. Numb deletion (stable knockdown by CRISPR/Cas9), however, did not affect proliferation, neither in a three-day follow up study by life cell imaging nor during a 14-day culture period, suggesting that Numb is not essential for the general control of keratinocyte division.

Dermal Contributions to Human Interfollicular Epidermal Architecture and Self-Renewal.

The human interfollicular epidermis is renewed throughout life by populations of proliferating basal keratinocytes. Though interfollicular keratinocyte stem cells have been identified, it is not known how self-renewal in this compartment is spatially organized. At the epidermal-dermal junction, keratinocytes sit atop a heterogeneous mix of dermal cells that may regulate keratinocyte self-renewal by influencing local tissue architecture and signalling microenvironments. Focusing on the rete ridges and complementary dermal papillae in human skin, we review the identity and organisation of abundant dermal cells types and present evidence for interactions between the dermal microenvironment and the interfollicular keratinocytes.

Using ABCG2 to identify clonogenic keratinocytes in human interfollicular epidermis for wound healing application

Using ABCG2 to identify clonogenic keratinocytes in human interfollicular epidermis for wound healing application

Breast cancer resistance protein identifies clonogenic keratinocytes in human interfollicular epidermis.

IntroductionThere is a practical need for the identification of robust cell-surface markers that can be used to enrich for living keratinocyte progenitor cells. Breast cancer resistance protein (ABCG2), a member of the ATP binding cassette (ABC) transporter family, is known to be a marker for stem/progenitor cells in many tissues and organs.MethodsWe investigated the expression of ABCG2 protein in normal human epidermis to evaluate its potential as a cell surface marker for identifying and enriching for clonogenic epidermal keratinocytes outside the pilosebaceous tract.ResultsImmunofluorescence and immunoblotting studies of human skin showed that ABCG2 is expressed in a subset of basal layer cells in the epidermis. Flow cytometry analysis showed approximately 2-3% of keratinocytes in non-hair-bearing epidermis expressing ABCG2; this population also expresses p63, β1 and α6 integrins and keratin 14, but not CD34, CD71, C-kit or involucrin. The ABCG2-positive keratinocytes showed significantly higher colony forming efficiency when co-cultured with mouse 3T3 feeder cells, and more extensive long-term proliferation capacity in vitro, than did ABCG2-negative keratinocytes. Upon clonal analysis, most of the freshly isolated ABCG2-positive keratinocytes formed holoclones and were capable of generating a stratified differentiating epidermis in organotypic culture models.ConclusionsThese data indicate that in skin, expression of the ABCG2 transporter is a characteristic of interfollicular keratinocyte progentior cells and suggest that ABCG2 may be useful for enriching keratinocyte stem cells in human interfollicular epidermis.

Mouse models of nonmelanoma skin cancer.

The skin is the largest organ of the mammalian body, made up of multiple layers, which include the epidermis, dermis, and subcutis (Alam and Ratner, N Engl J Med 344(13):975-983, 2001). The human interfollicular epidermis can be subdivided into five different layers: (1) stratum basale, (2) stratum spinosum, (3) stratum granulosum, (4) stratum lucidum, and (5) stratum corneum, all originating from basal keratinocytes by differentiation (Hameetman et al., BMC cancer 13:58, 2013; Ramirez et al., Differentiation 58(1):53-64, 1994). The epidermis is also able to generate different appendages: hair follicles (HF) and their associated sebaceous glands (Sibilia et al., Cell 102(2):211-220, 2000) as well as sweat glands (Luetteke et al., Genes Dev 8(4):399-413, 1994). The skin has important functions in several biological processes like environmental barrier, tissue regeneration, hair cycling, and wound repair. During these processes, stem cells from the interfollicular epidermis and from the hair follicle bulge are activated to renew the epidermis or hair. The epidermis and hair undergo continuous homeostatic regeneration and mutations, upon mutations which disturb the balance of homeostatic regeneration of epidermis and hair and lead to enhanced proliferation of keratinocytes, development of skin cancer is developed. Tumors that arise in the skin are mainly of three types: malignant melanoma, arising from melanocytes, basal cell carcinoma (BCC), and squamous cell carcinoma (SCC), the latter two both arising from keratinocytes or hair follicle cells. In this chapter, we will describe some genetically engineered mouse models (GEMM) that aim at modeling human BCC and SCC and their respective precancerous lesions. We will describe the experimental approaches used in our laboratory to analyze tumor-bearing mice focusing on methods necessary for the induction of tumor growth as well as for the molecular and histological analysis of tumor tissue.

Functional interplay between p63 and p53 controls RUNX1 function in the transition from proliferation to differentiation in human keratinocytes

The interfollicular epidermis is continuously renewed, thanks to a regulated balance between proliferation and differentiation. The ΔNp63 transcription factor has a key role in the control of this process. It has been shown that ΔNp63 directly regulates Runt-related transcription factor 1 (RUNX1) transcription factor expression in mouse keratinocytes. The present study showed for the first time that RUNX1 is expressed in normal human interfollicular epidermis and that its expression is tightly regulated during the transition from proliferation to differentiation. It demonstrated that ΔNp63 directly binds two different RUNX1 regulatory DNA sequences and modulates RUNX1 expression differentially in proliferative or differentiated human keratinocytes. It also showed that the regulation of RUNX1 expression by ΔNp63 is dependent on p53 and that this coregulation relies on differential binding and activation of RUNX1 regulatory sequences by ΔNp63 and p53. We also found that RUNX1 inhibits keratinocyte proliferation and activates directly the expression of KRT1, a critical actor in early keratinocyte differentiation. Finally, we described that RUNX1 expression, similar to ΔNp63 and p53, was strongly expressed and downregulated in basal cell carcinomas and squamous cell carcinomas respectively. Taken together, these data shed light on the importance of tight control of the functional interplay between ΔNp63 and p53 in regulating RUNX1 transcription factor expression for proper regulation of interfollicular epidermal homeostasis.

Cellular adhesion on collagen: a simple method to select human basal keratinocytes which preserves their high growth capacity

The regenerative capacity of human interfollicular epidermis is closely linked to the potential of immature keratinocytes present within its basal layer. The availability of selection methods and culture systems allowing precise assessment of basal keratinocyte characteristics is critical for increasing our knowledge of this cellular compartment. This report presents a multi-parametric comparative study of basal keratinocytes selected according to two different principles: 1) high adhesion capacity on a type-I collagen-coated substrate [Adh⁺⁺⁺], 2) high cell-surface expression of α6-integrin [Itg-α6 (high)]. Importantly, analysis performed at the single-cell level revealed similar primary clone-forming efficiency values of 45.5% ± 6.7% [Itg-α6(high)] and 43.7% ± 7.4% [Adh⁺⁺⁺], which were markedly higher than those previously reported. In addition, both methods selected keratinocytes exhibiting an extensive long-term growth potential exceeding 100 cell doublings and the capacity for generating a pluristratified epidermis. Our study also included a global transcriptome comparison. Genome-wide profiling indicated a strong similarity between [Adh⁺⁺⁺] and [Itg-α6(high)] keratinocytes, and revealed a common basal-associated transcriptional signature. In summary, cross-analysis of [Adh⁺⁺⁺] and [Itg-α6(high)] keratinocyte characteristics showed that these criteria identified highly equivalent cellular populations, both characterized by unexpectedly high growth capacities. These results may have broad impacts in the tissue engineering and cell therapy fields.

Exploration of the functional hierarchy of the basal layer of human epidermis at the single‐cell level using parallel clonal microcultures of keratinocytes

The basal layer of human epidermis contains both stem cells and keratinocyte progenitors. Because of this cellular heterogeneity, the development of methods suitable for investigations at a clonal level is dramatically needed. Here, we describe a new method that allows multi-parallel clonal cultures of basal keratinocytes. Immediately after extraction from tissue samples, cells are sorted by flow cytometry based on their high integrin-alpha 6 expression and plated individually in microculture wells. This automated cell deposition process enables large-scale characterization of primary clonogenic capacities. The resulting clonal growth profile provided a precise assessment of basal keratinocyte hierarchy, as the size distribution of 14-day-old clones ranged from abortive to highly proliferative clones containing 1.7 x 10(5) keratinocytes (17.4 cell doublings). Importantly, these 14-day-old primary clones could be used to generate three-dimensional reconstructed epidermis with the progeny of a single cell. In long-term cultures, a fraction of highly proliferative clones could sustain extensive expansion of >100 population doublings over 14 weeks and exhibited long-term epidermis reconstruction potency, thus fulfilling candidate stem cell functional criteria. In summary, parallel clonal microcultures provide a relevant model for single-cell studies on interfollicular keratinocytes, which could be also used in other epithelial models, including hair follicle and cornea. The data obtained using this system support the hierarchical model of basal keratinocyte organization in human interfollicular epidermis.

Visualization of Morphological and Molecular Features Associated with Chronic Ischemia in Bioengineered Human Skin

We present an in vitro model of human skin that, together with nonlinear optical microscopy, provides a useful system for characterizing morphological and structural changes in a living skin tissue microenvironment due to changes in oxygen status and proteolytic balance. We describe for the first time the effects of chronic oxygen deprivation on a bioengineered model of human interfollicular epidermis. Histological analysis and multiphoton imaging revealed a progressively degenerating ballooning phenotype of the keratinocytes that manifested after 48 h of hypoxic exposure. Multiphoton images of the dermal compartment revealed a decrease in collagen structural order. Immunofluorescence analysis showed changes in matrix metalloproteinase (MMP)-2 protein spatial localization in the epidermis with a shift to the basal layer, and loss of Ki67 expression in proliferative basal cells after 192 h of hypoxic exposure. Upon reoxygenation MMP-2 mRNA levels showed a biphasic response, with restoration of MMP-2 levels and localization. These results indicate that chronic oxygen deprivation causes an overall degeneration in tissue architecture, combined with an imbalance in proteolytic expression and a decrease in proliferative capacity. We propose that these tissue changes are representative of the ischemic condition and that our experimental model system is appropriate for addressing mechanisms of susceptibility to chronic wounds.

A Stable Niche Supports Long-Term Maintenance of Human Epidermal Stem Cells in Organotypic Cultures

Stem cells in human interfollicular epidermis are still difficult to identify, mainly because of a lack of definitive markers and the inability to label human beings for label-retaining cells (LRCs). Here, we report that LRCs could be identified and localized in organotypic cultures (OTCs) made with human cells. Labeling cultures for 2 weeks with iododeoxyuridine (IdU) and then chasing for 6-10 weeks left <1% of basal cells retaining IdU label. Whole mounts demonstrated that LRCs were individually dispersed in the epidermal basal layer. Some LRCs, but not all, colocalized with cells expressing melanoma chondroitin sulfate proteoglycan, a putative stem cell marker. Although we found LRCs in both collagen- and scaffold-based OTCs, only the scaffold-OTCs supported long-term survival and regeneration. LRCs ' short survival in collagen-OTCs was not due to loss of appropriate growth factors from fibroblasts. Instead, it was due to expression of metalloproteinases, especially matrix metalloproteinase (MMP)-2 and MMP-14, which caused collagen fragmentation, matrix degradation, and dislocation of specific basement membrane components bound to epidermal integrins. Blocking MMP activation not only abrogated MMP-dependent matrix degradation but also increased longevity of the epidermis and the LRCs in these cultures. Such findings indicate that the stem cell niche, the microenvironment surrounding and influencing the stem cell, is essential for stem cell survival and function, including long-term tissue regeneration. Disclosure of potential conflicts of interest is found at the end of this article.

Role of the Notch Ligand Delta1 in Embryonic and Adult Mouse Epidermis

The Notch ligand Delta1 (Dll1) is expressed in human interfollicular epidermis (IFE) and regulates differentiation and adhesion of cultured human keratinocytes. However, the consequences of deleting Dll1 in mouse epidermis have not been examined. Here, we report that in embryonic mouse skin Dll1 is expressed by patches of keratinocytes in the basal layer of the IFE and in the dermal papilla and hair bulb. In a Dll1 hypomorph mutant that survives until birth, hair follicles formed normally but proliferation and thickness of the IFE were increased. Deletion of Dll1 using Cre recombinase expressed under the control of the keratin-5 (K5) promoter resulted in a delay in the first postnatal anagen, but subsequent hair cycles were normal. As in the hypomorph, IFE proliferation was stimulated and expression of K10 and K17 was disturbed. Older mice developed tumors with elements of IFE differentiation. Keratinocytes cultured from K5Cre x Dll1(flox/flox) epidermis showed a transient increase in proliferation, with a subsequent decrease in integrin expression and increased terminal differentiation. These results demonstrate that Dll1 contributes to the control of proliferation and differentiation in IFE, whereas Jagged1 regulates hair follicle differentiation.

FGF2 signalling is critical for DNA repair in human epidermal stem cells

The sensitivity of stem cells to DNA damaging agents is a matter of debate. For mouse somatic stem cells, both radiosensitive and radioresistant stem cells have been described. By contrast, the response of human stem cells to ionizing radiation has been poorly studied. We evaluated in the present work the radiosensitivity of the epithelial stem cells from human interfollicular epidermis. We used flow cytometry and antibodies against cell surface markers (a6 integrin and CD71) to isolate keratinocyte stem cells and progenitors from human foreskin. Using a short-term cell survival assay (XTT at 72 h) and a long-term cell survival assay (CFE at 2 weeks), we demonstrated that keratinocyte progenitors were radiosensitive whereas the stem cells were more radioresistant (1). Using microarrays, we found that radiation exposure induced specifically several cytokines and growth factors genes in the stem cells, and notably the FGF2 pathway. Furthermore, DNA repair genes were found overexpressed in the stem cells. We thus postulated that the stem cells might have a more efficient DNA repair than the progenitors and might acquire an activated stress signalling response compared to the progenitors. We first studied the DNA repair of double strand breaks with the ?H2AX foci assay to evaluate the repair of DNA double strand breaks. After irradiation, the number of foci per cell decreased much more rapidly in the stem cells, suggesting a faster repair of DNA breaks. Moreover, using new bioarrays that measure DNA repair enzyme activities, we found that several repair enzymes were more active in the stem cells. Thereafter, to address the relationship between FGF2 and DNA repair, we inhibited this pathway at the level of the FGF2 receptor. Blocking FGF2 signalling inhibited the rapid decrease of ?H2AX foci in stem cells, suggesting a direct role of FGF2 in DNA repair. These results show that keratinocyte stem cells are a radioresistant cell population with a high DNA repair capacity. Moreover, we demonstrate that the FGF2 pathway plays a direct and specific role in DNA repair in epidermis stem cells. As stem cells are a long-term reservoir, these processes may be important for epidermis renewal and for carcinoma formation. (1) Rachidi W, Radiother Oncol, 2007, 83, 267

Cellules souches de l'épiderme interfolliculaire humain : phénotypes et potentialités

Stem cells from different tissue origins share common characteristics, including selfrenewal capacity and tissue regeneration potential. Finding criteria to identify particular stem cell types, and understanding signaling pathways responsible for stemness, represent major research areas that will lead to a better characterization of the normal state of stem cells, thus improving our capability to use them for regenerative therapies. We will review here different approaches and experimental models liable to increase our knowledge of stem cells from human interfollicular epidermis. One of them, based on transcriptional profiling performed at the level of the global genome, consisted in searching universal molecular markers of stem cells. In other approaches, stem cells were studied at the level of specific characteristics. Understanding somatic stem cell properties such as quiescence or slow cycling state, and detoxification potential, led to the identification of phenotypes suitable for the selection of epidermal keratinocyte sub-populations with stem cell properties. The specific interests of these different research strategies will be discussed.

Syntenin mediates Delta1-induced cohesiveness of epidermal stem cells in culture

In human interfollicular epidermis, stem cell clusters express high levels of the Notch ligand Delta1. Delta1 stimulates neighbouring cells to differentiate and also promotes stem cell clustering. Although Notch signalling is known to stimulate epidermal differentiation, little is known about the mechanism by which Delta1 promotes epidermal cell cohesiveness. This is an important issue, because the location of stem cells determines the local microenvironmental signals they receive. We now show that mutation of the Delta1 PDZ-binding domain abolishes Delta1-mediated keratinocyte cohesiveness, stimulates Notch transcriptional activity and promotes epidermal differentiation. A yeast two-hybrid screen revealed that Delta1 binds to the adaptor protein syntenin - an interaction dependent on the Delta1 PDZ-binding domain. Syntenin, like Delta1, is upregulated in the stem cell clusters of human interfollicular epidermis. Knockdown of syntenin in cells overexpressing full-length Delta1 had the same effects on Notch signalling, epidermal differentiation and adhesion as overexpressing Delta1 with a mutated PDZ-binding domain. Syntenin has previously been reported to regulate membrane traffic, and mutation of the Delta1 PDZ-binding domain or knockdown of syntenin led to rapid internalisation of Delta1. We propose that syntenin binding to Delta1 plays a dual role in promoting intercellular adhesion and regulating Notch signalling.

Single-cell expression profiling of human epidermal stem and transit-amplifying cells: Lrig1 is a regulator of stem cell quiescence

Considerable progress has been made in characterizing epidermal stem cells by microarray analysis of FACS-selected populations. One limitation of this approach is that the gene expression profiles represent the average of the cell population, potentially masking cellular heterogeneity of functional significance. To overcome this problem, we have performed single-cell expression profiling. We have generated cDNA libraries from single human epidermal cells, designated as stem or transit-amplifying cells on the basis of Delta1 and melanoma-associated chondroitin sulfate proteoglycan expression. Of the 14 putative stem cell markers identified, we selected one, the EGF receptor antagonist leucine-rich repeats and immunoglobulin-like domains 1 (Lrig1), for further study. Lrig1 was expressed in groups of basal cells in human interfollicular epidermis previously identified as enriched for stem cells. Overexpression of Lrig1 decreased keratinocyte proliferation but did not affect the proportion of stem and transit-amplifying cells, as judged by clonal growth characteristics. Down-regulation of Lrig1 using siRNA increased cell-surface EGF receptor levels, enhanced activation of downstream pathways, and stimulated proliferation. Lrig1 acted in part by negatively regulating the Myc promoter. We propose that Lrig1 maintains epidermal stem cells in a quiescent nondividing state, and that Lrig1 down-regulation triggers proliferation.

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.