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Intrinsic Patterns of Behavior of Epithelial Stem Cells

2004; Elsevier BV; Volume: 9; Issue: 3 Linguagem: Inglês

10.1111/j.1087-0024.2004.09310.x

1529-1774

Debbie Tudor, Matthew Locke, Eleri Owen-Jones, Ian C. Mackenzie,

Mesenchymal stem cell research

The early concepts concerning hematopoietic and epithelial stem cells that were derived from kinetic studies have been greatly enhanced by new information about a range of other properties of somatic and embryonic stem cells. Firstly, the stem and amplifying pattern characteristically established by epithelial lineages has been found to represent an intrinsic pattern that is generated by somatic epithelial stem cells without the need for additional environmental information. Secondly, it is now apparent that somatic epithelial stem cells are plastic and can be directed into a range of new pathways of differentiation by heterotypic interactions. The mechanisms of this plasticity need to be reconciled with the normally stable commitment of these cells to production only of progeny entering a tightly restricted range of phenotypic pathways. The present review discusses the intrinsic properties of epithelial stem cells and how they may be acted upon by connective tissues to generate a wide range of phenotypically different epithelial structures. The early concepts concerning hematopoietic and epithelial stem cells that were derived from kinetic studies have been greatly enhanced by new information about a range of other properties of somatic and embryonic stem cells. Firstly, the stem and amplifying pattern characteristically established by epithelial lineages has been found to represent an intrinsic pattern that is generated by somatic epithelial stem cells without the need for additional environmental information. Secondly, it is now apparent that somatic epithelial stem cells are plastic and can be directed into a range of new pathways of differentiation by heterotypic interactions. The mechanisms of this plasticity need to be reconciled with the normally stable commitment of these cells to production only of progeny entering a tightly restricted range of phenotypic pathways. The present review discusses the intrinsic properties of epithelial stem cells and how they may be acted upon by connective tissues to generate a wide range of phenotypically different epithelial structures. In renewing tissues such as stratifying squamous epithelia, the mechanisms controlling cell proliferation, differentiation and death are of central importance to the maintenance of normal structure, to physiological repair, and to a range of neoplastic and other pathological changes. The early view that basal cells are equipotential, and either proliferate or differentiate as chance events, is incompatible with the patterns of mitosis since found in epithelia and it is now clear that basal cells are heterogeneous in regard to their proliferative capacities (Leblond et al., 1964Leblond C.P. Greulich R.C. Marques-Pereira J.P. Relationship of cell formation and cell migration in the renewal of stratified squamous epithelia.Adv Biol Skin. 1964; 5: 39-67Google Scholar). The concept of epithelial stem cells was first proposed to explain the small relatively simple proliferative units found in rodent epidermis (Mackenzie, 1969Mackenzie I.C. The ordered structure of the stratum corneum of mammalian skin.Nature. 1969; 222: 881-882Crossref PubMed Scopus (128) Google Scholar,Mackenzie, 1970Mackenzie I.C. Relationship between mitosis and the ordered structure of the stratum corneum in mouse epidermis.Nature. 1970; 226: 653-655Crossref PubMed Scopus (149) Google Scholar;Potten, 1974Potten C.S. The epidermal proliferative unit: The possible role of the central basal cell.Cell Tissue Kinet. 1974; 7: 77-78PubMed Google Scholar) and it has since become apparent that similar proliferative hierarchies of stem, amplifying and post-mitotic differentiating cells are associated with the renewal of most, perhaps all, epithelia (Lavker and Sun, 1982Lavker R.M. Sun T.T. Heterogeneity in epidermal basal keratinocytes: Morphological and functional correlations.Science. 1982; 215: 1239-1241Crossref PubMed Scopus (300) Google Scholar;Potten, 1983Potten C.S. Stem Cells: Their Identification and Characterization. Cambridge, Churchill Livingstone1983Google Scholar;Hall and Watt, 1989Hall P.A. Watt F.M. Stem cells: The generation and maintenance of cellular diversity.Development. 1989; 106: 619-633PubMed Google Scholar;Cotsarelis et al., 1999Cotsarelis G. Kaur P. Dhouailly D. Hengge U. Bickenbach J. Epithelial stem cells in the skin: Definition, markers, localization and functions.Exp Dermatol. 1999; 8: 80-88Crossref PubMed Scopus (162) Google Scholar). Basic defining features initially proposed for stem cells included a high capacity for self-renewal and an ability to produce cells that differentiate to maintain tissue function (Lajtha, 1979Lajtha G. Stem cell concepts.Differentiation. 1979; 14: 23-34Crossref PubMed Scopus (398) Google Scholar). The validity of these concepts for epithelial stem cells has now been supported by a wide range of studies, but these studies have also shown that the behavior and distribution of stem cells can differ quite markedly between various types of epithelia (Cotsarelis et al., 1999Cotsarelis G. Kaur P. Dhouailly D. Hengge U. Bickenbach J. Epithelial stem cells in the skin: Definition, markers, localization and functions.Exp Dermatol. 1999; 8: 80-88Crossref PubMed Scopus (162) Google Scholar;Lavker and Sun, 2000Lavker R.M. Sun T.T. Epidermal stem cells: Properties, markers, and location.Proc Natl Acad Sci USA. 2000; 97: 13473-13475Crossref PubMed Scopus (352) Google Scholar;Alonso and Fuchs, 2003Alonso L. Fuchs E. Stem cells of the skin epithelium.Proc Natl Acad Sci USA. 2003; 100: 11830-11835Crossref PubMed Scopus (394) Google Scholar;Fuchs and Watt, 2003Fuchs E. Watt F.M. Cell differentiation. Focus on epithelia.Curr Opin Cell Biol. 2003: 738-739Google Scholar). Stem cells in murine epithelia were anticipated to be slowly cycling (Potten, 1974Potten C.S. The epidermal proliferative unit: The possible role of the central basal cell.Cell Tissue Kinet. 1974; 7: 77-78PubMed Google Scholar) and this property enabled their initial localization in tissues by “label retention”, a method that involves labeling epithelia in vivo with tritiated thymidine and sampling tissues a month or more later when division had diluted label from cycling cells (Bickenbach, 1981Bickenbach J.R. Identification and behavior of label-retaining cells in oral mucosa and skin.J Dent Res 60 (Spec No C). 1981: 1611-1620Crossref Google Scholar). “Label-retaining cells” (LRC) in murine epithelia have a range of features supporting their identity as stem cells (Bickenbach, 1981Bickenbach J.R. Identification and behavior of label-retaining cells in oral mucosa and skin.J Dent Res 60 (Spec No C). 1981: 1611-1620Crossref Google Scholar;Mackenzie and Bickenbach, 1985Mackenzie I.C. Bickenbach J.R. Label-retaining keratinocytes and Langerhans cells in mouse epithelia.Cell Tissue Res. 1985; 242: 551-556Crossref PubMed Scopus (79) Google Scholar). For example, they are found beneath the central regions of columnar units in epidermis, at the deep tips of epithelial rete, and at the base of the anterior and posterior columns of tongue papillae, all sites predicted to be stem cell locations by other kinetic studies (Hume, 1983Hume W.J. Stem cells in oral epithelia.in: Potten C.S. Stem Cells: Their Identification and Characterization. New York, Churchill Livingstone1983: 233-270Google Scholar;Potten, 1983Potten C.S. Stem Cells: Their Identification and Characterization. Cambridge, Churchill Livingstone1983Google Scholar). Further evidence comes from demonstration that LRC are more clonogenic than other cells (Morris and Potten, 1994Morris R.J. Potten C.S. Slowly cycling (label-retaining) epidermal cells behave like clonogenic stem cells in vitro.Cell Prolif. 1994; 27: 279-289Crossref PubMed Scopus (193) Google Scholar) and from lineage studies suggesting that the units of structure in murine epidermis are clonal, each being renewed by a single stem cell (Mackenzie, 1995;Ghazizadeh and Taichman, 2001Ghazizadeh S. Taichman L.B. Multiple classes of stem cells in cutaneous epithelium: a lineage analysis of adult mouse skin.EMBO J. 2001; 20: 1215-1222Crossref PubMed Scopus (300) Google Scholar). For more complex epithelial structures the patterns of stem cell distribution are not yet fully clear but LRC have been localized to the “bulge” region of hair follicles and to the limbal region of the cornea, regions shown to contain cells with high clonogenic potential (Cotsarelis et al., 1989Cotsarelis G. Cheng S.Z. Dong G. Sun T.T. Lavker R.M. Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: Implications on epithelial stem cells.Cell. 1989; 57: 201-209Abstract Full Text PDF PubMed Scopus (1168) Google Scholar,Cotsarelis et al., 1990Cotsarelis G. Sun T.T. Lavker R.M. Label-retaining cells reside in the bulge area of pilosebaceous unit: Implications for follicular stem cells, hair cycle, and skin carcinogenesis.Cell. 1990; 61: 1329-1337Abstract Full Text PDF PubMed Scopus (1898) Google Scholar;Rochat et al., 1994Rochat A. Kobayashi K. Barrandon Y. Location of stem cells of human hair follicles by clonal analysis.Cell. 1994; 76: 1063-1073Abstract Full Text PDF PubMed Scopus (462) Google Scholar). In human epidermis, which is thicker than rodent epidermis, patterns of staining for β1 integrins, combined with staining for markers of proliferation and differentiation, indicate that stem cells lie among the basal cells in zones overlying the tips of the connective tissue papillae (Jensen et al., 1999Jensen U.B. Lowell S. Watt F.M. 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.Development. 1999; 126: 2409-2418PubMed Google Scholar) but the number of stem cells in these locations is uncertain. The behavior of epithelial stem cells is of significance to a broad range of clinical problems. For example, interest in stem cell properties has arisen from their predicted roles in the initiation and progression of malignancy (Morris, 2000Morris R.J. Keratinocyte stem cells: Targets for cutaneous carcinogens.J Clin Invest. 2000; 106: 3-8Crossref PubMed Scopus (87) Google Scholar) and particularly how the persistence of stem and amplifying patterns in malignancy would influence the behavior of malignant lesions and their responses to therapy (Denekamp, 1994; Kummermehr, 2001). Further interest in somatic stem cells has been stimulated by the need for stem cell transduction in gene therapy and by the requirements for stem cell manipulation during tissue engineering procedures (Bickenbach and Roop, 1999Bickenbach J.R. Roop D.R. Transduction of a preselected population of human epidermal stem cells: Consequences for gene therapy.Proc Assoc Am Phys. 1999; 111: 184-189Crossref PubMed Scopus (21) Google Scholar;Bickenbach and Dunnwald, 2000Bickenbach J.R. Dunnwald M. Epidermal stem cells: Characteristics and use in tissue engineering and gene therapy.Adv Dermatol. 2000; 16: 159-183PubMed Google Scholar). Surprisingly, it has now been shown that a range adult somatic stem cells, including those for keratinocytes, can be induced by appropriate developmental signals to participate in the formation of a much wider range of tissues than had previously been believed (Liang and Bickenbach, 2002Liang L. Bickenbach J.R. Somatic epidermal stem cells can produce multiple cell lineages during development.Stem Cells. 2002; 20: 21-31Crossref PubMed Scopus (138) Google Scholar;Prockop, 2002Prockop D.J. Adult stem cells gradually come of age.Nat Biotechnol. 2002; 20: 791-792Crossref PubMed Scopus (19) Google Scholar). One of the aims of tissue engineering is to amplify stem cells and direct their progeny into patterns of differentiation and morphogenesis appropriate to the regeneration of new tissues. We have been particularly interested in two questions of relevance to these processes: (a) the extent to which somatic stem cell behavior is intrinsically determined or is dependent on interactions with the environment and (b) the degree to which the particular patterns of differentiation acquired by stem cell progeny are determined by a pre-existing commitment of the somatic stem cells producing them. In the present review, we discuss experimental work related to these topics and we conclude from current evidence that; (a) that the basic stem and amplifying pattern associated with epithelial renewal is an intrinsic epithelial property; (b) somatic stem cells are normally committed to generate progeny that enter restricted pathways of differentiation; (c) mesenchymal signals can direct stem cell progeny into new pathways of differentiation; and (d) mesenchymal modulation of the basic stem and amplification pattern is required to produce and maintain most epithelial structures. As it is not possible to undertake in situ labeling studies of human epidermis, information about human epithelial stem cells has been derived mostly from in vitro studies, primarily those assaying the “stemness” of cells as measured by clonogenicity. The widely differing proliferative potentials of keratinocytes isolated from normal human epithelia can be demonstrated by plating them for growth at clonal densities (Barrandon and Green, 1985Barrandon Y. Green H. Cell size as a determinant of the clone-forming ability of human keratinocytes.Proc Natl Acad Sci USA. 1985; 82: 5390-5394Crossref PubMed Scopus (286) Google Scholar,Barrandon and Green, 1987Barrandon Y. Green H. Three clonal types of keratinocyte with different capacities for multiplication.Proc Natl Acad Sci USA. 1987; 84: 2302-2306Crossref PubMed Scopus (1093) Google Scholar). Some cells give rise to round colonies composed of small compact cells that can be repeatedly passaged; others form irregular colonies capable of less extensive growth; yet others form colonies of large flattened cells that do not passage well. These colony forms, referred to as holoclones, meroclones and paraclones, are thought to be derived from, and consist of, stem cells and early and late amplifying cells, respectively (Barrandon and Green, 1987Barrandon Y. Green H. Three clonal types of keratinocyte with different capacities for multiplication.Proc Natl Acad Sci USA. 1987; 84: 2302-2306Crossref PubMed Scopus (1093) Google Scholar). The ability, however, of normal human keratinocytes to grow in vitro is limited and after extensive passage all colonies begin to assume the appearance of paraclones and cease division, a change that appears to be associated with depletion of stem cells. For example, when human keratinocytes marked by retroviral transduction are mixed with unmarked keratinocytes, the patterns of clonal extinction observed at late passages suggest that very few cells are functioning as stem cells and that the cultures consist essentially of amplifying cells (Mathor et al., 1996Mathor M.B. Ferrari G. Dellambra E. Cilli M. Mavilio F. Cancedda R. De Luca M. Clonal analysis of stably transduced human epidermal stem cells in culture.Proc Natl Acad Sci USA. 1996; 93: 10371-10376Crossref PubMed Scopus (153) Google Scholar). It is therefore interesting that, after their in vitro expansion, transplantion of epidermal and mucosal keratinocytes to in vivo sites results in reformation and maintenance of a functional epithelium for many years (De Luca et al., 1990De Luca M. Albanese E. Megna M. Cancedda R. Mangiante P.E. Cadoni A. Franzi A.T. Evidence that human oral epithelium reconstituted in vitro and transplanted onto patients with defects in the oral mucosa retains properties of the original donor site.Transplantation. 1990; 50: 454-459Crossref PubMed Scopus (119) Google Scholar;Compton et al., 1998Compton C.C. Nadire K.B. Regauer S. et al.Cultured human sole-derived keratinocyte grafts re-express site-specific differentiation after transplantation.Differentiation. 1998; 64: 45-53Crossref PubMed Google Scholar). Similar conclusions can be drawn from studies dealing with the characterization, culture, and transplantation of limbal stem cells for corneal regeneration following chemical burns and other injuries (Rama et al., 2001Rama P. Bonini S. Lambiase A. Golisano O. Paterna P. DeLuca M. Pellegrini G. Autologous fibrin-cultured limbal stem cells permanently restore the corneal surface of patients with total limbal stem cell deficiency.Transplantation. 2001; 72: 1478-1485Crossref PubMed Scopus (431) Google Scholar;Espana et al., 2003Espana E.M. Kawakita T. Romano A. Di Pascuale M. Smiddy R. Liu C.Y. Tseng S.C. Stromal niche controls the plasticity of limbal and corneal epithelial differentiation in a rabbit model of recombined tissue.Invest Ophthalmol Vis Sci. 2003; 44: 5130-5135Crossref PubMed Scopus (97) Google Scholar). Cell cultures thus seem to contain cells that are capable of maintaining or regaining the stem cell function of indefinite renewal when returned to the in vivo environment. An understanding of the way that the in vivo and in vitro environments differ in their ability to maintain stem cells appears central to further development of many of the tissue-engineering procedures now being proposed. The present inability to identify and examine directly the fate of human epithelial stem cells in vitro hinders investigation of these mechanisms. One way that the in vitro and in vivo environments differ is that epithelia normally exist in association with mesenchyme and both the initial development of epithelial structure and its maintenance throughout adult life depend on interactions between epithelium and mesenchyme (Cunha et al., 1985Cunha G.R. Bigsby R.M. Cooke P.S. Sugimura Y. Stromal–epithelial interactions in adult organs.Cell Differ. 1985; 17: 137-148Crossref PubMed Scopus (251) Google Scholar;Mackenzie, 1994Mackenzie I.C. Epithelial-mesenchymal interactions in the development and maintenance of epithelial tissues.in: Lane E.B. Leigh I. Watt F.M. The Keratinocyte Handbook. Cambridge, Cambridge University Press1994Google Scholar). Work with compound cultures has enabled analysis of interactions occurring between adult cells and a series of interesting experiments (Maas-Szabowski et al., 1999Maas-Szabowski N. Shimotoyodome A. Fusenig N.E. Keratinocyte growth regulation in fibroblast cocultures via a double paracrine mechanism.J Cell Sci. 1999; 112: 1843-1853PubMed Google Scholar;Maas-Szabowski et al., 2001Maas-Szabowski N. Szabowski A. Stark H.J. et al.Organotypic cocultures with genetically modified mouse fibroblasts as a tool to dissect molecular mechanisms regulating keratinocyte growth and differentiation.J Invest Dermatol. 2001; 116: 816-820Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) has shown that reciprocal signals are generated by paracrine acting cytokines; e.g., keratinocyte growth factor is released by fibroblasts in response to interleukin 1 production by the epithelium. These interactions are necessary to maintain good patterns of growth and differentiation of epithelia. The general pattern is that regionally different keratinocytes grown on plastic retain some phenotypic differences, grown as organotypic cultures they express good regional differentiation patterns and, typically, when returned to an in vivo environment, fully regain their original phenotype. Such phenotypic stability is the basis of current tissue-engineering procedures where, after in vitro generation and transplantation of human epidermis, the particular phenotype of the epidermal region of origin remains expressed for years (Compton et al., 1998Compton C.C. Nadire K.B. Regauer S. et al.Cultured human sole-derived keratinocyte grafts re-express site-specific differentiation after transplantation.Differentiation. 1998; 64: 45-53Crossref PubMed Google Scholar). The commitment of somatic epithelial stem cells to regionally various differentiation pathways thus seems to be quite rigid but it has been experimentally demonstrated that epithelia can be directed into new phenotypic patterns under the influence of regionally differing embryonic, and even adult, mesenchymes (Mackenzie, 1994Mackenzie I.C. Epithelial-mesenchymal interactions in the development and maintenance of epithelial tissues.in: Lane E.B. Leigh I. Watt F.M. The Keratinocyte Handbook. Cambridge, Cambridge University Press1994Google Scholar). In response to embryonic developmental signals, somatic stem cells from skin can give rise to an even wide range of new lineages (Liang and Bickenbach, 2002Liang L. Bickenbach J.R. Somatic epidermal stem cells can produce multiple cell lineages during development.Stem Cells. 2002; 20: 21-31Crossref PubMed Scopus (138) Google Scholar). Information about the nature of these signals, and about how the progeny of somatic stem cells can be directed into particular differentiation pathways, is becoming of major interest to tissue engineering (Prockop et al., 2003Prockop D.J. Gregory C.A. Spees J.L. One strategy for cell and gene therapy: harnessing the power of adult stem cells to repair tissues.Proc Natl Acad Sci USA. 2003; 100: 11917-11923Crossref PubMed Scopus (371) Google Scholar). Clonal growth of murine epidermal keratinocytes in vitro initially required complex growth media together with feeder cells (Morris et al., 1987Morris R.J. Tacker K.C. Baldwin J.K. Fischer S.M. Slaga T.J. A new medium for primary cultures of adult murine epidermal cells: Application to experimental carcinogenesis.Cancer Lett. 1987; 34: 297-304Abstract Full Text PDF PubMed Scopus (32) Google Scholar) but simpler media that permit expansive clonal growth in the absence of feeder cells have now been reported (Hager et al., 1999Hager B. Bickenbach J.R. Fleckman P. Long-term culture of murine epidermal keratinocytes.J Invest Dermatol. 1999; 112: 971-976Crossref PubMed Scopus (107) Google Scholar;Caldelari et al., 2000Caldelari R. Suter M.M. Baumann D. De Bruin A. Muller E. Long-term culture of murine epidermal keratinocytes.J Invest Dermatol. 2000; 114: 1064-1065Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Using a modification of the methods of Caldelari and co-workers, we have been able to grow and extensively passage keratinocytes isolated from a range of murine skin and mucosal tissues. We have found that murine keratinocytes, like human keratinocytes, show varying clonogenic abilities, but that they differ from human keratinocytes in the morphologies and behaviors of the colonies they produce. Figure 1 illustrates the series of morphologically distinct colony types formed by murine keratinocytes when they are isolated and plated at clonal densities. We have termed these colonies Type I, II, and III in reference to the number of cell types they contain. Type I colonies consist only of large flattened (Type 1) cells and show little growth (Figure 1c). Type II colonies consist of smaller relatively uniform (Type 2) cells surrounded by a peripheral zone of cells corresponding to the larger flattened Type 1 cells (Figure 1b). Type III colonies have an additional central zone consisting of small and closely packed (Type 3) cells (Figure 1a). The boundaries established between these zones of differing cells are usually quite well delineated (Figure 1n). On repeated bulk passage these cell morphologies and colony forms are recapitulated but if the cells of individual Type I, II, and III colonies are individually isolated and plated for growth, it is found that only Type III colonies contain cells that are capable of expansive growth and reformation of new Type III colonies. The clonogenic potential required to support extensive passage is thus restricted to the central Type 3 cells. Despite differences of these colony forms from those developed by human keratinocytes, particularly in the concentric distribution of cells in Type III colonies, it appears that the Type I, II, and III colonies are the functional murine equivalents of, respectively, the paraclone, meroclone, and holoclone forms of human colonies. These mutine colony morphologies are developed by keratinocytes from both epidermis and mucosa with functional stem cells apparently restricted to the Type III colonies. Some additional support for this notion has been provided by other findings. For example, the central Type 3 cells in colonies developed from oral mucosa fail to stain for cytokeratin 6 (Figure 1d), a marker of early differentiation in murine mucosal cells (unpublished observations), but stain positively for cytokeratin 15 (Figure 1e), a putative stem cell marker both in hair (Lyle et al., 1999Lyle S. Christofidou-Solomidou M. Liu Y. Elder D.E. Albelda S. Cotsarelis G. Human hair follicle bulge cells are biochemically distinct and possess an epithelial stem cell phenotype.J Investig Dermatol Symp Proc. 1999; 4: 296-301Abstract Full Text PDF PubMed Scopus (106) Google Scholar) and human oral mucosa (Figure 1l). There appear to be two distinct mechanisms by which expansion of the population of Type 3 cells is controlled. Firstly, time-lapse video recording shows that proliferation of Type 3 cells results in centrifugal movement and transition into Type 2 cells. Secondly, morphological observations and staining for Caspase 3 indicate that apoptosis occurs relatively infrequently among the Type 2 cells but quite frequently among the Type 3 cells of the central region (Figure 1f, g). There are few data concerning differential apoptotic sensitivities of stem and amplifying cells of stratifying epithelia but possibly sensitivity of these cells to apoptotic stimuli may reflect the similar sensitivity of stem cells of some regions of the gut (Potten et al., 2003Potten C.S. Booth C. Hargreaves D. The small intestine as a model for evaluating adult tissue stem cell drug targets.Cell Prolif. 2003; 36: 115-129Crossref PubMed Scopus (95) Google Scholar). The clonal patterns developed in vitro by isolated human and murine keratinocytes indicate the intrinsic nature of their ability to generate stem and amplifying patterns. Lineage studies of murine epidermis indicate that individual stem cells generate clonal units in vivo and that these correspond morphologically to the small columnar units normally present (Mackenzie, 1995;Ghazizadeh and Taichman, 2001Ghazizadeh S. Taichman L.B. Multiple classes of stem cells in cutaneous epithelium: A lineage analysis of adult mouse skin.EMBO J. 2001; 20: 1215-1222Crossref PubMed Scopus (243) Google Scholar). To test whether establishment of this in vivo clonal pattern also represents an intrinsic ability of epidermal stem cells, epidermal keratinocytes were recombined with connective tissues from regions without columnar epithelial. The reformation of clonal columnar units in these preparations indicated that the epidermal pattern can be regenerated in the absence of regionally specific mesenchymal signals (Mackenzie, 1995). We have since extended these types of lineage studies to human epithelia by transduction of epidermal keratinocytes to produce populations containing a mixture of marked and unmarked cells. These were then used to generate organotypic cultures for transplantation to SCID mice for up to 12 wk. The results obtained with human epidermis were similar to those for murine epidermis. Two wk after transplantation, the reformed epidermis was hyperplastic and contained large irregular clusters of labeled cells that appeared to be differentiating but, by 12 wk, hyperplasia had diminished and both the number and size of the labeled cell clusters was greatly reduced (Figure 1h). The labeled cell clusters that remained corresponded morphologically to the columnar units present in human epidermis (Mackenzie et al., 1981Mackenzie I.C. Zimmerman K. Peterson L. The pattern of cellular organization of human epidermis.J Invest Dermatol. 1981; 76: 459-461Abstract Full Text PDF PubMed Scopus (35) Google Scholar) suggesting that thin human epidermis, like mouse epidermis, consists of small clonal units of structure. The small clonal units found in rodent epidermis are not typical of most epithelia. For example, stem cells in hair follicles are restricted to the bulge region, stem cells in cornea are found in the surrounding limbal region, and stem cells in gut lie at the base of the villi (Cotsarelis et al., 1989Cotsarelis G. Cheng S.Z. Dong G. Sun T.T. Lavker R.M. Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: Implications on epithelial stem cells.Cell. 1989; 57: 201-209Abstract Full Text PDF PubMed Scopus (1168) Google Scholar,Cotsarelis et al., 1990Cotsarelis G. Sun T.T. Lavker R.M. Label-retaining cells reside in the bulge area of pilosebaceous unit: Implications for follicular stem cells, hair cycle, and skin carcinogenesis.Cell. 1990; 61: 1329-1337Abstract Full Text PDF PubMed Scopus (1898) Google Scholar;Potten and Loeffler, 1990Potten C.S. Loeffler M. Stem cells: Attributes, cycles, spirals, pitfalls and uncertainties. Lessons for and from the crypt. Development.. 1990; 110: 1001-1020Google Scholar). Even in fairly simple epithelial structures, such as rete ridges, stem cells appear to be restricted to certain zones but, strangely, the position of such zones differs between skin and mucosal epithelia: In the epidermis, stem cells are clustered over the tips of the connective tissue papillae (Jensen et al., 1999Jensen U.B. Lowell S. Watt F.M. 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.Development. 1999; 126: 2409-2418PubMed Google Scholar) but lie at the deep tips of the epithelial rete of palmar epidermis (Lavker and Sun, 1982Lavker R.M. Sun T.T. Heterogeneity in epidermal basal keratinocytes: Morphological and functional correlations.Science. 1982; 215: 1239-1241Crossref PubMed Scopus (300) Google Scholar) and in mucos