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Epithelial Stem Cells: Turning over New Leaves

2007; Cell Press; Volume: 128; Issue: 3 Linguagem: Inglês

10.1016/j.cell.2007.01.014

1097-4172

Cédric Blanpain, Valerie Horsley, Elaine Fuchs,

Plant tissue culture and regeneration

Most epithelial tissues self-renew throughout adult life due to the presence of multipotent stem cells and/or unipotent progenitor cells. Epithelial stem cells are specified during development and are controlled by epithelial-mesenchymal interactions. Despite morphological and functional differences among epithelia, common signaling pathways appear to control epithelial stem cell maintenance, activation, lineage determination, and differentiation. Additionally, deregulation of these pathways can lead to human disorders including cancer. Understanding epithelial stem cell biology has major clinical implications for the diagnosis, prevention, and treatment of human diseases, as well as for regenerative medicine. Most epithelial tissues self-renew throughout adult life due to the presence of multipotent stem cells and/or unipotent progenitor cells. Epithelial stem cells are specified during development and are controlled by epithelial-mesenchymal interactions. Despite morphological and functional differences among epithelia, common signaling pathways appear to control epithelial stem cell maintenance, activation, lineage determination, and differentiation. Additionally, deregulation of these pathways can lead to human disorders including cancer. Understanding epithelial stem cell biology has major clinical implications for the diagnosis, prevention, and treatment of human diseases, as well as for regenerative medicine. Epithelia are continuous sheets of tightly linked cells that constitute the surfaces (such as the epidermis and corneal epithelium) and linings (such as the digestive, respiratory, and uro-genital epithelia) of the body. At these locations, epithelia not only provide a protective envelope against the external environment but also regulate water and nutrient absorption as well as glandular secretions. Although epithelia can be multilayered (stratified) or single-layered (simple) and may be derived from ectoderm, mesoderm, or endoderm, the epithelial tissues of the body share several molecular and cellular characteristics. In development, epithelia begin as a sheet of cells that adhere to a basement membrane, rich in extracellular matrix (ECM) and growth factors that are produced and deposited at the interface of the epithelium and the underlying mesenchyme. Epithelial cells express transmembrane integrin heterodimers that bind to ECM ligands in the basement membrane and provide a link to the cellular cytoskeleton (Giancotti and Tarone, 2003Giancotti F.G. Tarone G. Positional control of cell fate through joint integrin/receptor protein kinase signaling.Annu. Rev. Cell Dev. Biol. 2003; 19: 173-206Crossref PubMed Scopus (299) Google Scholar). In addition, focal adhesions and hemidesmosomes regulate the dynamics of adhesion and detachment of cells and their underlying ECM, thus mediating cell migration, stratification, and differentiation (Fuchs and Raghavan, 2002Fuchs E. Raghavan S. Getting under the skin of epidermal morphogenesis.Nat. Rev. Genet. 2002; 3: 199-209Crossref PubMed Scopus (537) Google Scholar, Watt, 2002Watt F.M. Role of integrins in regulating epidermal adhesion, growth and differentiation.EMBO J. 2002; 21: 3919-3926Crossref PubMed Scopus (508) Google Scholar). Epithelial cells also make intercellular connections through the formation of adherens junctions, tight junctions, and desmosomes that enable epithelial cells to communicate and function as a sheet. In conjunction with integrins, intercellular junctions and associated components distinguish the apical, basal, and lateral surfaces of the cell, which are essential for establishing epithelial cell polarity (Shin et al., 2006Shin K. Fogg V.C. Margolis B. Tight junctions and cell polarity.Annu. Rev. Cell Dev. Biol. 2006; 22: 207-235Crossref PubMed Scopus (531) Google Scholar). Most epithelia need to constantly replace damaged or dead cells throughout the life of the animal. The process of continual cell replacement is called tissue homeostasis and is critical for the maintenance of adult tissues. Typically, epithelial tissue homeostasis is maintained through the presence of stem cells. Stem cells are functionally defined by their ability to self-renew and to differentiate into the cell lineages of their tissue of origin (Moore and Lemischka, 2006Moore K.A. Lemischka I.R. Stem cells and their niches.Science. 2006; 311: 1880-1885Crossref PubMed Scopus (1226) Google Scholar). Once activated, epithelial stem cells can generate proliferating progeny, which are often referred to as transiently amplifying (TA) cells. In their normal environment, TA cells will divide actively for a restricted period of time, expanding the cellular pool that will then differentiate along a particular cell lineage to make the tissue. The homeostatic replacement of cells varies substantially among different epithelia. The epithelium of the intestine completely self-renews within ∼5 days. By contrast, interfollicular epidermis takes ∼4 weeks to renew, whereas the lung epithelium can take as long as 6 months to be replaced. In addition, some epithelia present a cyclic mode of tissue homeostasis. Hair follicles, for example, cycle continuously through bouts of hair growth (anagen), degeneration (catagen), and rest (telogen) (Blanpain and Fuchs, 2006Blanpain C. Fuchs E. Epidermal stem cells of the skin.Annu. Rev. Cell Dev. Biol. 2006; 22: 339-373Crossref PubMed Scopus (498) Google Scholar). Similarly, the mammary gland proceeds through cycles of growth and degeneration during and following pregnancy (Hennighausen and Robinson, 2005Hennighausen L. Robinson G.W. Information networks in the mammary gland.Nat. Rev. Mol. Cell Biol. 2005; 6: 715-725Crossref PubMed Scopus (347) Google Scholar). Unless the epithelial stem cells and associated mesenchyme are permanently damaged, most epithelia are also able to repair their tissues following injuries. Typically, tissue regeneration upon wounding involves recruitment of epithelial stem cells to replace the damaged cells. The adult liver offers an unusual example: although its epithelial cells do not turn over significantly under physiological conditions, they have an impressive capacity to regenerate tissue after injury. Oddly, following acute injuries, this tissue repair appears to occur without obvious participation of multipotent stem cells. Rather, liver regeneration after a partial hepatectomy occurs through proliferation of hepatocytes (Taub, 2004Taub R. Liver regeneration: from myth to mechanism.Nat. Rev. Mol. Cell Biol. 2004; 5: 836-847Crossref PubMed Scopus (1157) Google Scholar). A similar mode of regeneration called self-duplication has also been proposed to account for the renewal of pancreatic islet cells (Dor et al., 2004Dor Y. Brown J. Martinez O.I. Melton D.A. Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation.Nature. 2004; 429: 41-46Crossref PubMed Scopus (1801) Google Scholar). There is still no consensus as to whether adult cells with the capacity to regenerate and/or contribute to only a single lineage should be referred to as unipotent stem cells, unipotent progenitors, or simply cells that have retained proliferative potential. Epithelial stem cells can generate tissues that display a fascinating array of cellular architectures, each of which are specifically tailored for distinct functions (Figure 1). In this review, we will focus on four well-characterized epithelial stem cells whose tissues possess diverse architectural designs and physiology: intestine, epidermis, mammary gland, and cornea. We will briefly introduce the organization of these tissues into their functional units, delineate which cell lineages compose the unit, and discuss where the stem cells are located within each unit and their routes of activation and differentiation. The small intestine is a simple epithelium composed of crypt-villus units (Radtke and Clevers, 2005Radtke F. Clevers H. Self-renewal and cancer of the gut: two sides of a coin.Science. 2005; 307: 1904-1909Crossref PubMed Scopus (560) Google Scholar), which function to absorb water and nutrients and to form a functional barrier to protect against ingested pathogens. In the adult, the stem cells and their TA progeny reside in a region near the base of the crypt region, and as they migrate out of their niche, they cease to proliferate and initiate differentiation into the different cell lineages of the mature villi. Intestinal stem cells can differentiate into four different cell lineages: the absorptive enterocytes, mucin-secreting-goblet cells, peptide hormone-secreting neuroendocrine cells, and microbicide-secreting Paneth cells. The majority of cells in the villi are enterocytes with a few goblet and neuroendocrine cells located at various intervals. These three lineages form and mature as they migrate up the crypt to the tip of the villus. During tissue homeostasis, these cells are replaced and subsequently exfoliated into the intestinal lumen. Paneth cells differentiate as they travel down to the base of the crypt (Figure 1). Encasing the body, the skin epidermis is composed of pilo-sebaceous units containing a hair follicle, sebaceous gland, and interfollicular epidermis (Blanpain and Fuchs, 2006Blanpain C. Fuchs E. Epidermal stem cells of the skin.Annu. Rev. Cell Dev. Biol. 2006; 22: 339-373Crossref PubMed Scopus (498) Google Scholar). The interfollicular epidermis is a stratified squamous epithelium consisting of an innermost (basal) layer of proliferative cells that differentiate outward to form the distinctive suprabasal layers of the differentiated tissue: spinous, granular, and finally outermost stratum corneum layers to form an impermeable body surface. Resident basal stem cells strongly adhere to their underlying basement membrane and maintain homeostasis of the interfollicular epidermis by continually replenishing the suprabasal terminally differentiating cells as dead stratum corneum cells (squames) eventually reach and are sloughed from the skin surface. The hair follicle is formed during embryogenesis as an appendage of the epidermis. A condensation of specialized mesenchymal cells (dermal papilla) in the dermis stimulates cells within the overlying epidermal basal layer. The epidermal cells respond by changing their shape to develop a bud, or “placode,” of hair progenitor cells that subsequently proliferate and grow downward. Postnatally, the base of the mature follicle contains rapidly proliferating (TA) matrix cells, which after several rounds of cell division differentiate upward to form the hair and its surrounding channel, the inner root sheath. Eventually, the supply of differentiating matrix cells ceases, and the follicle regresses and draws the dermal papillae upward. After the lower two-thirds of the follicle have degenerated, the dermal papillae come to rest at the base of the remaining permanent follicle segment, called the “bulge.” At the start of the next hair cycle, quiescent stem cells residing at the base of the bulge are stimulated to migrate and proliferate to supply the cells needed for hair-follicle regeneration and hair growth. The surface of the eye is covered by the corneal epithelium, which acts as a protective outer barrier and helps to focus light into the retina. Like the epidermis, the cornea is a stratified epithelium. Adjacent to the corneal epithelium is the limbal region, a junctional zone between the cornea and the conjunctival epithelium. Corneal stem cells are thought to reside in this zone, giving rise to cells that migrate toward the center of the cornea, where they stratify and differentiate (Sun and Lavker, 2004Sun T.T. Lavker R.M. Corneal epithelial stem cells: past, present, and future.J. Investig. Dermatol. Symp. Proc. 2004; 9: 202-207Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Initially, mammary gland morphogenesis proceeds in a fashion that bears a marked resemblance to hair-follicle development. Ectodermal cells are instructed by their underlying mesenchyme to adopt a mammary epithelial fate and to grow downward as a germ. The germ then penetrates into the specialized breast mesenchyme, the fat pad, to form a rudimentary ductal tree that terminates into club-shaped structures, called terminal end buds (Hennighausen and Robinson, 2005Hennighausen L. Robinson G.W. Information networks in the mammary gland.Nat. Rev. Mol. Cell Biol. 2005; 6: 715-725Crossref PubMed Scopus (347) Google Scholar). Mammary stem cells and their TA progeny reside in this structure along the basement membrane and have the capacity to form epithelial precursors that are committed to either a ductal or alveolar fate. During puberty, cells of terminal end buds proliferate rapidly to produce cells for the growth and branching of new ducts, which fill the entire fat pad. During pregnancy, alveoli are formed by the differentiation of alveoli precursors to form an external, loose network of myoepithelial cells. Differentiated alveoli epithelial cells then secrete milk into the lumen of the ducts during lactation. Contraction of the myoepithelial cells surrounding the ducts subsequently allows milk release. At the conclusion of lactation, alveoli structures undergo involution, and the mammary epithelium returns to its virgin appearance, ready for another cycle of alveolar growth and differentiation during the next pregnancy. The location of stem cells within each of these epithelia was identified by taking advantage of the relative quiescence of stem cells within epithelial tissues. Despite their reduced mitotic index, epithelial stem cells can be labeled by continuous administration of nucleotide analogs (pulse) such as BrdU or 3H-TdR for a prolonged period. During the subsequent chase period, cells dilute their label through cell divisions. Those cells that divide less frequently during the chase period retain the label and hence have been referred to as label-retaining cells (Bickenbach, 1981Bickenbach, J.R. (1981). Identification and behavior of label-retaining cells in oral mucosa and skin. J. Dent. Res. 60 Spec No C, 1611–1620.Google Scholar). Label-retaining cells have been found in discrete locations within these epithelia. These sites of putative stem cells include the limbal region of the cornea (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 (1078) Google Scholar), the bulge of the hair follicle (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 (1767) Google Scholar), the terminal end bud of the mammary gland (Zeps et al., 1998Zeps N. Bentel J.M. Papadimitriou J.M. D'Antuono M.F. Dawkins H.J. Estrogen receptor-negative epithelial cells in mouse mammary gland development and growth.Differentiation. 1998; 62: 221-226Crossref PubMed Google Scholar), and a narrow band near the bottom of the intestinal crypts (Potten et al., 2002Potten C.S. Owen G. Booth D. Intestinal stem cells protect their genome by selective segregation of template DNA strands.J. Cell Sci. 2002; 115: 2381-2388PubMed Google Scholar). To circumvent the use of BrdU or 3H-TdR in order to mark, isolate, and characterize living label-retaining cells, Tumbar and colleagues engineered transgenic mice expressing histone 2B-green fluorescent protein (H2B-GFP) under the control of a tetracycline-regulated enhancer element (Tumbar et al., 2004Tumbar T. Guasch G. Greco V. Blanpain C. Lowry W.E. Rendl M. Fuchs E. Defining the epithelial stem cell niche in skin.Science. 2004; 303: 359-363Crossref PubMed Scopus (1548) Google Scholar). To isolate label-retaining cells from stratified and glandular epithelia, they mated them to mice expressing the tetracycline-activatable repressor under control of a keratin 5 promoter (K5-tetoff) (Tumbar et al., 2004Tumbar T. Guasch G. Greco V. Blanpain C. Lowry W.E. Rendl M. Fuchs E. Defining the epithelial stem cell niche in skin.Science. 2004; 303: 359-363Crossref PubMed Scopus (1548) Google Scholar). In the absence of tetracycline, H2B-GFP is synthesized and incorporated into the nucleus of proliferating cells, but upon tetracycline addition, H2B-GFP expression is repressed. Assuming that chromosomally packaged H2B-GFP is very stable, GFP fluorescence intensity is likely to correlate with the number of cell divisions during the chase period. In mouse skin epithelium, a chase period of a month specifically marks bulge cells and has been used to isolate and characterize living bulge label-retaining cells. This method may be applicable to other epithelia, allowing the characterization and isolation of epithelial stem cells for which few or no cell-surface markers are currently known. One defining characteristic of an epithelium is the close contact these cells have with the underlying mesenchyme. A number of classical tissue recombination experiments have demonstrated the inductive ability of mesenchymal tissues in the control of epithelial cell-fate determination. In the embryo, incubation of mesenchyme from different tissues can induce epithelia to adopt the cell fate of the mesenchymal tissue of origin. For instance, murine backskin dermis can induce nonhairy epidermis to form hair follicles (Hardy, 1992Hardy M.H. The secret life of the hair follicle.Trends Genet. 1992; 8: 55-61Abstract Full Text PDF PubMed Scopus (751) Google Scholar). Similarly, epithelial lung buds are instructed to form gastric glands when placed in contact with stomach mesenchyme, villi epithelium when recombined with intestinal mesenchyme, and hepatic cords when incubated with liver mesenchyme (Birchmeier and Birchmeier, 1993Birchmeier C. Birchmeier W. Molecular aspects of mesenchymal-epithelial interactions.Annu. Rev. Cell Biol. 1993; 9: 511-540Crossref PubMed Scopus (208) Google Scholar). Prospective mammary mesenchyme can induce dorsal epidermis to differentiate into mammary epithelium (Cunha et al., 1995Cunha G.R. Young P. Christov K. Guzman R. Nandi S. Talamantes F. Thordarson G. Mammary phenotypic expression induced in epidermal cells by embryonic mammary mesenchyme.Acta Anat. (Basel). 1995; 152: 195-204Crossref PubMed Scopus (69) Google Scholar). Central corneal cells can be reprogrammed to form pilosebaceous units when transplanted with embryonic back-skin dermis (Ferraris et al., 2000Ferraris C. Chevalier G. Favier B. Jahoda C.A. Dhouailly D. Adult corneal epithelium basal cells possess the capacity to activate epidermal, pilosebaceous and sweat gland genetic programs in response to embryonic dermal stimuli.Development. 2000; 127: 5487-5495PubMed Google Scholar). These experiments support the notion that epithelial appendage and tissue formation is instructed by mesenchymal signals. Whether the inductive potential is enhanced and maintained in adult stem cells is an intriguing question, as yet unexplored. Additional epithelial-mesenchymal crosstalk is required to complete morphogenesis and differentiation. When combined with mouse, chicken, or lizard epidermis, murine dermis can induce hair follicle, feather, or scale placodes, respectively, but these placodes do not grow or differentiate further, due to the absence of dermal papillae. These results suggest that a second signal emanating from the epidermis is required for the differentiation of mesenchymal cells into a proper organizing center (Hardy, 1992Hardy M.H. The secret life of the hair follicle.Trends Genet. 1992; 8: 55-61Abstract Full Text PDF PubMed Scopus (751) Google Scholar). When taken together, these tissue recombination studies demonstrate that the reciprocal inductive interactions between the mesenchyme and the overlying epithelium are essential for the fate induction of various epithelia. Although the precise mediators of these epithelial-mesenchymal interactions are not well defined, secreted Wnt ligands may be one of the first signals involved in this communication as discussed later. Epithelial homeostasis is typically maintained by unipotent progenitor cells, which have the ability to differentiate into one particular cell lineage. The direct demonstration of the existence of these progenitors and their lineage potential relies on clonal transplantation of isolated progenitors and genetic lineage tracing experiments. The interfollicular epidermis and sebaceous glands contain unipotent progenitors that can maintain homeostasis of their respective tissue. Histological analyses of mouse skin reveals that the epidermis is organized in stacks of cells with a hexagonal surface area covering about ten basal cells (Potten, 1974Potten C.S. The epidermal proliferative unit: the possible role of the central basal cell.Cell Tissue Kinet. 1974; 7: 77-88PubMed Google Scholar). This structure has been proposed to function as an epidermal proliferative unit, harboring one putative stem cell per unit. More recently, this notion has been demonstrated experimentally by lineage tracing analysis, either by grafting virally tagged keratinocytes onto the backs of Nude mice (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 (293) Google Scholar, Kolodka et al., 1998Kolodka T.M. Garlick J.A. Taichman L.B. Evidence for keratinocyte stem cells in vitro: long term engraftment and persistence of transgene expression from retrovirus-transduced keratinocytes.Proc. Natl. Acad. Sci. USA. 1998; 95: 4356-4361Crossref PubMed Scopus (176) Google Scholar, Mackenzie, 1997Mackenzie I.C. Retroviral transduction of murine epidermal stem cells demonstrates clonal units of epidermal structure.J. Invest. Dermatol. 1997; 109: 377-383Crossref PubMed Scopus (112) Google Scholar) or by inducing permanent expression of GFP clonally in the interfollicular epidermis and monitoring the fate of marked cells over time (Ro and Rannala, 2004Ro S. Rannala B. A stop-EGFP transgenic mouse to detect clonal cell lineages generated by mutation.EMBO Rep. 2004; 5: 914-920Crossref PubMed Scopus (35) Google Scholar). Stem cells from cultured human interfollicular epidermis have been isolated based on elevated expression of integrins (Jones and Watt, 1993Jones P.H. Watt F.M. Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression.Cell. 1993; 73: 713-724Abstract Full Text PDF PubMed Scopus (956) Google Scholar). Recent fate-mapping analysis has also revealed the existence of a small group of unipotent sebaceous progenitor cells residing at the base of the gland that express the transcriptional repressor Blimp1 (Horsley et al., 2006Horsley V. O'Carroll D. Tooze R. Ohinata Y. Saitou M. Obukhanych T. Nussenzweig M. Tarakhovsky A. Fuchs E. Blimp1 defines a progenitor population that governs cellular input to the sebaceous gland.Cell. 2006; 126: 597-609Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). In addition to these unipotent progenitors, multipotent stem cells reside in the bulge (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 (1767) Google Scholar, Oshima et al., 2001Oshima H. Rochat A. Kedzia C. Kobayashi K. Barrandon Y. Morphogenesis and renewal of hair follicles from adult multipotent stem cells.Cell. 2001; 104: 233-245Abstract Full Text Full Text PDF PubMed Scopus (833) Google Scholar, Taylor et al., 2000Taylor G. Lehrer M.S. Jensen P.J. Sun T.T. Lavker R.M. Involvement of follicular stem cells in forming not only the follicle but also the epidermis.Cell. 2000; 102: 451-461Abstract Full Text Full Text PDF PubMed Scopus (910) Google Scholar). Multipotent stem cells can differentiate into all epithelial cell lineages residing in the tissue. In skin, they not only serve as a reservoir of cells for regeneration during the normal cyclic periods of hair growth but also in conditions of hyperproliferation of sebaceous glands (Horsley et al., 2006Horsley V. O'Carroll D. Tooze R. Ohinata Y. Saitou M. Obukhanych T. Nussenzweig M. Tarakhovsky A. Fuchs E. Blimp1 defines a progenitor population that governs cellular input to the sebaceous gland.Cell. 2006; 126: 597-609Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar) and in the repair of the interfollicular epidermis following wounding (Ito et al., 2005Ito M. Liu Y. Yang Z. Nguyen J. Liang F. Morris R.J. Cotsarelis G. Stem cells in the hair follicle bulge contribute to wound repair but not to homeostasis of the epidermis.Nat. Med. 2005; 11: 1351-1354Crossref PubMed Scopus (918) Google Scholar, Levy et al., 2005Levy V. Lindon C. Harfe B.D. Morgan B.A. Distinct stem cell populations regenerate the follicle and interfollicular epidermis.Dev. Cell. 2005; 9: 855-861Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, Taylor et al., 2000Taylor G. Lehrer M.S. Jensen P.J. Sun T.T. Lavker R.M. Involvement of follicular stem cells in forming not only the follicle but also the epidermis.Cell. 2000; 102: 451-461Abstract Full Text Full Text PDF PubMed Scopus (910) Google Scholar, Tumbar et al., 2004Tumbar T. Guasch G. Greco V. Blanpain C. Lowry W.E. Rendl M. Fuchs E. Defining the epithelial stem cell niche in skin.Science. 2004; 303: 359-363Crossref PubMed Scopus (1548) Google Scholar) (Figure 2). Whether progenitors resident in the interfollicular epidermis or sebaceous glands can similarly become multipotent when the situation mandates is an intriguing question for future studies. The multipotency of stem cells within the bulge was first suggested from transplantation experiments in which a dissected bulge region was grafted onto immunodeficient mice (Oshima et al., 2001Oshima H. Rochat A. Kedzia C. Kobayashi K. Barrandon Y. Morphogenesis and renewal of hair follicles from adult multipotent stem cells.Cell. 2001; 104: 233-245Abstract Full Text Full Text PDF PubMed Scopus (833) Google Scholar). Transplanted bulge cells were able to differentiate into the complete repertoire of skin epithelial cells: the interfollicular epidermis, sebaceous glands, and the eight cell lineages constituting mature hair follicles. Bulge cells purified by fluorescence-activated cell sorting (FACS) on the basis of their preferred expression of a GFP transgene also differentiated into these lineages upon transplantation (Morris et al., 2004Morris R.J. Liu Y. Marles L. Yang Z. Trempus C. Li S. Lin J.S. Sawicki J.A. Cotsarelis G. Capturing and profiling adult hair follicle stem cells.Nat. Biotechnol. 2004; 22: 411-417Crossref PubMed Scopus (1005) Google Scholar). Clonal analyses were essential to demonstrate that bulge cells are multipotent stem cells and not simply a mixture of unipotent progenitors. This was accomplished by first culturing progeny derived from single FACS-purified bulge cells (Blanpain et al., 2004Blanpain C. Lowry W.E. Geoghegan A. Polak L. Fuchs E. Self-renewal, multipotency, and the existence of two cell populations within an epithelial stem cell niche.Cell. 2004; 118: 635-648Abstract Full Text Full Text PDF PubMed Scopus (1055) Google Scholar) or from microdissected tissue (Claudinot et al., 2005Claudinot S. Nicolas M. Oshima H. Rochat A. Barrandon Y. Long-term renewal of hair follicles from clonogenic multipotent stem cells.Proc. Natl. Acad. Sci. USA. 2005; 102: 14677-14682Crossref PubMed Scopus (244) Google Scholar) and then transplantation. The existence of multipotent stem cells in the mammary gland was posited from experiments in which fragments of mammary tissue containing cells harboring randomly inserted MMTV retroviruses were first transplanted into the epithelium-free mammary fat pads of an uninfected recipient mouse and then serially transplanting clonally derived outgrowths to create second-generation glands (Kordon and Smith, 1998Kordon E.C. Smith G.H. An entire functional mammary gland may comprise the progeny from a single cell.Development. 1998; 125: 1921-1930Crossref PubMed Google Scholar). With each round of outgrowth, the viral integration site was mapped and shown to be identical, suggesting that an entire functional gland may arise from the progeny of a single mammary epithelial stem cell, which displays long-term self-renewal properties. Two different groups recently demonstrated this rigorously, first by identifying and purifying a population of mammary cells (with the marker profile Lin−, α6 or β1High, and CD24+), which are enriched for stem cells, and then by transplantation studies (Shackleton et al., 2006Shackleton M. Vaillant F. Simpson K.J. Stingl J. Smyth G.K. Asselin-Labat M.L. Wu L. Lindeman G.J. Visvader J.E. Generation of a functional mammary gland from a single stem cell.Nature. 2006; 439: 84-88Crossref PubMed Scopus (1493) Google Scholar, Stingl et al., 2006Stingl J. Eirew P. Ricketson I. Shackleton M. Vaillant F. Choi D. Li H.I. Eaves C.J. Purification and unique properties of mammary epithelial stem cells.Nature. 2006; 439: 993-997Crossref PubMed Scopus (1174) Google Scholar). By tagging these cells with a lacZ transgene, the researchers were able to directly demonstrate that individual mammary stem cells are able to generate a functional mammary gland upon transplantation (Figure 2). Similar to bulge stem cells, mammary stem cells display elevated surface integrins as well as keratins 5 and 14, which are characteristics of stratified and glandular epithelial cells that reside along a base