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Keratinocyte Stem Cell Assays: An Evolving Science

      Although the existence of epithelial stem cells in the skin has been known for some decades from cell kinetic studies performed in vivo, attempts to prospectively isolate these cells for further biological characterization have been made possible relatively recently facilitated by the availability of antibodies that detect cell surface markers on epidermal cells. Elegant gene marking studies in vivo have provided confirmation of the patterns of epithelial tissue replacement predicted by classical cell turnover studies. But, the identification of candidate epidermal stem cells ex vivo remains an area of great controversy, requiring the re-evaluation of current experimental approaches that rely of necessity on predicted epidermal stem cell behavior in culture. Here we review the diverse experimental approaches utilized to identify keratinocyte stem cells and their underlying assumptions. We conclude that hair follicles and interfollicular epidermis each have their own self-renewing stem cell populations, contributing to distinct regions of the epithelium during homeostasis, although this is perturbed during wound healing. The need for the development of more rigorous assays for stem cell activity is highlighted given our recent observations using current assays and the discovery of new surface markers that identify putative epidermal stem cells.

      Keywords

      Abbreviations:

      EPU
      epidermal proliferative unit
      FACS
      fluorescence activated cell sorting
      HF
      hair follicle
      IF
      interfollicular
      KSC
      keratinocyte stem cell
      LRC
      label-retaining cell
      PMD
      post-mitotic differentiating
      SP
      side population
      TA
      transient-amplifying
      The extensive capacity of the epidermis for cell renewal in vivo has been demonstrated by their ability to survive, expand, and generate cultured human epidermal sheets capable of rescuing patients with full thickness burns covering up to 98% of their body surface that are maintained for over a decade (
      • Gallico III, G.G.
      • O'Connor N.E.
      • Compton C.C.
      • Kehinde O.
      • Green H.
      Permanent coverage of large burn wounds with autologous cultured human epithelium.
      ;
      • Compton C.C.
      • Gill J.M.
      • Bradford D.A.
      • Regauer S.
      • Gallico G.G.
      • O'Connor N.E.
      Skin regenerated from cultured epithelial autografts on full-thickness burn wounds from 6 days to 5 years after grafting. A light, electron microscopic and immunohistochemical study.
      ). Although these studies demonstrate the immense regenerative capacity of keratinocytes, the stem cells in this tissue remain uncharacterized. Extensive investigation combining close histological analyses and elegant in vivo cell kinetic studies conducted worldwide some 25 y ago firmly established that murine interfollicular (IF) epidermis is a highly organized, stratified tissue with an impressive cell turnover rate which is ultimately dependent on a minor population of resident stem cells (
      • Mackenzie I.C.
      Relationship between mitosis and the ordered structure of the stratum corneum in mouse epidermis.
      ;
      • Allen T.D.
      • Potten C.S.
      Fine structural identification and organization of the epidermal proliferative unit.
      ;
      • Potten C.S.
      Stem cells in epidermis from the back of the mouse.
      ;
      • Morris R.J.
      • Fischer S.M.
      • Slaga T.J.
      Evidence that the centrally and peripherally located cells in the murine epidermal proliferative unit are two distinct populations.
      ). Thus, mature cells or squames lost from the skin surface are continuously replaced by a carefully orchestrated process of cell proliferation within the basal layer, which lies adjacent to the basement membrane. Similar in vivo cell kinetic analysis in mice subsequently established the presence and role of epithelial stem cells in cell renewal at other anatomical sites including the bulge region of the hair follicle (HF), limbus of the cornea, the basal layer of oral mucosa, and in the base of small intestinal crypts (reviewed by
      • Miller S.J.
      • Lavker R.M.
      • Sun T-T.
      Keratinocyte stem cells of cornea, skin and hair follicle: Common and distinguishing features.
      ). The development of cell culture techniques permitting the in vitro propagation of epithelial cells was a further critical step advancing the study of epithelial stem cells (
      • Rheinwald J.G.
      • Green H.
      Serial cultivation of strains of human epidermal keratinocytes: The formation of keratinizing colonies from single cells.
      ) providing the impetus for skin biologists to identify, isolate and assay epidermal stem cells from explanted tissue using the experimental approaches and criteria developed in dissecting and ordering the hemopoietic stem and progenitor cell hierarchy as a template.
      Although the functional attributes of epithelial stem cells and hemopoietic stem cells will be different, reflecting their diverse biological roles in the body, they share many operational characteristics of adult stem cells of all continuously renewing cell populations: low incidence, low probability of cycling, slow turnover rate, the ability to self-renew, and the ability to regenerate and repair tissue in the steady state and following damage. Thus, colony-forming assays, alone or in combination with fluorescence activated cell sorting (FACS), and gene marking combined with the analysis of tissue regeneration in vitro or in vivo (
      • Barrandon Y.
      • Green H.
      Three clonal types of keratinocytes with different capacities for multiplication.
      ;
      • Jones P.H.
      • Watt F.M.
      Separation of human epidermal stem cells from transit amplifying cells on the basis of difference in integrin function and expression.
      ;
      • Jones P.H.
      • Harper S.
      • Watt F.M.
      Stem cell patterning and fate in human epidermis.
      ;
      • Mackenzie I.C.
      Retroviral transduction of murine epidermal stem cells demonstrates clonal units of epidermal structure.
      ;
      • Kolodka 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.
      ;
      • Li A.
      • Simmons P.J.
      • Kaur P.
      Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype.
      ;
      • Tani H.
      • Morris R.J.
      • Kaur P.
      Enrichment for murine keratinocyte stem cells based on cell surface phenotype.
      ;
      • Ghazizadeh S.
      • Taichman L.B.
      Multiple classes of stem cells in cutaneous epithelium: A lineage analysis of adult mouse skin.
      ) have proved extremely useful in beginning to extend our knowledge of epidermal stem cells.
      As in the hemopoietic system, the continuing challenge for epithelial stem cell biologists is the development and refinement of predictive surrogate assays which provide a measure of stem cell activity in cell populations removed from their native microenvironment. The controversy currently hotly debated in scientific forums is almost entirely centered around the validity of specific molecular markers and assays employed to identify epidermal stem cells either in situ or ex vivo, and the validity of assays which purportedly distinguish stem cells from their more committed progeny.
      In the absence of (a) a rigorous epidermal stem cell assay analogous to bone marrow reconstitution with candidate hemopoietic stem cells in lethally irradiated mice; (b) a unique phenotypic marker repertoire; and (c) recombinant cytokines that have enabled hemopoietic stem cell biologists to hierarchically order closely related marrow stem and progenitor cell populations by in vitro surrogate assays (
      • Bertoncello I.
      • Bradford G.B.
      Surrogate assays for hematopoietic stem cell activity.
      ), epithelial stem cell biologists have had to resort to readouts which are largely based on untested assumptions about the expected properties and behavior of isolated epidermal or keratinocyte stem cells (KSC).
      Consequently, individual laboratories have favored the use of different surrogate assays which they feel best measure the characteristic(s) they regard as their gold-standard criteria defining a stem cell. These include, colony-forming efficiency, colony size and morphology, epithelial regeneration, and long-term proliferative potential in vitro. In this review, we examine the evidence and assumptions supporting the various experimental approaches taken to date in evaluating epidermal stem cell potential in vivo and in vitro and discuss their limitations.

      Evidence Supporting the Presence of Stem Cells in the IF Epidermis In Vivo

      The dorsal epithelium in mice is a complex tissue specialized into HF, sebaceous glands, and the IF epithelium, each characterized by its own distinct program of differentiation. Recent developments have led to re-examination of the long-held view put forward by Potten, Mackenzie, Bickenbach, and Morris that the IF epidermis is a self-renewing tissue even in hairy skin. These investigators generated a vast body of compelling cell kinetic data which demonstrated unequivocally that cell replacement in the IF epidermis occurs within small packets of self-renewing epidermis termed the epidermal proliferative unit or EPU comprising about ten basal cells and their suprabasal progeny lying directly above them. Three classes of basal keratinocytes have been identified by cell turnover studies and painstaking spatial analysis in situ.
      The long-lived KSC comprise a minor subpopulation (∼1%–10% of basal cells) within the center of the EPU that are relatively quiescent, and identified as single label-retaining cells (LRC), after a prolonged chase period (6–8 wk) following repeated administration of 3H-Tdr. Short-lived transient-amplifying (TA) cells (∼60% of basal cells) located peripherally to the KSC are rapidly proliferating cells that readily incorporate 3H-Tdr, but are lost from the basal layer to terminal differentiation within 4–5 d (
      • Mackenzie I.C.
      Relationship between mitosis and the ordered structure of the stratum corneum in mouse epidermis.
      ;
      • Potten C.S.
      The epidermal proliferative unit: The possible role of the central basal cell.
      ;
      • MacKenzie I.C.
      • Bickenbach J.R.
      Localization of label-retaining cells in mouse epithelia.
      ;
      • Morris R.J.
      • Fischer S.M.
      • Slaga T.J.
      Evidence that the centrally and peripherally located cells in the murine epidermal proliferative unit are two distinct populations.
      ;
      • Bickenbach J.R.
      • McCutecheon J.
      • MacKenzie I.C.
      Rate of loss of tritiated thymidine label in basal cells in mouse epithelial tissue.
      ; see
      • Miller S.J.
      • Lavker R.M.
      • Sun T-T.
      Keratinocyte stem cells of cornea, skin and hair follicle: Common and distinguishing features.
      for detailed review). A third class of basal post-mitotic differentiating (PMD) keratinocytes located at the edges of the EPU, exhibit the early stages of keratinization as judged by morphology and ultrastructure (
      • Christophers E.
      Cellular architecture of the stratum corneum.
      ;
      • Allen T.D.
      • Potten C.S.
      Fine structural identification and organization of the epidermal proliferative unit.
      ). These PMD cells retain some contact with the basement membrane but have a shape suggestive of their being in the process of migrating out of the basal layer. They can be visualized as K10-positive “hand-mirror”-shaped cells found in both murine and human epidermis (
      • Schweizer J.
      • Kinjo M.
      • Furstenberger G.
      • Winter H.
      Sequential expression of mRNA-encoded keratin sets in neonatal mouse epidermis: Basal cells with properties of terminally differentiating cells.
      ;
      • Mackenzie I.C.
      • Mackenzie S.L.
      • Rittman G.A.
      Isolation of subpopulations of murine epidermal cells using monoclonal antibodies against differentiation-related cell surface molecules.
      ;
      • Kaur P.
      • Li A.
      Adhesive properties of human basal epidermal cells: An analysis of keratinocyte stem cells, transit amplifying cells, and postmitotic differentiating cells.
      ).
      It is well accepted that these three subsets of murine basal epidermal cells exist in vivo at specific anatomical sites, i.e., the IF epidermis of the dorsum, ear, and tail. The interrelationship of these subsets (i.e., KSC → TA → PMD cells) is also apparent and a reasonable model for cell renewal. Thus KSC, defined here as LRC generated in newborn mice, are long-lived and persist into adulthood, suggesting that they are permanent residents in these tissues. TA cells have a short lifespan in vivo and are lost to terminal differentiation within weeks. And, migration studies tracking the progress of 3H-Tdr-labelled cells from the basal layer into suprabasal layers, provide strong evidence for the maturation of TA cells into basal PMD cells, and then terminally differentiating suprabasal cells. Importantly, the suspected progression of KSC → TA → PMD cells has recently been confirmed by elegant gene marking and lineage analysis studies in two independent laboratories. Thus, cultured murine and human keratinocytes comprised of a mixture of β-galactosidase transduced and untransduced cells, transplanted in vivo, regenerated a chimeric epithelium with interspersed columns/EPU of transduced (blue) cells (
      • Mackenzie I.C.
      Retroviral transduction of murine epidermal stem cells demonstrates clonal units of epidermal structure.
      ;
      • Kolodka 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.
      ), providing strong evidence for the clonal derivation of the epidermis from IF stem cells as originally proposed by Mackenzie and Potten three decades ago (
      • Mackenzie I.C.
      Relationship between mitosis and the ordered structure of the stratum corneum in mouse epidermis.
      ;
      • Allen T.D.
      • Potten C.S.
      Fine structural identification and organization of the epidermal proliferative unit.
      ). Most recently, lineage analysis studies of epithelial cells retrovirally marked in situ have provided compelling evidence supporting the self-sustaining units of epidermal cell renewal or EPU even in hairy dorsal skin followed over 33 wk (
      • Ghazizadeh S.
      • Taichman L.B.
      Multiple classes of stem cells in cutaneous epithelium: A lineage analysis of adult mouse skin.
      ). These data collectively point to a central role for resident IF stem cells in homeostatic cell renewal and the generation of mature keratinocytes for the lifetime of a mouse in vivo.

      Physiological Role of HF Stem Cells

      Kinetic studies have revealed the presence of stem cells visualized as slow-cycling cells/LRC in the bulge region of HF as well as the limbal region of the cornea and the dorsal tongue mucosa of adult mice (
      • Bickenbach J.R.
      Identification and behavior of label-retaining cells in oral mucosa and skin.
      ;
      • Morris R.J.
      • Fischer S.M.
      • Slaga T.J.
      Evidence that the centrally and peripherally located cells in the murine epidermal proliferative unit are two distinct populations.
      ;
      • Bickenbach J.R.
      • McCutecheon J.
      • MacKenzie I.C.
      Rate of loss of tritiated thymidine label in basal cells in mouse epithelial tissue.
      ;
      • Cutsarelis 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.
      ,
      • Cotsarelis 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.
      ;
      • Lavker R.M.
      • Dong G.
      • Cheng S.Z.
      • Kudoh K.
      • Cotsarelis G.
      • Sun T-T.
      Relative proliferative rates of limbal and corneal epithelia. Implications of corneal epithelial migration, circadian rhythm, and suprabasally located DNA-synthesizing keratinocytes.
      ; see
      • Miller S.J.
      • Lavker R.M.
      • Sun T-T.
      Keratinocyte stem cells of cornea, skin and hair follicle: Common and distinguishing features.
      for detailed review). The central role of the bulge region in cell renewal is supported by studies of human HF demonstrating that epithelial cells within this region have greater proliferative potential than that of IF epidermal cells in culture (
      • Yang J.S.
      • Lavker R.M.
      • Sun T-T.
      Upper human hair follicle contains a subpopulation of keratinocytes with superior in vitro proliferative potential.
      ;
      • Rochat A.
      • Kobayashi K.
      • Barrandon Y.
      Location of stem cells of human hair follicles by clonal analysis.
      ). A number of recent observations have led to the concept that the HF stem cells found in the bulge region are the ultimate precursors of the IF epidermis under homeostatic conditions, and further that the IF epidermis regenerating cells can be viewed as TA cells (
      • Lavker R.M.
      • Sun T-T.
      Epidermal stem cells: Properties, markers, and location.
      ;
      • Taylor 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.
      ). Specifically, a number of exquisite in vivo studies tracking the migration of β-galactosidase- or BrDU-marked cells out of the bulge region have demonstrated the unequivocal role of these stem cells to give rise to all HF cell lineages, the sebaceous gland, and importantly to those regions of the IF epidermis adjacent to the HF. Whereas the contribution of follicle-derived cells to IF epidermis was seen after implanting marked cells into wounded skin (
      • Oshima H.
      • Rochat A.
      • Kedzia C.
      • Kobayashi K.
      • Barrandon Y.
      Morphogenesis and renewal of hair follicles from adult multipotent stem cells.
      ) or in neonatal skin (
      • Taylor 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.
      ), the work of
      • Ghazizadeh S.
      • Taichman L.B.
      Multiple classes of stem cells in cutaneous epithelium: A lineage analysis of adult mouse skin.
      clearly addressed the contribution of adult follicular cells to the IF epidermis under physiological conditions, i.e., up to 33-wk post-wounding. The latter study concurred that IF epidermal cells adjacent to HF originated from follicular stem cells. But importantly, self-sustaining units of epidermal cells not associated with HF were consistently observed (
      • Ghazizadeh S.
      • Taichman L.B.
      Multiple classes of stem cells in cutaneous epithelium: A lineage analysis of adult mouse skin.
      ). Collectively, these studies demonstrate that the HF is an important reservoir for emergency repopulation of the IF epidermis following wounding and in the neonate as suggested previously (
      • Eisen A.Z.
      • Holyoke J.B.
      • Lobitz W.C.
      Responses of the superficial portion of the human pilosebaceous apparatus to controlled injury.
      ;
      • Krawczyk W.S.
      A pattern of epidermal migration during wound healing.
      ;
      • Al-Barwari S.E.
      • Potten C.S.
      Regeneration and dose response characteristics of irradiated mouse dorsal epidermal cells.
      ;
      • Cotsarelis 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.
      ). Further, parts of the IF epidermis are clearly derived from the HF routinely, but there is a class of IF epidermal stem cells that are capable of self-renewal throughout the lifetime of the animal, independent of the HF.
      Another confounding factor in the HF versus IF stem cell debate has been the observation that some of the slowest cycling cells, i.e., LRC, are readily found in the bulge region and not in the IF epidermis. It is important to bear in mind that IF stem cells exist as single cells at the center of EPUs and it is therefore harder to locate these single cells in a sea of unlabelled cells in randomly cut sections. In contrast, the follicular stem cells exist in a geographically distinct site in clusters of slowly cycling cells. Further, in common with the hemopoietic system (
      • Bradford G.B.
      • Williams B.
      • Rossi R.
      • Bertoncello I.
      Quiescence, cycling, and turnover in the primitive hematopoietic stem cell compartment.
      ), it is critical to note that the epithelial stem cell compartment is a dynamic population of proliferative cells that cycle at a reasonable rate, although clearly less frequently than TA cells. Thus, the numbers of LRC found in any tissue at a given time is likely to reflect the number of divisions it has undergone since being labelled, the turnover rate of that tissue, and therefore the demands on the LRC for cell regeneration. That is to say, DNA label retention is a dynamic property. A study by Bickenbach quantitating the steady decline in numbers of LRC at different rates in different epithelia over time elegantly illustrates this point (
      • Bickenbach J.R.
      • McCutecheon J.
      • MacKenzie I.C.
      Rate of loss of tritiated thymidine label in basal cells in mouse epithelial tissue.
      ). Thus, LRC have to be evaluated in the context of the time over which label retention is estimated. The difficulty in finding LRC in the IF epidermis after 8–10-wk post-labelling then, can most likely be attributed to the rate at which these cells have to divide to maintain the epidermis, resulting in a more rapid loss of label and that they are much harder to find. So, how slowly does a cell have to cycle to qualify as a stem cell? The point is that one cannot compete for stem cell status based on the rate at which a DNA label is retained because this merely reflects the cellular kinetics of a particular epithelium. In fact, if a cell retains its DNA label for a prolonged period of time, such as the single LRC found in the bulge region a year post-labelling (
      • Morris R.J.
      • Potten C.S.
      Highly persistent label-retaining cells in the hair follicles of mice and their fate following induction of anagen.
      ), one could argue that it has contributed little to cell regeneration.

      In Vitro Approaches to Defining Epithelial Stem Cells

      It can be confidently stated that all attempts to study epidermal stem cells in vitro are based on very specific assumptions about the intrinsic properties of stem cells made by the many investigators who have dared to venture into this minefield of experimental biology. But it should also be noted that the context in which stem cells are assayed is an equally, if not more important determinant of the assay readout. Not only does the act of removing candidate stem cells from their microenvironmental niche potentially alter their behavior, but the failure of surrogate assays to recapitulate the conditions under which stem cells grow and function in situ exerts artificial proliferative and differentiative pressures on these cells which will irreversibly alter their properties and influence their fate (
      • Lavker R.M.
      • Sun T-T.
      Epidermal stem cells: Properties, markers, and location.
      ). This philosophy is best encapsulated in the following statement made by Potten: “Stem cells are defined by virtue of their functional attributes. This immediately imposes difficulties since in order to identify whether a cell is a stem cell or not its function has to be tested. This inevitably demands that the cell must be manipulated experimentally, which may alter its properties” (
      • Potten C.S.
      • Loeffler M.
      Stem cells: Attributes, cycles, spirals, pitfalls and uncertainties. Lessons for and from the crypt.
      ). In support of this,
      • Morris R.J.
      • Potten C.S.
      Slowly-cycling (label-retaining) epidermal cells behave like clonogenic stem cells in vitro.
      have elegantly demonstrated that murine epidermal 3H-Tdr LRC (putative KSC) can be recruited into proliferation upon placing them into culture. Thus clearly, stem cells do not remain quiescent when cultured; however, an unmistakable assumption in some of the pioneering work in the epidermal stem cell area is the notion that other subpopulations of keratinocyte progenitors, namely TA cells and differentiating cells, exhibit similar restricted proliferative potential when explanted in vitro as they do in vivo. For example,
      • Barrandon Y.
      • Green H.
      Three clonal types of keratinocytes with different capacities for multiplication.
      placed individual human keratinocytes in culture and could subsequently identify three classes of clonal cells: holoclones, which gave rise to large, rapidly growing colonies; paraclones, which formed small colonies that underwent terminal differentiation after a few cell divisions; and meroclones, which gave rise to a mixture of growing and abortive colonies. These workers concluded that holoclones were likely to be founded by KSC and paraclones by TA cells. Importantly, this interpretation was based on the assumptions that: (a) TA cells when explanted into culture would not be able to proliferate extensively in vitro and (b) analogous to the hemopoietic colonies with high proliferative potential, the large keratinocyte colonies (holoclones) must be founded by stem cells. It remains unclear exactly which class of keratinocyte progenitor is represented by meroclones. In favor of the interpretation that holoclones are at least enriched for stem cells is the work from De Luca and Pellegrini showing that holoclones are capable of extensive long-term proliferation measured over many weeks in culture both in skin (
      • Mathor M.B.
      • Ferrari G.
      • Dellambra M.
      • Cilli M.
      • Mavilio F.
      • Cancedda R.
      • De Luca M.
      Clonal analysis of stably transduced human epidermal stem cells in culture.
      ) and in the cornea (
      • Pellegrini G.
      • Golisano O.
      • Paterna P.
      • et al.
      Location and clonal analysis of stem cells and their differentiated progeny in the human ocular surface.
      ). Importantly, the limbal region of the cornea known to be enriched for LRC gives rise to holoclones, whereas the central cornea does not (
      • Pellegrini G.
      • Golisano O.
      • Paterna P.
      • et al.
      Location and clonal analysis of stem cells and their differentiated progeny in the human ocular surface.
      ).
      An equally reasonable interpretation of these observations could be that holoclones arise from both stem and TA cells, meroclones arise from more committed TA cells and that paraclones arise from keratinocytes that have initiated differentiation. Alternately, it could be argued that dispersing keratinocytes and placing them in culture perturbs the system so greatly that their behavior cannot reasonably be equated to that of any specific keratinocyte progenitor in vivo under homeostatic conditions, or as stated by
      • Barrandon Y.
      • Green H.
      Three clonal types of keratinocytes with different capacities for multiplication.
      , “the relation, if any, between the clonal types we have described and the multiple cell types defined by thymidine labelling kinetics remains to be clarified”.

      Identification of Markers for the Detection of KSC

      Watt and colleagues described the first attempt to prospectively define and isolate IF epidermal stem cells using cell surface markers and FACS techniques, an important prerequisite for further characterization of these cells that had best been studied in situ until then.
      • Jones P.H.
      • Watt F.M.
      Separation of human epidermal stem cells from transit amplifying cells on the basis of difference in integrin function and expression.
      demonstrated that cultured human foreskin keratinocytes expressing high levels of β1 integrin had a higher colony forming efficiency (CFE) determined over 2 wk in culture, whereas those keratinocytes with lower levels of β1 integrin exhibited poorer CFE. These studies were extended to freshly isolated human neonatal keratinocytes, which demonstrated that cells expressing high levels of β1 integrin could generate an epithelium when grafted onto mice (
      • Jones P.H.
      • Harper S.
      • Watt F.M.
      Stem cell patterning and fate in human epidermis.
      ), suggesting that this subpopulation of basal epidermal cells is enriched for KSC. These workers noted that one class of keratinocytes expressing low levels of β1 integrin divided one to five times before undergoing terminal differentiation in vitro and that this appeared to correspond with the TA population described in vivo by cell kinetic studies (
      • Jones P.H.
      • Watt F.M.
      Separation of human epidermal stem cells from transit amplifying cells on the basis of difference in integrin function and expression.
      ). This assumption once again underscores the reasoning that whereas epidermal stem cells are recruited to proliferate when cultured, TA cells continue to exhibit limited proliferation in vitro. Recent experiments in our laboratory indicate that TA cells (our definitions of which are described in detail below) and even keratinocytes that have downregulated integrin expression and begun to express K10 are capable of extensive proliferation in vitro (
      • Li A.
      • Simmons P.J.
      • Kaur P.
      Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype.
      ) and can form an epidermis in an organotypic culture given the right microenvironment (
      • Li A.
      • Pouliot N.
      • Redvers R.
      • Kaur P.
      Extensive tissue-regenerative capacity of neonatal human keratinocyte stem cells and their progeny.
      ).
      Further work from Watt and colleagues has sought to localize the epidermal stem cells in vivo using β1 integrin as a marker (
      • Jones P.H.
      • Harper S.
      • Watt F.M.
      Stem cell patterning and fate in human epidermis.
      ;
      • Jensen 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.
      ). Similarly, other investigators have attempted to show that K15 and K19 may be markers of HF stem cells (
      • Michel M.
      • Torok N.
      • Godbout M-J.
      • Lussier M.
      • Gaudreau P.
      • Royal A.
      • Germain L.
      Keratin 19 as a biochemical marker of skin stem cells in vivo and in vitro: Keratin 19 expressing cells are differentially localized in function of anatomic sites, and their number varies with donor age and culture stage.
      ;
      • Lyle S.
      • Christofidou-Solomidou M.
      • Liu Y.
      • Elder D.E.
      • Albelda S.
      • Cotsarelis G.
      The C8/144B monoclonal antibody recognizes cytokeratin 15 and defines the location of human hair follicle stem cells.
      ), and that p63 may be a stem cell marker for the IF epidermis (
      • Pellegrini G.
      • Dellambra E.
      • Golisano O.
      • et al.
      p63 identifies keratinocyte stem cells.
      ). In this context, it is worth discussing the factors that need to be borne in mind when assigning a stem cell marker. Most notably, the incidence and location of stem cells has been well characterized by many groups who have described LRC. Thus, a correlation with expected numbers of stem cells and expected sites of stem cell location should be important validating criteria. The epitopes recognized by a particular antibody may also influence the observed staining pattern, as well as the accessibility of epitopes particularly when looking at cell surface antigens in situ. Without exception, although many of the purported stem cell markers react with cells in the appropriate location, they exhibit too broad a specificity to be considered as stem cell markers. Estimates of epidermal stem cell numbers from in vivo studies indicate that they constitute between 1% and 10% of the basal layer depending on the methodology used (
      • Potten C.S.
      • Hendry J.H.
      Clonogenic cells and stem cells in epidermis.
      ;
      • MacKenzie I.C.
      • Bickenbach J.R.
      Localization of label-retaining cells in mouse epithelia.
      ;
      • Morris R.J.
      • Fischer S.M.
      • Slaga T.J.
      Evidence that the centrally and peripherally located cells in the murine epidermal proliferative unit are two distinct populations.
      ;
      • Bickenbach J.R.
      • McCutecheon J.
      • MacKenzie I.C.
      Rate of loss of tritiated thymidine label in basal cells in mouse epithelial tissue.
      ;
      • Morris R.J.
      • Potten C.S.
      Slowly-cycling (label-retaining) epidermal cells behave like clonogenic stem cells in vitro.
      ). Given their observation that approximately 40% of the basal layer in human foreskin exhibits high levels of β1 integrin in vivo,
      • Jones P.H.
      • Harper S.
      • Watt F.M.
      Stem cell patterning and fate in human epidermis.
      concluded that not all basal keratinocytes with this phenotype can be KSC, although they postulate that stem cells of the foreskin may exist as clusters of β1 integrin bright cells (
      • Jensen 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.
      ). It is possible that the incidence of stem cells may be greater in a neonatal epithelium, or that the spatial arrangement in human foreskin is different from that in murine epidermal tissues.

      Combining In Vitro Assays and In Vivo Approaches to Identify and Isolate Epidermal Stem Cells for Further Characterization

      Recent work from our laboratory has sought to devise ways to identify and isolate epidermal KSC based on multiple criteria combining what is known from cell kinetic studies in vivo with in vitro assays adopting the FACS approaches utilized by hemopoietic stem cell biologists. We have sought to demonstrate that we have selected epidermal stem cells based on the incidence of the population, cell size, nuclear to cytoplasmic ratio, in vivo cell kinetic properties, cell cycle analysis, long-term proliferative output (measured over 80–90 d), localization to an accepted niche in vivo, i.e., the HF bulge region, and lack of differentiation markers. We have also ascribed great importance to working with freshly isolated primary epidermal cells suspecting that culturing the cells first would lead to a change in the very markers that we would like to assign to stem cells in vivo. For example, integrins are upregulated during wound healing in vivo (
      • Hertle M.D.
      • Kubler M.D.
      • Leigh I.M.
      • Watt F.M.
      Aberrant integrin expression during epidermal wound healing and in psoriatic epidermis.
      ) and therefore may well be altered once keratinocytes are dispersed and placed in culture. We elected to use the α6 integrin and the 10G7 antigen now known to be the transferrin receptor or CD71 as cell surface markers to fractionate primary epidermal cells initially from neonatal foreskin epidermis (Figure 1). High integrin expression serves well as a marker for basal cells, permitting their separation from more committed cells that have initiated their program of differentiation and suprabasal cells that inevitably form a part of the isolated primary epidermal preparations. We also selected α6 rather than β1 integrin because the latter is expressed on non-epithelial cells that are also present in the primary cell preparations. Although it was not evident to us at the time, CD71 was a good choice to separate actively cycling TA cells from quiescent stem cells, given that it is highly expressed by many proliferating cells (
      • Trowbridge I.S.
      • Omary M.B.
      Human cell surface glycoprotein related to cell proliferation is the receptor for transferrin.
      ). This is further confirmed by studies in the hemopoietic system where the actively proliferating committed progenitors (CD34+CD71high) can be distinguished from the stem cell compartment (CD34+CD71dim) on the basis of CD71 expression (
      • Lansdorp P.M.
      • Dragowska W.
      Maintenance of hematopoiesis in serum-free bone marrow cultures involves sequential recruitment of quiescent progenitors.
      ). Consistent with this work, we were able to distinguish a minor population of basal keratinocytes that expressed high levels of α6 and low levels of CD71 (α6briCD71dim; Figure 1) in human epidermis that fulfilled three important stem cell criteria predicted from in vitro cell kinetic studies: (a) low incidence; (b) relative quiescence determined by cell cycle analysis; and (c) greatest long-term proliferative capacity in vitro (
      • Li A.
      • Simmons P.J.
      • Kaur P.
      Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype.
      ). In contrast, the remaining α6bri cells which expressed high levels of CD71 (α6briCD71bri; Figure 1) fulfilled criteria predicted of TA cells: (a) high incidence in the basal layer; (b) actively cycling; and (c) lower long-term proliferative potential relative to the α6briCD71dim cells. A third population of primary human keratinocytes that expressed low levels of α6 integrin termed α6dim, was also consistently observed in cell preparations (Figure 1). These cells probably represent a mixture of basal and suprabasal cells that exhibited a limited proliferative potential in vitro, expressed K10 (
      • Li A.
      • Simmons P.J.
      • Kaur P.
      Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype.
      ) and involucrin (
      • Kaur P.
      • Li A.
      Adhesive properties of human basal epidermal cells: An analysis of keratinocyte stem cells, transit amplifying cells, and postmitotic differentiating cells.
      ), which we termed PMD cells based on the relatively low number of cells in this population in S phase (
      • Li A.
      • Simmons P.J.
      • Kaur P.
      Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype.
      ). We expected that the candidate stem cell population would be maintained in culture longer than the TA and PMD cells given its inherent proliferative potential, but observed that the lifespan of all three populations was comparable. We reasoned that critical growth factors or agents required for self-renewal were missing from the culture conditions, resulting in terminal differentiation of the stem cells. This is consistent with the notion that stem cells retain their characteristic features in vivo due to the occupation of a “stem cell niche” or microenvironment (
      • Schofield R.
      The relationship between the spleen colony-forming cell and the haemopoietic stem cell.
      ). Thus, removal from that niche may well result in differentiation in vitro.
      Figure thumbnail gr1
      Figure 1Phenotype assigned to keratinocyte stem cells (KSC), transit-amplifying (TA) cells and early differentiating cells (D) derived from primary neonatal human foreskin epidermis on the basis of two cell surface markers: α6 integrin and transferrin receptor/CD71. Functional assays utilized to distinguish these three phenotypically discrete compartments are summarized in .
      Further verification that the α6briCD71dim cells were likely to be epidermal stem cells was obtained by showing an enrichment for LRC within this phenotypic fraction, and conversely localizing pulse-labelled cells to the α6briCD71bri phenotypic fraction (
      • Tani H.
      • Morris R.J.
      • Kaur P.
      Enrichment for murine keratinocyte stem cells based on cell surface phenotype.
      ), isolated from murine dorsal epidermis. This direct correlation between phenotypically distinct populations, their distinct kinetic turnover rates in vivo and their long-term proliferative output in vitro represents the first attempt to reconcile stem cells identified in situ with their behavior in culture. Further confirmation came from determining the smaller size and higher nuclear: cytoplasmic ratio of the α6briCD71dim cells compared with the α6briCD71bri cells. In addition, we were able to demonstrate that the HF bulge region known to contain LRC was CD71dim whilst expressing high levels of α6 confirming that the phenotype we had ascribed to epidermal stem cells on isolated cells could be verified in vivo. These data have been confirmed by recent studies from the lab of Watt and Fuchs localizing slow-cycling cells to this region in whole mounts (
      • Braun K.M.
      • Niemann C.
      • Jensen U.B.
      • Sundberg J.P.
      • Silva-Vargas V.
      • Watt F.M.
      Manipulation of stem cell proliferation and lineage commitment: Visualisation of label-retaining cells in wholemounts of mouse epidermis.
      ) expressing low levels of CD71 (
      • Tumbar T.
      • Guasch G.
      • Greco V.
      • Blanpain C.
      • Lowry W.E.
      • Rendl M.
      • Fuchs E.
      Defining the epithelial stem cell niche.
      ). Our results indicate that stem cells of both the IF and HF epidermis can be identified by the same phenotype given our study in non-hairy human skin, although we have yet to demonstrate the presence of CD71dim cells in the IF epidermis. This is a demanding technical challenge given the spatial arrangement of these single isolated cells in the epidermis that lowers the probability of detecting them; however, consistent with this an abundance of CD71bri cells are readily detected in the epidermis (
      • Tani H.
      • Morris R.J.
      • Kaur P.
      Enrichment for murine keratinocyte stem cells based on cell surface phenotype.
      ).
      The multiple stem cell characteristics described by us for both murine and human epidermal α6briCD71dim cells summarized in Table I and Table II represent the broadest definition encompassed by any candidate epidermal stem cell population, although sustained tissue regenerative capacity both in vitro and in vivo needs to be examined, an area that we are actively pursuing. In addition to the criteria we have published, we have also sought to determine whether holoclones (large colony-forming cells) can be prospectively identified within the α6briCD71dim fraction. In several replicate experiments, comparing the size of colonies formed by α6briCD71dim and α6briCD71bri keratinocytes there was no enrichment for larger colonies in the KSC fraction—rather different size colonies were obtained in both fractions. These data suggest that holoclones may arise from stem cells and TA cells; however, it is important to bear in mind that in our experiments, keratinocytes have been subjected to extensive manipulation (FACS) prior to assessing their colony-forming ability. Thus a direct comparison with Barrandon's work is difficult.
      Table IFeatures of human basal keratinocytes derived from neonatal foreskin fractionated according to their cell surface phenotype
      α6briCD71dim KSCα6briCD71bri TAα6dim PMD
      Short-term CFE (2 weeks)++++++++
      Long-term proliferative output (12 weeks)+++++++
      Cell cycle analysis (% cells in S+G2M)LowHighLow
      Incidence4%–7%MajorityVariable
      Keratin 14 expression++++++++
      Keratin 10 expression--++
      Involucrin expression--++
      KSC, keratinocyte stem cell; TA, transient amplifying; PMD, post-mitotic differentiation.
      Table IIFeatures of murine basal keratinocytes derived from dorsal skin fractionated according to their cell surface phenotype
      α6briCD71dim KSCα6briCD71bri TA
      3H-Tdr label-retaining cells71.95%12.6%
      3H-Tdr pulse labelled cells16.24%69.73%
      Cell cycle analysis (% cells in S+G2M)LowHigh
      Incidence8.1%58.6%
      Nuclear:cytoplasmic ratioHighLow
      Cell sizeSmallBigger
      Localized to hair follicle bulge region+-
      Keratin 14 expression++++++
      Keratin 10 expression--
      KSC, keratinocyte stem cell; TA, transient amplifying.

      Limitations of the α6briCD71dim Phenotype as a Marker for KSC

      Whereas the α6briCD71dim phenotype is a reliable means of isolating epidermal stem cells from fresh primary skin tissue, it is unsuitable for studying the behavior of stem cells in situ during carcinogenesis, wound healing, or in culture. As discussed before, dispersing keratinocytes from their epithelial sheets in vivo and placing them in culture is akin to wounding the skin. The result of placing keratinocytes in culture even for a single passage is an upregulation of α6 integrin and CD71. Thus, irrespective of the original phenotype of isolated epidermal cells, we have observed upregulation of both the α6 integrin and the transferrin receptor/CD71 soon after placing them in culture. Specifically, cells of the phenotype α6briCD71dim, α6briCD71bri, or α6dim gain the TA phenotype (α6briCD71bri) within one passage in vitro (Figure 2). This is consistent with in vivo studies showing upregulation of integrins in wound healing (
      • Hertle M.D.
      • Kubler M.D.
      • Leigh I.M.
      • Watt F.M.
      Aberrant integrin expression during epidermal wound healing and in psoriatic epidermis.
      ), and upregulation of the transferrin receptor by cultured keratinocytes (
      • Kaur P.
      • Paton S.
      • Furze J.
      • Wrin J.
      • Olsen S.
      • Danks J.
      • Scurry J.
      Identification of a cell surface protein with a role in stimulating human keratinocyte proliferation, expressed during development and carcinogenesis.
      ). The expression of CD71 can be downregulated in culture as keratinocytes become confluent and stop proliferating (
      • Kaur P.
      • Paton S.
      • Furze J.
      • Wrin J.
      • Olsen S.
      • Danks J.
      • Scurry J.
      Identification of a cell surface protein with a role in stimulating human keratinocyte proliferation, expressed during development and carcinogenesis.
      ). Thus, the presence of CD71dim cells in cultures is not a reliable indicator of stem cells in vitro, but could indicate differentiation. α6briCD71dim stem cells also become activated to proliferate in culture and adopt a TA phenotype (Figure 2), and therefore cannot be distinguished from TA cells. Similarly, CD71 expression is upregulated on epithelial cells in vivo during development and in transformed cells in both benign and malignant tumors in vivo (
      • Kaur P.
      • Paton S.
      • Furze J.
      • Wrin J.
      • Olsen S.
      • Danks J.
      • Scurry J.
      Identification of a cell surface protein with a role in stimulating human keratinocyte proliferation, expressed during development and carcinogenesis.
      ). Thus, given that CD71 expression is high on proliferating stem cells and on TA cells, it is impossible to phenotypically distinguish TA cells from stem cells in many biologically interesting conditions which result in hyperproliferation (cancer, wound healing, in vitro). Thus, the identification of new markers that recognize KSC which are not altered by culture is important for their study in these particular situations, if indeed such markers exist.
      Figure thumbnail gr2
      Figure 2Keratinocyte stem cells (KSC) do not maintain the α6briCD71dim phenotype in culture. Primary human neonatal foreskin keratinocytes (panel A) were labelled with antibodies to α6 integrin (x-axis; fluorescein isothiocyanate) and CD71 (y-axis; phycoerthrin) and subjected to fluorescence activated cell sorting (FACS) to obtain KSC (α6briCD71dim), transit-amplifying (TA) (α6briCD71bri), and early differentiating (D) (α6dim) cells. These fractions were placed in culture for a single passage, harvested and their α6/CD71 cell surface phenotype determined by flow cytometry. The data demonstrate that all fractions of primary keratinocytes (KSC: panel E; TA: panel D; D cells: panel C) and unsorted keratinocytes (panel B) upregulate expression of both α6 and CD71 following placement in culture, exhibiting the α6briCD71bri phenotype characteristic of TA cells.

      Can Keratinocyte Stem Cells be Identified by Differential Dye Effluxing Properties?

      The “diminishing stem-ness spiral model” proposed by
      • Potten C.S.
      • Loeffler M.
      Stem cells: Attributes, cycles, spirals, pitfalls and uncertainties. Lessons for and from the crypt.
      suggests a continuum between stem cells and TA cells, accompanied by a gradual loss in self-renewal capacity and increasing probability of differentiation, implying heterogeneity even within the stem cell fraction. In fact, direct evidence for such a hierarchy within the murine hemopoietic stem cell compartment has been provided by recent studies using Hoechst 33342, (Sigma, St. Louis, Missouri) which resolves a “side population” (SP) of hemopoietic progenitors, when fluorescence from this dye is displayed simultaneously at two emission wavelengths: i.e., red and blue (
      • Goodell M.A.
      • Brose K.
      • Paradis G.
      • Conner A.S.
      • Mulligan R.C.
      Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo.
      ). The SP cells represent 0.1% of total bone marrow, exhibit cell surface markers characteristic of stem cells, and are enriched for in vivo reconstitution activity. Importantly, the SP is a subset (∼10%) of the Sca-1+/linneg/low fraction enriched for hemopoietic stem cells. Subsequently, this hemopoietic SP has been fractionated into three subsets of Hoechst-dull/Rhodamine-dull populations (RI, RII, and RIII), which form a hierarchy of successively less quiescent and less potent populations, as determined by their long-term repopulating ability in ablated mice (
      • Bradford G.B.
      • Williams B.
      • Rossi R.
      • Bertoncello I.
      Quiescence, cycling, and turnover in the primitive hematopoietic stem cell compartment.
      ). Importantly, SP populations can be resolved in hemopoietic tissues from human, rhesus, and miniature swine bone marrow (
      • Goodell M.A.
      • Rosenzweig M.
      • Kim H.
      • et al.
      Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species.
      ), and from non-hemopoietic tissues including muscle (
      • Gussoni E.
      • Soneoka Y.
      • Strickland C.D.
      • et al.
      Dystrophin expression in the mdx mouse restored by stem cell transplantation.
      ). These data suggest that this method which appears to rely on the differential ability of stem cells to efflux the Hoechst dye, can be applied to isolate them from various tissues.
      • Dunnwald M.
      • Tomanek-Chalkley A.
      • Alexandrunas D.
      • Fishbaugh J.
      • Bickenbach J.R.
      Isolating a pure population of epidermal stem cells for use in tissue engineering.
      have recently reported a modification of the method for detecting a purified population of murine epidermal stem cells, apparently combining staining with Hoechst 33342 and propidium iodide, with selection for small cells to identify LRC. These data suggest that the epidermis does not contain an SP analogous to those reported in the bone marrow and muscle; however, it is difficult to reconcile these data with that of other laboratories using the Hoechst red versus blue fluorescence emission to define SP cells as the SP descriptor is exclusively defined by the dye specificity of membrane efflux pumps and by the unique spectral characteristics of Hoechst 33342.
      Recent work from our laboratory demonstrates that a Hoechst-dull SP very similar to that reported for hemopoietic and other tissues can be detected in both murine and human epidermis (Figure 3). We have determined that the SP cells isolated from the epidermis are of epithelial origin by cytokeratin staining, and that they are a minor subset of the α6briCD71dim compartment of murine dorsal epithelium (Li, Redvers & Kaur, unpublished data). By analogy with the hemopoietic system, it is likely that the Hoecsht SP of the epidermis are the most primitive stem cells of this tissue. Without functional evidence to support this notion, it remains for the present an attractive hypothesis requiring experimental validation.
      Figure thumbnail gr3
      Figure 3Hoechst 33342 staining reveals an epidermal side population (SP) in adult murine tail keratinocytes. A typical Hoechst red versus blue fluorescence profile of basal keratinocytes harvested from 9-wk mouse tail epidermis (stained with 15 μM Hoechst 33342 for 90 min at 37°C) demonstrating the detection of an SP (boxed region) that typically represent 0.3%–0.5% of total primary keratinocytes isolated from murine adult tail epidermis.

      Tissue Regeneration Assays for Epidermal Stem Cells

      Stem cells are ultimately defined by their ability to sustain life-long production of mature functional tissue cells in the steady state and following tissue injury. Integral to this definition is the ability of the stem cell pool to undergo continuous renewal, regeneration and expansion on demand. The hemopoietic system is arguably the most instructive model for the development of cell-separative strategies and surrogate assays for the prospective isolation and characterization of stem and progenitor cell cohorts of defined potentiality and differentiative capacity.
      The refinement of cell separative strategies for the prospective isolation of hemopoietic stem cells by an iterative process of correlating phenotypic marker expression in target cells co-fractionating with transplantable stem cell activity in vivo, and/or surrogate assay readouts in vitro has revealed that more primitive stem and progenitor cells require multiple cytokines acting in synergy, often in the presence of a supportive stromal microenvironment in order to express their developmental potential in vitro. This notwithstanding, it has become evident that in vitro surrogate assays are imperfect predictive tools for dissecting the stem cell compartment. Significantly, transplantation is the only definitive gold-standard assay which identifies the primogenitive stem cell pool. This process has provided investigators with a hierarchy of assays (Figure 4) that not only resolve stem cells and TA progenitor cells of more restricted potential, but also enable us to discern a hierarchy of engraftable cells with differing proliferative capacity and potentiality within the stem cell compartment which are responsible for long- and short-term engraftment following transplantation.
      Figure thumbnail gr4
      Figure 4Schematic representation of the hierarchical organization of hemopoietic stem and progenitor cell compartments. The hemopoietic system comprises a concatenated series of primitive cells of progressively restricted potentiality and proliferative capacity which can be resolved and characterized using a panel of specific surrogate in vivo and in vitro functional assays (
      • Bertoncello I.
      • Bradford G.B.
      Surrogate assays for hematopoietic stem cell activity.
      ). LTRA, transplantable stem cells with long-term reconstituting ability in vivo; LTCIC, long-term culture initiating cell; CFU-S8 and CFU-S12, colony-forming unit-spleen measured 8 or 12 d post-transplantation; HPP-CFC1 and HPP-CFC2, high-proliferative potential colony-forming cells stimulated by IL1α+IL3+CSF-1 and IL3+CSF-1, respectively; CFU-GEMM, colony-forming unit-granulocyte/erythrocyte/macrophage/megakaryocyte; CFU-GM, colony-forming unit-granulocyte/macrophage; BFU-E, burst-forming unit-erythroid; CFU-E, colony-forming unit-erythroid; LPP-CFC, low proliferative potential colony-forming cell.
      Although much attention has been focussed on clonogenic assays to determine stem cell activity, tissue regeneration from candidate epidermal stem cells isolated prospectively has not been studied extensively, although
      • Jones P.H.
      • Harper S.
      • Watt F.M.
      Stem cell patterning and fate in human epidermis.
      have reported on the ability of β1 integrin bright keratinocytes to reform an epidermis in vivo following transplantation. The underlying assumption in this study was that the ability to regenerate an epidermis was limited to stem-cell-enriched fractions of basal keratinocytes and these workers did not compare the relative tissue regenerative ability of β1bri cells with non-stem-cell-enriched fractions. Recent data from our laboratory indicate a strong need to re-evaluate this assumption and take stock of current experimental approaches utilized to determine stem cell activity. We have found that short-term tissue regeneration in organotypic cultures is a function of both (i) the intrinsic capabilities of the keratinocyte progenitors being assayed and (ii) extrinsic factors in the microenvironment included in the assay such as dermally derived growth factors and extracellular matrix components (
      • Li A.
      • Pouliot N.
      • Redvers R.
      • Kaur P.
      Extensive tissue-regenerative capacity of neonatal human keratinocyte stem cells and their progeny.
      ). Surprisingly, we could demonstrate that the ability to reform an epidermis is not an exclusive property of the epidermal stem cell fraction, but can also be elicited from the TA and even the PMD population which have initiated differentiation, when provided with the right signals. These data highlight the value of comparing all fractions of cells so as to ascertain the suitability of current assay systems to truly distinguish stem cells from their more committed progeny. It further points to the strong regulatory role of the microenvironment in influencing the outcome of the experiment. It has previously been demonstrated by many investigators that transplantation of epidermal cells results in improved keratinocyte growth and differentiation over the organotypic model, and that transplanted human keratinocytes can be maintained in mice for periods of up to a year (
      • Kolodka 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.
      ).
      On the basis of these findings, and informed by the understanding gained from the analysis of the hemopoietic system, we propose that current assays used to evaluate epidermal stem cell potential are inadequate for distinguishing stem cells from their more committed progeny, and that measurement of stem cell activity requires the development of long-term assays that measure sustained epithelial tissue regeneration. In this context, we have chosen to develop an in vivo transplantation model for human keratinocytes that will permit comparative analysis of keratinocyte progenitors including but not restricted to stem cells. This assay has been optimized to permit the analysis of small numbers of primary FACS-isolated human and murine keratinocytes and can also be utilized to study the competitive repopulation ability of specific classes of genetically tagged keratinocyte progenitors to establish the potency of KSC versus TA versus PMD cells (Pouliot et al, submitted).
      In conclusion, given that the field of epidermal stem cell biology is in its infancy, no single parameter is sufficient to designate a particular subset of cells as stem cells—rather the collective attributes of quiescence, low incidence, sustained and actual (as opposed to potential) proliferative output and tissue-regenerative capacity are essential. Thus, vital steps in the complete biological characterization of KSC encompass the ability to isolate these cells, propagate them in vitro and determine their proliferative and tissue-regenerative potential in various in vitro and in vivo assays. The establishment of transplantation assays is critical to understanding the full capability of not just the stem cell compartment, but also their progeny which may well turn out to have greater proliferative and tissue regenerative capacity than previously suspected. In this context,
      • Schneider T.E.
      • Barland C.
      • Alex A.M.
      • et al.
      Measuring stem cell frequency in epidermis: A quantitative in vivo functional assay for long-term repopulating cells.
      have recently reported a transplantation technique that provides an excellent means to assess the competitive repopulation ability of putative KSC. In particular, when we venture collectively into ex vivo isolation and manipulation of epithelial stem cells, it is critical to note that dispersing epithelial cells and placing them into culture is an exercise in wound healing illustrated by dramatic changes in gene expression. In this context, our observations that all classes of basal keratinocytes (KSC, TA, and PMD) exhibit extensive growth potential in vitro (
      • Li A.
      • Simmons P.J.
      • Kaur P.
      Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype.
      ,
      • Li A.
      • Pouliot N.
      • Redvers R.
      • Kaur P.
      Extensive tissue-regenerative capacity of neonatal human keratinocyte stem cells and their progeny.
      ) suggest that keratinocyte progenitors may retain more flexibility to revert to a proliferative state when called upon. In this respect, skin cells may be intrinsically programmed to retain extensive growth capacity in order to fulfill their overriding function to cover the body and provide barrier function.
      The arguments about the dependence of the IF epidermis on HF stem cells are fuelled by the demonstration that HF stem cells can give rise to the former. But it is equally clear that self-renewing stem cells exist in the IF epidermis. Notably, genetically knocking out β-catenin has been shown to impair HF stem cell differentiation, ablating HF formation in these mice (
      • Huelsken J.
      • Vogel R.
      • Erdmann B.
      • Cotsarelis G.
      • Birchmeier W.
      Beta-catenin controls hair follicle morphogenesis and stem cell differentiation in the skin.
      ). Interestingly, the IF epidermis in these mice remains normal suggesting that in the absence of HF, the epidermis is capable of self-renewal. Conversely over-expressing β-catenin in skin gives rise to de novo HF formation (
      • Gat U.
      • DasGupta R.
      • Degenstein L.
      • Fuchs E.
      De novo hair follicle morphogenesis and hair tumors in mice expressing a truncated beta-catenin in skin.
      ) providing insight into the regulation of fate specification of epidermal progenitors. It is quite possible that the biology of skin has evolved such that barrier function is more important than hair growth—that is to say, we can do without our hair, but not our skin.
      The microenvironment in which KSC reside needs to be defined and the role of specific elements: dermal cells, growth factors, extracellular matrix components, etc., in maintaining stem cells in an undifferentiated state, yet allowing their neighbors to progress through multiple rounds of proliferation and terminal differentiation, is a major unanswered question in stem cell biology.

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