25 years of epidermal stem cell research.

This is a chronicle of concepts in the field of epidermal stem cell biology and a historic look at their development over time. The past 25 years have seen the evolution of epidermal stem cell science, from first fundamental studies to a sophisticated science. The study of epithelial stem cell biology was aided by the ability to visualize the distribution of stem cells and their progeny through lineage analysis studies. The excellent progress we have made in understanding epidermal stem cell biology is discussed in this article. The challenges we still face in understanding epidermal stem cells include defining molecular markers for stem and progenitor sub-populations, determining the locations and contributions of the different stem cell niches, and mapping regulatory pathways of epidermal stem cell proliferation and differentiation. However, our rapidly evolving understanding of epidermal stem cells has many potential uses that promise to translate into improved patient therapy.

INTRODUCTION

The last 25 years (1985 – present) have been the time during which epidermal stem cell biology evolved from first fundamental studies to a sophisticated science. The last 25 years have seen an exponential growth in the field of epidermal stem cells. A literature search of “epidermis” and “stem cell” revealed 0 to 5 articles per year in the years from 1975 to 1985, followed by a rapid increase to over 150 articles per year for the last 4 years (Figure 1). In the 60-70’s careful study of epidermal morphology and of cell kinetics gave insight into epidermal proliferation units and of epidermal cell kinetics. This laid a groundwork for our understanding of epidermal stem cells. From the 1980’s to the present our understanding of cutaneous stem cell biology has undergone tremendous progress due to the large body of work that has been conducted, enhanced by knowledge gained from other tissues. This timeline makes the last 25 years a perfect interval in which to journey through and reflect on how our concepts of epidermal stem cells have evolved over time. In Figure 2 approximations of the occurrence of evolving concepts and scientific evidence for these concepts are illustrated on a timeline.

FIGURE 1

There was an exponential increase in epidermal stem cell publications from 1985 to 2010.

FIGURE 2

While the exponential growth is impressive, it can be seen in Figure 3 how growth in the science of epidermal stem cells began approximately 20 years after that in hematopoiesis. It can also be seen that, due to the size of our specialty, the numbers of papers and presumably the volume of work/experiments conducted is of an order of magnitude less than hematopoietic stem cells. However, the bright side is that, following in these steps, we have learned from concepts and knowledge already gained and progressed at an accelerated pace toward a more thorough understanding of epidermal stem cell biology and the ability to use epidermal stem cells for clinical advantage. Furthermore, other fields can learn from the epidermal stem cell field, because skin stem cell work has focused on lineage analysis in tissue sections, allowing visualization of stem cells and their immediate progeny, something bone marrow and blood do not lend themselves to easily.

FIGURE 3

The field of epidermal stem cell research was born 20 years after that of hematopoietic stem cell research.

For this article I have examined the progress of stem cell research from a historical perspective, looking at the evolution of concepts in epidermal stem cell biology over time. In this quest, given the size of the literature and the large amount of progress, I have surely omitted excellent and concept-changing work by many of my epidermal stem cell biologist colleagues, and for this I apologize before I begin.

Till and McCulloch: Hematopoiesis leads the way (1961- )

In 1961 Till and McCulloch published a seminal paper, that was published in its original form again this year, providing a quantitative method for analyzing hematopoietic cells capable of continued proliferation in vivo and providing a singularly important observation; that single cells could give rise to all hematopoietic lineages in vivo (Till and McCulloch, 1961; Till and McCulloch, 2011; Weissman, 2011). Supralethally irradiated mice were injected with nucleated bone marrow cells and the spleen colony forming units (CFU-S) quantified. The number of macroscopic spleen colonies was directly proportional to the number of cells injected and the colonies were noted to be heterogeneous in size. Further conceptually important experiments studies showed that the clones were heterogeneous in their self-renewal ability (Siminovitch et al, 1963). This was the beginning of quantitative assessment of stem cell proliferation in vivo and the quest for methods to study defining characteristics of stem cells – believed to be long term proliferation in vivo and self-renewal. Thus, active work in the field of hematopoietic stem cells began almost 20 years ahead of active epidermal stem cell research as can be seen in Figure 3.

Colony formation in vivo following skin irradiation (1967- )

The hematopoietic colony forming unit assay from Till and McCulloch determined a dose-survival relationship for injected hematopoietic cells. This was followed by the Withers colony forming assay after skin irradiation, in 1967 (Withers, 1967a; Withers, 1967b). This assay used Poisson limiting dilution statistics to determine the frequency of cells capable of initiating colonies following skin irradiation. Following irradiation small nodules of keratinocytes could be observed by 10-20 days and the colony forming assay allowed determination of the dose-survival relationship (surviving cells per unit area at different doses of X-rays). This demonstrated the survival of a subset of cells with clonogenic ability post-radiation.

The epidermal proliferation unit – Epidermis is arranged in columns with a precise stacking arrangement and a clonogenic central basal cell (1969-)

The superficial corneocytes, suprabasal cells and basal cells together can be regarded as one epidermal proliferation unit and cells at the base of a cell column produce the more superficial cells. Each epidermal proliferation unit had, on average, 10.6 basal nuclei beneath a column of cells. There are usually 10 - 11 basal cells, 3 flattened nucleated differentiating suprabasal cells, and 5 - 7 cornified cells making a total of 18 - 21 cells spanning on the order of 0.05 millimeter (Mackenzie, 1969)(Mackenzie, 1970). Thus, in the epidermis of many body regions of several species there is a basic structural unit composed of approximately 20 cells.

Potten and Hendry reviewed data from three studies of keratinocyte survival curves post radiation (Potten and Hendry, 1973). The data showed that the average number of clonogenic cells in murine epidermis ranged from 309 to 1107 clonogenic cells per mm2. Meanwhile, the number of basal cells in a given area has been reported as 1.4 to 2 × 104 basal cells/mm2 (Potten and Hendry, 1973). From these data it can be calculated that 1.5% to 14% of basal cells are clonogenic post-radiation. Later, using [3H]thymidine labeling and a one month chase period it was shown that cell division is restricted to the basal cells and the central one may cycle at a slightly slower rate (Morris et al, 1985). In subsequent years, with the success of keratinocyte culture methods, studies of colony-forming efficiency in vitro confirmed that the frequency of clonogenic keratinocytes in culture also ranges from 2 – 8% in mouse (Morris et al, 1988; Bickenbach and Chism, 1998; Popova and Morris, 2004). Thus, there is good evidence that epidermal proliferation units have a clonogenic cell at their origin.

Morphologic definition of these proliferative units in epidermis greatly aided our understanding of cell replacement in interfollicular epidermis, while the issue of the contributions of the hair follicle stem cells to interfollicular epidermis was only to be addressed at a later date.

Lajtha: A hierarchy of proliferative epithelial cells (1979)

A thoughtful outline of concepts of stem cell proliferation and differentiation succinctly summarized, including the concept of a hierarchy of proliferative epithelial cells, with only a small fraction of them acting as stem cells. This work remains much read and regularly cited today (Lajtha, 1979).

Label retaining cells as stem cells in murine epidermis (1981- )

While human studies of epidermal stem cells have been largely restricted to in vitro colony studies, studies in mice have assayed label-retaining cells as stem cells. Mice were continuously or pulse labeled, with either tritiated thymidine or 5-bromo-2’-deoxyuridine (BrdU), to generate labeled cells and then the kinetics of accumulation/loss of label examined (Morris et al, 1985; Potten, 1974; Bickenbach, 1981; Bickenbach et al, 1986; Potten and Morris, 1988). These studies relied on the concept that an epidermal stem cell might be identified by a slow cell-cycle duration. Injections of nucleotide analogues were given repeatedly at a time when the tissue is hyperproliferative, so that all dividing cells were labeled. Weeks to months later, cells that rarely divide and therefore retain their label were considered stem cells (Morris et al, 1985; Bickenbach, 1981; Bickenbach et al, 1986). Slower cycling cells, in the center of epidermal proliferation units, could be identified using label retaining ability, in contrast to the more rapidly proliferating cells located in the peripheral region of the epidermal proliferation unit that do not retain DNA label (Bickenbach, 1981). The number of labeled mitoses in the epidermis examined over time indicated the existence of at least two distinct populations of cells with different cell cycle durations, as well as a post-mitotic population (Potten and Morris, 1988). Later (1990), it was shown that in murine hair follicle, label retaining cells were confined to the bulge region (Cotsarelis et al, 1990).

The issues with this method include the concern that since very few stem cells are dividing at any time only a small subset of stem cells would be labeled and the most primitive stem cells may remain unlabeled. Another issue was that after damage caused by incorporation of a nucleotide analogue the cells may not be capable of dividing. This however, does not appear to be the case, as label-retaining cells have been stimulated to divide (Bickenbach, 1981)(Braun et al, 2003). Also in more recent studies fluorescently labeled slow-cycling cells were produced in a keratinocytes. This method was used to purify the label-retaining cells that mark the skin stem cell niche and to follow the progeny of label retaining cells in the murine bulge (Tumbar, 2004). For excellent reviews of label retaining epidermal cells see Braun and Watt (Braun and Watt, 2004) and Fuchs (Fuchs, 2009).

Clonal analysis of human epidermal stem cells establishes a hierarchy of proliferative keratinocytes (1987- )

Study of human epidermal stem cells has been largely based on studies in short-term in vitro culture. In an effort to identify cells with a high proliferative capacity in human epidermis Barrandon and Green studied the self-renewal ability of human epidermal cells in replating assays (Barrandon and Green, 1987). For clonal analysis of a population of keratinocytes, 50-100 cells were individually plated. More than 50% of cultured human keratinocytes formed colonies. Six days later colonies were resuspended and each plated into a 100 mm Petri dish. Different cell types founded different types of colonies in secondary culture (Barrandon and Green, 1987). Cells that form mostly large smooth colonies with less than 5% of small abortive colonies are termed holoclones. Paraclones are terminally differentiating cells that form small and abortive colonies. Cells intermediate between stem and differentiated cells form meroclones, intermediate in appearance and reproductive capacity. In the studies of Barrandon and Green (Barrandon and Green, 1987) cultured keratinocytes produced 28% holoclones, 49% meroclones, and 23% paraclones. It is believed that the holoclones are stem cells and that the paraclones are transit amplifying cells (Barrandon and Green, 1987), thereby suggesting a hierarchy of epidermal stem cells beginning with an epidermal stem cell which gives rise to a continuum of cell populations with progressively diminishing capacity to proliferate and self-renew. These studies provided a new way to examine for keratinocytes capable of self-renewal in vitro.

Isolation of a nearly pure population of murine hematopoietic stem cells (1988- )

Mouse bone marrow hematopoietic stem cells were isolated with the use of a variety of phenotypic markers. Thirty of these cells were sufficient to save 50 percent of lethally irradiated mice, and to reconstitute all blood cell types in the survivors (Spangrude et al, 1988; Weissman, 2002). Being able to obtain a nearly pure population of stem cells was key to more rapid progress in the understanding of hematopoietic stem cell self-renewal and differentiation.

The bulge, not the bulb, is the site of hair follicle stem cells in the mouse (1990- )

It was previously believed that hair follicle stem cells resided in the bulbar area of hair follicles. However, studies in the 60’s had shown that after surgical removal of the matrix/hair bulb an entire hair follicle could still be regenerated (Oliver, 1966a; Oliver, 1966b). While the bulbar region of the hair follicle was known to contain a pool of relatively undifferentiated epithelial cells, termed matrix cells (that give rise to the hair and the inner root sheath), studies of label retaining cells in mice indicated that cells responsible for the cyclical regeneration of the lower follicle are located in the hair follicle bulge (Cotsarelis et al, 1990). Furthermore, when rat follicles were microdissected that cells from the bulge had the highest colony forming efficiency (Oshima et al, 2001). These studies established that the bulge is a site of murine hair follicle cells capable of cyclic regeneration of the hair follicle.

Colony forming efficiency as a surrogate test for stem cell behavior (1993- )

In vitro studies showed that the population of cultured keratinocytes that adheres most rapidly to collagen IV is enriched in colony forming cells (Jones and Watt, 1993). Whether this population of rapidly adherent cells includes a greater proportion of holoclones was not studied. In rats, 95% of colonies formed in culture were from cells of the vibrissa bulge region (Kobayashi et al, 1993). In human hair follicles, the greatest colony forming efficiency ability was in the lower outer root sheath (Rochat et al, 1994), while label retaining cells were localized in the human bulge area (Lyle et al, 1998). The stage of the human follicle at the time of isolation may influence colony forming efficiency in humans, given that hair follicles are not synchronized in their growth (Rochat et al, 1994) (Oshima et al, 2001). Proliferation in vitro/ colony forming ability became a popular surrogate assay by which to analyze for the stem cell like nature of a population of cells, [for example (Braun and Watt, 2004; Tani et al, 2000; Inoue et al, 2009; Nijhof et al, 2006; Ohyama et al, 2006)], while the more arduous method of clonal analysis for self-renewal ability was less utilized.

The search for a molecular signature for epidermal stem cells begins (1993- )

Jones and Watt showed that high levels of β1 integrins on the surface of human epidermal keratinocytes correlated with high proliferative potential in vitro (Jones and Watt, 1993). This work was followed by an extensive amount of work by many groups with the goal of isolating a keratinocyte population enriched for epidermal stem cells. Since functional assays to identify stem cells were lacking for epidermal stem cells, surrogate assays including colony formation, cell cycle status, cell size, etc. were used. As a result an extensive number of strategies have been proposed to enrich for epidermal stem cells, but controversy exists regarding the best epidermal stem cell marker and the degree of the enrichment provided by each method is unknown.

A summary of many of the putative epidermal stem cell markers is included in Table 1. The results from these various studies are difficult to compare as different populations of cells (human, murine, follicular, interfollicular, non-follicular/glaborous) were studied using different methods. Only quantifying the degree to which functional stem cells are isolated by these different methods, through the use of a standardized approach, will allow the molecular signature of the epidermal stem cell to be determined, and thus provide us with the best approach(es) for enriching populations of epidermal stem cells. Based on findings in the hematopoetic system we can anticipate that a combination of mutiple positive and negative surface markers will be required to identify the epidermal stem cell.

Table 1

Putative Stem Cell Markers in Epidermis

MARKER(function)	STEM CELL PROPERTIES*
α6integrinhi(Adhesion)	α6 integrinhi human keratinocytes were label retaining, quiescent, exhibited high nuclear tocytoplasmic ratio, high colony formation capacity and had the greatest tissue regenerationcapacity (Amy Li et al. 2004; P Kaur and Li 2000).
β1 integrins(Adhesion)	Human keratinocytes that adhere rapidly to type IV collagen, a β1 integrin ligand, exhibitedhigh proliferative potential in vitro (P H Jones, Harper, and Watt 1995).
BrdU/LRC(Incorporated into DNA)	Murine label retaining cells exhibited high colony forming efficiency in vitro and the highestintegrin levels (Braun and Watt 2004).
CD133(Prominin)	Co-expressed with β1 integrin in the basal layer of neonatal human epidermis (Yu et al. 2002).
CD200+(Auto immunity)	Located in murine follicular bulge. High colony forming efficiency and in G0/G1 phase (Inoue et al. 2009).
Side population(SP)(Efflux Hoechst 33342)	Using a modified Hoechst 33342 technique, more than 90% of putative murine stem cellswere in G0/G1 and these cells formed larger, more expandable colonies in vitro, than otherfractions (Dunnwald et al. 2001). Human SP cells were enriched in quiescent cells, but werenot label retaining cells and had low expression of surface antigens traditionally thought tomark stem cells (Terunuma et al. 2003). Murine SP cells were found to express keratin 14, β1integrin, and p63 (J.-X. Zhou et al. 2004). Both murine and human SP cells were a subset ofthe α6 integrin positive cells and human SP cells expressed the drug transporter ABCG2(Triel et al. 2004). SP cells exhibited high short- and long-term proliferative potential andformed a pluristratified epidermis (Larderet et al. 2006).
ABCG2+(ATP binding cassetteprotein)	Human side population cells expressed the drug transporter ABCG2 (Triel et al. 2004).
Keratin 19(Structural protein)	Co-localized with label retaining cells in mice (Michel et al. 1996).
CD34+(Cell-cell adhesion factor)	Marked murine bulge keratinocytes (but not human). Predominantly in G0/G1, and expressedhigher a6 integrin (Inoue et al. 2009). Refractory to differentiation in culture (Sasahara et al. 2009). Predominantly in G0/G1and co-localized with label retaining cells and keratin 15positive cells (Trempus et al. 2003).
Aldehydedehydrogenase (ALDH+)(Cytosolic enzyme forbiotransformation ofalcohols and aldehydes)	Expressed in human epidermis (Cheung et al. 1999) (150). Marker of hematopoietic(Armstrong et al. 2004; Hess et al. 2004; Hess et al. 2006) and mammary stem cells(Ginestier et al. 2007).
CD44+(hyaluronic acid receptor)	Marker of mammary stem cells (Al-Hajj et al. 2003) and head and neck squamous cell cancerstem cells (Prince et al. 2007).
CD90+(156) anchored cellsurface protein)	Human CD90+ cells formed larger clusters compared to CD90- cells, when injected inNODSCID mice (Nakamura et al. 2006).
Membrane potential (Δ)(Voltage difference[interior and exterior ofmitochondria])	Δlo murine embryonic stem cells possessed enhanced differentiation capacity compared to Δhicells (Schieke et al. 2008).
Lgr5(leucine-rich G protein-coupled receptor)	Lgr5+ murine keratinocytes were actively proliferating and multipotent stem cells able to giverise to new hair follicles for the long-term (Jaks et al. 2008)
Lgr6(leucine-rich G protein-coupled receptor)	Adult Lgr6+ murine keratinocytes were capable of long-term wound repair including theformation of new hair follicles (Snippert et al. 2010).
MTS24(glycoprotein)	MTS24+ murine cells expressed a6 integrin and keratin 14 and exhibited a two-fold increasein colony formation and colony size compared to MTS24-cells (Nijhof et al. 2006).
Lgr1(leucine-rich G protein-coupled receptor)	In murine epidermis Lgr1+ cells gave rise to all of the adult epidermal lineages in skinreconstitution assays (K. B. Jensen et al. 2009). In human epidermis Lgr1 was a regulator ofstem cell quiescence (K. B. Jensen and Watt 2006).
Delta1(Notch ligand)	Delta1 expression was confined to the basal layer of human epidermis with highestexpression where stem cells reside (Lowell et al. 2000). Deletion of Delta1 under the controlof keratin 5 promoter in mice, resulted in delay of the first anagen (Estrach et al. 2008).
p63(p53 homologue)	Holoclones formed using human keratinocytes (in vitro clones that show less than 5%terminal colonies) showed high expression of p63 (G Pellegrini et al. 2001).
EGFRlo(Epidermal growth factorreceptor)	Human EGFRlo cells generated a pluristratified epidermis in a model of skin reconstructionafter long-term expansion (Fortunel et al. 2003).
CD24lo(glycoprotein)	CD24− was a more primitive mammary cell than CD24lo or CD24hi. CD24 is a marker ofpostmitotic human keratinocytes (Bergoglio et al. 2007).
(MHC) Class I-HLAlow/negative(Self-nonselfdiscrimination)	Low/negative expression in a subpopulation of basal human keratinocytes (Matic 2005).Embryonic stem cells lack MHC class I antigens.
Connexin43 (Cx43dim)(Gap junction protein)	10% of human basal keratinocytes were Cx43 negative, as determined by flow cytometry.The cells were small and low in granularity, and most murine label-retaining cells did notexpress Cx43 (Z. Chen et al. 2006). Cx43dim human limbal epithelial cells are small cells, lowgranularity, contain high percentage of LRCs, and are positive for p63, ABCG2, and integrinβ1 (Z. Chen et al. 2006).
Desmoglein3 (Dsg3dim)(Intercellular junctionprotein)	High β1 integrin-expressing human keratinocytes had low levels of Dsg3. Dsg3dimkeratinocytes had greater long-term proliferative capacity in vitro than Dsg3bri. Primary adultDsg3dim cells showed comparable clonogenicity to α6hiCD71lo cells (Wan et al. 2003; Wan et al. 2007).
CD71lo(Transferrin receptor)	Human α6 integrinhi CD71hi keratinocytes have the greatest tissue regeneration capacity (Li, Simmons, and Kaur 1998).
CD146 MCAM(Probable adhesionmolecule)	Using multiple markers along with CD146lo, selected for human hair follicle cells with highcolony forming efficiency (Ohyama et al. 2006).

The hair follicle bulge as a site of hair follicle stem cells in humans (1996- )

The bulge is a less morphologically distinct structure in the adult human compared to the adult mouse. However, studies of label-retaining cells indicated that human hair follicle stem cells were located in the bulge of the human hair follicle (Lyle et al, 1998; Ohyama et al, 2006). Human bulge cells were found to express Keratin 15 and Keratin 15 expressing human keratinocytes were slow cycling, proliferated at the onset of follicle growth, expressed a high level of B1 integrin (Lyle et al, 1998; Lyle et al, 1999). Moreover, bulge cells isolated by laser capture microdissection were highly clonogenic in vitro (Ohyama et al, 2006). Recently, a cell surface marker, CD34, was reported as a specific marker of murine bulge keratinocytes (Trempus et al, 2003). CD34 positive cells were predominantly in G0/G1, and had higher α6 integrin expression than CD34 negative cells (Trempus et al, 2003). However, CD34 was not expressed in human bulge cells (Ohyama et al, 2006; Cotsarelis, 2006a). Having confirmed the bulge as a site of hair follicle stem cells in humans, in a recent study, human bulge cell markers were used to determine that human hair follicle stem cells were maintained in men with androgenetic alopecia, but that CD200-rich and CD34 positive progenitors were decreased (Garza et al, 2011).

Not all colony forming cells are stem cells (1998- )

Phenotypic analysis of hematopoietic stem cells provided the ability to separate long-term proliferating cell from cells detected in colony forming assays (Hodgson and Bradley, 1979; Van Zant, 1984; Lerner and Harrison, 1990; Spangrude and Johnson, 1990; Morrison and Weissman, 1994; Weissman, 2002; Trevisan and Iscove, 1995; Randall et al, 1996). Hematopoietic stem cells have also been shown to have minimal to no clonal capacity (Sutherland et al, 1990; Haylock et al, 1992). While tissues may differ in their biology, it behooves us not to ignore the knowledge obtained through extensive and thorough research in hematopoiesis.

Concerns with the use of colony forming efficiency to assess epidermal stem cells include that 1) culture conditions commonly used have been designed to produce cells as fast as possible in the short-term and therefore may favor proliferation of committed progenitors; 2) cell culture may affect cell division creating an alteration in the number of stem cells (for review see (Cotsarelis, 2006b) 3) removing cells from tissue for in vitro analysis requires perturbing the system so that some events may be the result of a response to wounding and not homeostasis (Kaur, 2006); 4) the population of keratinocytes enriched in short-term colony forming cells (rapidly adherent to collagen IV) in vitro contains significantly fewer long-term repopulating cells in vivo compared to the population of keratinocytes not enriched in colony forming cells (not rapidly adherent to collagen IV) (Strachan et al, 2008) and; 5) there is prior evidence that stem cell-rich fractions exhibit substantially lower CFE than the source total population in adult rat epidermis (Pavlovitch et al, 1991), neonatal human foreskin (Li et al, 1998) and limbal epithelium (Budak et al, 2005; Selver et al, 2011).

Whether colony forming units reflect true long-term functional capabilities in the epidermis is not known, but it seems likely that long-term repopulating stem cells are either a subset of, or a different set of cells from, the colony-forming cell. Thus the majority of colony-forming cells in short-term cultures may represent intermediate but committed progenitors rather than true epidermal stem cells (given their relatively high frequency and high level of cell cycling). All together these observations indicate that in vitro colony forming assays are not an adequate surrogate for in vivo assays and much work is still needed to understand how in vitro growth relates to epidermal progenitor type and stem cell behavior in vivo.

Notch signaling is important, both for cell-fate decisions and in lineage commitment (2000- )

Notch signaling is important in determination of stem cell self-renewal versus differentiation. Signaling via the Notch receptor, delivered by ligands Delta and Serrate, plays a key role in cell fate decisions in both Drosophila and vertebrate development (Artavanis-Tsakonas et al, 1995). In human epidermis expression of Delta (Notch ligand) in stem cells has been proposed to induce differentiation of the neighboring (Notch1 expressing) cells (Lowell et al, 2000). Expression of the Jagged 1 and 2 ligands and Notch 1 and 2 receptors increases in differentiating keratinocytes of the suprabasal layers and is thought to be important for synchronization of differentiation and epidermal border formation (Luo et al, 1997)(Rangarajan et al, 2001). The deletion of the Notch 1 and 2 and/or RBP-Jκ genes indicated a role for Notch signaling in lineage commitment along the hair follicle lineages versus interfollicular lineage (Yamamoto et al, 2003; Pan et al, 2004; Blanpain et al, 2006). For two comprehensive reviews see (Dotto, 2008; Watt et al, 2008).

Evidence for an interfollicular stem cell: Lineage tracing in vivo - Seeing is believing (Table 2) (2001- )

While the ability to illuminate epidermal clones with retroviral transduction of keratinocytes was elegantly demonstrated in 1997 (Mackenzie, 1997), it was a few years before evidence for a long-term repopulating cell in the interfollicular epidermis was provided by in vivo lineage tracing (Ghazizadeh and Taichman, 2001; Niemann and Watt, 2002; Schneider et al, 2003; Ghazizadeh and Taichman, 2005; Ito et al, 2005; Langton et al, 2008). In these types of studies a label is integrated into the cell’s genome and this label is stably inherited by all progeny. Such cell fate mapping techniques allow investigation of the distribution of progeny arising from a single precursor cell. Prior to this time there was uncertainty whether the interfollicular stem cell represented a true stem cell or an early progenitor derived from follicular stem cells. However, with the ability to perform genetic label lineage tracing studies strong evidence was provided that under homeostatic conditions an independent pool of stem cells maintains the interfollicular epidermis. Previous studies of label retaining cells had also indicated that interfollicular epidermis is maintained by a stem cell compartment distinct from the follicle stem cell compartment (Braun et al, 2003). Cleverly designed studies to ablate hair follicles in hairy skin provided further evidence for an independent stem cell population (Ito et al, 2005). Finally, although many studies show that hair follicle-derived stem cells participate in acute wound closure, analysis of wound healing in the Edaradd mutant mouse (with developmental defects in hair follicle formation) showed that hair follicle stem cells are important but not necessary for normal wound healing or interfollicular epidermis homeostasis (Langton et al, 2008).

There is also evidence for multipotency of ‘interfollicular’ stem cells. There is evidence that hairless skin possesses pluripotent stem cells that, under the appropriate conditions, can produce hair follicles. Even though there are no hair follicles in the foreskin (the source of human keratinocytes for most in vitro studies), implantation of dermal papillae results in hair development from keratinocytes of these non-hairy sites (Ehama et al, 2007). It should be noted however, that these studies investigate whether non-hairy skin, not interfollicular skin, is multipotent. The question of whether interfollicular cells are multipotent is somewhat different, since presumably the non-hairy/glaborous skin contains a primitive stem cell, while perhaps (if the pluripotent epidermal stem cell in hairy epidermis is restricted to the hair follicle) the interfollicular epidermis may not. Thus, while non-hairy skin is multipotent, interfollicular epidermis may or may not be. This issue is germane for stem cell isolation and gene therapy. However, together, the above data provide strong evidence for the presence of an distinct stem cell in the interfollicular epidermis.

Long term repopulation in vivo as a functional assay of epidermal stem cells (2003- )

It is evident that methods to evaluate epidermal stem cells are inadequate and long-term repopulation assays that measure sustained epithelial tissue regeneration, analogous to “gold standard” assays in hematopoiesis, are essential for progress in epidermal stem cell biology.

Dr. Kaur’s group designed a transplantation assay based on repopulation of epidermis inside a rat trachea implanted subcutaneously in a SCID mouse (Li et al, 2004; Pouliot et al, 2005). This assay allows long-term maintenance of transplanted epidermal cells in vivo.

Similarly, our group developed a in vivo transplantation assay for epidermal stem cells (Schneider et al, 2003) based on well-established functional assays for hematopoietic stem cells (Chang et al, 2000; Harrison, 1980; Szilvassy et al, 1990; Taswell, 1981). This assay was combined with limiting dilution analysis to enable the quantization of the frequency of long term repopulating cells in a population of keratinocytes.

Using long-term repopulation combined with a limiting dilution design, stem cell frequency in the bone marrow was determined to be 1 in 10,000 nucleated cells (Szilvassy et al, 1990; Taswell, 1981). This type of assay is now a standard in hematopoiesis, for the evaluation of stem cell markers (Weissman, 2002; Spangrude et al, 1988) and for evaluation of stem cell differentiation and regulation (Mikkola et al, 2003; Park et al, 2003; Smith et al, 1991) and has been key in studying other populations of hematopoietic stem cells quantitatively (e.g. aging/ disease/ cancer) (Chen et al, 1999; Dick, 2003; Lapidot et al, 1994; Sudo et al, 2000). Similar in vivo transplantation assays showed that the frequency of long term repopulating mammary gland stem cells was 1 in 4,900 lin cells (Shackleton et al, 2006).

The in vivo transplantation assay for epidermal stem cells allows similar - quantization of epidermal stem cells. This assay has been used to demonstrate that the frequency of murine epidermal stem cell in this model is less in vivo than previously predicted and more in line with hematopoietic stem cell frequency, on the order of 1 in 10,000 (Schneider et al, 2003), that rapidly adherent colony forming cells are not enriched in long-term repopulating cells (Strachan et al, 2008) and while no significant difference in aged versus young epidermal stem cell (long-term repopulating cell, 9 weeks of repopulation) frequency could be detected, transient amplifying cell (short-term repopulating cell, 3 weeks of repopulation) frequency was greater in the aged (Charruyer et al, 2009).

Epidermal Immigrants. Bone marrow derived stem cells in the epidermis during epidermal regeneration (2004- )

Studies have shown that with regeneration, bone marrow derived cells not only contributed to dermal but also epidermal cells (Fathke et al, 2004; Deng et al, 2005; Brittan et al, 2005; Badiavas et al, 2003; Borue et al, 2004). Although it was suggested that bone marrow derived cells could form fusion cells with keratinocytes in an ex vivo study (Baxter et al, 2004), other studies indicated that fusion does not occur in vivo (Borue et al, 2004; Wu et al, 2007; Harris et al, 2004) and indicate that that bone marrow derived stem cells differentiate into epidermal cells. For a detailed review see Wu, Y et al (Wu et al, 2010).

2004-Stem cell differentiation: p63 is responsible for initiation of epithelial stratification

One protein that is critical for transit amplifying cell regulation is p63. It has been suggested that ΔNp63 is involved in maintaining transit amplifying cells and in preventing premature onset of differentiation (Truong et al, 2006; Honeycutt et al, 2004; Koster et al, 2005) and ΔNp63 was found to be important for maintaining the proliferative state of transit amplifying cells (Truong et al, 2006). p63 is strongly expressed in epithelial cells with high clonogenic and proliferative capacity (Senoo et al, 2007) and mice with reduced ΔNp63 expression show excessive keratinocyte proliferation while lacking normal differentiated layers (Koster et al, 2007). The role of p63 in epidermal stratification is discussed in detail in a review by Dr. Koster (Koster, 2009).

2005- Expansion versus maintenance of the epidermis – symmetric versus asymmetric stem cell division

Stem cells in the niche undergo symmetric self-renewal divisions leading to two daughter cells identical to the original cell, symmetric differentiation divisions releasing two committed cells, and asymmetric self-renewal divisions leading to one daughter cell identical to the original stem cell and another non-stem daughter cell that leaves the niche and undergoes differentiation (Xie and Spradling, 2000; Potten and Loeffler, 1990). When stem cells divide asymmetrically, tissues maintain a constant number of stem cells while, depending on the division rate, allowing a progressive increase in the number of transient amplifying cells. Symmetric self-renewal divisions allow maintenance and/or expansion of the immature precursor pool and symmetric differentiation divisions allow only differentiated progeny to be generated.

In Drosophila, during asymmetric division (using an intrinsic mechanism), stem cells set up an axis of polarity during interphase that orients the mitotic spindle and ensures the asymmetric segregation of self-renewal determinants (for review see (Horvitz and Herskowitz, 1992; Knoblich, 2008; Lechler and Fuchs, 2005). A second (extrinsic) mechanism places the axis of polarity perpendicular to the basement membrane so only one daughter cell remains in contact with the stem cell niche and maintains the ability to self renew. In this case the basal cell remains attached by integrins to the basement membrane (Blanpain and Fuchs, 2009). These two types of asymmetric division have been observed both in epidermal stratification during development and homeostasis of adult epidermis (Lechler and Fuchs, 2005). The extrinsic mechanism is observed in adult stem cells mainly, while intrinsic (and parallel to the basement membrane asymmetric division is more common during epidermal development [for review see (Knoblich, 2008; Blanpain and Fuchs, 2009)].

Although involvement of mitotic spindle orientation in asymmetric fate has yet to be determined, the ability of spindle orientation to change during epidermal development has been shown in epidermis (Lechler and Fuchs, 2005). Basal progenitors first divide in a single layer parallel to the basement membrane. After, during epidermal stratification, they divide perpendicular to the basement membrane. The increased proportion of perpendicular cell divisions in the late versus early embryonic stages allows a balancing of the generation of suprabasal layers with lateral skin expansion. In adult skin the proportion of this asymmetric division is thought to decrease (Lechler and Fuchs, 2005). Recently it was shown that compromising asymmetric cell divisions results in profound defects in stratification, differentiation and barrier formation and Notch signaling is implicated as an important effector (Williams et al, 2011).

Understanding the different mechanisms used by stem cells to divide and proliferate will allow the development of strategies by which to correct diseases such as cancer and psoriasis, in which normal stem cell divisions may be altered.

2007- A single epidermal progenitor versus a hierarchy of progenitors

Proliferating cells of the epidermis have been shown to be heterogeneous, by presence of a subset of label retaining cells (Braun and Watt, 2004), the association of actively cycling cells and terminal differentiation (23), and the heterogeneity of clonal growth in culture (Barrandon and Green, 1987). Classically the epidermis has been thought to be sustained by an epidermal stem cell/transient amplifying cell hierarchy of progenitors, in which the epidermal stem cell divides only occasionally (asymmetrically) to give rise to both a daughter stem cell (identical to the mother stem cell) and a daughter cell called a transit amplifying cell, which terminally differentiates after several rounds of cell division. Recently, however, some observations have contradicted the epidermal stem cell/transient amplifying cell model (Clayton et al, 2007; Jones et al, 2007). Using inducible genetic labeling, the authors were able to trace the fate of progenitor cells in murine tail epidermis in vivo at single cell resolution with a time interval of one year. If the epidermal stem cell/transient amplifying cell model is true and individual stem cells retain their self-renewal capacity, this model would predict that the basal-layer clone size distribution would remain time-independent and follow a “epidermal proliferation unit” pattern. However lineage tracing revealed that the average number of basal layer cells per clone increased with time in a linear manner. Clones remained cohesive and expanded in size over time, such that clone-size distributions were consistent with a new model of homeostasis involving only one type of progenitor cell which may undergo an unlimited number of divisions. If this new model proves correct this will be a major paradigm shift in our understanding of epidermal homeostasis.

2009- Toward regenerative medicine for the epidermis

Although we lack a complete understanding of much epidermal stem cell biology, we already graft both autologous and allogenic keratinocytes for healing of burns and ulcers.

Ideally, rather than culturing autologous cells in vitro it might be more effective to expand the pool of epidermal stem cells ex vivo and return an expanded pool of stem cells to the wound (Charruyer and Ghadially, 2009). This would be the pool of cells with the greatest proliferative potential for the long-term. Progenitor cells were found superior to more differentiated keratinocytes for generation of tissue-engineered skin (Pellegrini et al, 1999; Dunnwald et al, 2001). Stem cell and transit-amplifying cell populations were used to bioengineer an epidermis in combination with a collagen type I gel containing neonatal mouse dermal fibroblasts. Both populations formed an epidermis, but after two months only the stem cell-derived skin maintained a normal epidermis, while the epidermis formed from transit-amplifying cells had completely differentiated (Dunnwald et al, 2001). A Hoechst and propidium iodide cell-sorting strategy with improved sorting conditions was used to isolate an epidermal stem cell population. This population, when used as a source for tissue-engineered skin, was able to maintain an epidermis and expression of an integrated recombinant gene (Dunnwald et al, 2001). These findings suggest that it may be advantageous to use primitive tissue progenitors for tissue engineering. Finally, successful complete epidermal regeneration on both legs of a patient throughout a 1-year follow-up after transducing primary keratinocytes with laminin B3 cDNA was attributed to transduction of stem cells in the regenerated epidermis (Mavilio et al, 2006; Ferrari et al, 2006).

Another issue is the production of a tissue engineered graft with epidermal appendages. Recent advances are bringing science close to the day where we will be able to produce a three dimensional graft bioengineered graft that contains dermis as well as epidermis with hair follicles and other cutaneous appendages (Marazzi et al, 2011; Mahjour et al, 2011).

2011- Rebuttal to the single progenitor theory from multiple stem cell researchers

For an excellent commentary in response to the single progenitor theory see Kaur and Potten (Kaur and Potten, 2011). Evidence for a proliferative hierarchy in epidermis is based on extensive cell kinetic and mathematical modeling data based on the percentage of labeled mitoses, continuous labeling studies, cell cluster analysis and clonogenic studies following irradiation. The observed spectrum of keratinocyte behavior could result from a continuum of proliferation ability, as seen in hematopoiesis, and problems identifying transient amplifying cells (committed progenitors) may result from a lack of adequate methods, rather than their non-existence. Finally, the studies of Jones et al were performed on tail skin (Clayton et al, 2007; Jones et al, 2007) and these findings may not be representative of skin from other sites.

2011- From skin to iPs cells and back again: Keratinocytes as a source of iPS cells and iPS cells as a source of keratinocytes

Under appropriate conditions induced pluripotent stem (iPS) cells (derived from fibroblasts or keratinocytes) have the potential to differentiate into to a multitude of cell types. In 2008 iPS cells were generated from human keratinocytes (Aasen et al, 2008) (Carey et al, 2009). Keratinocyte-derived human iPS cells could be differentiated into pancreatic endoderm with an efficiency that was comparable to that for human embryonic stem cells (Santamaria et al, 2010). Recent reports demonstrate that iPS cells can differentiate into keratinocytes (Bilousova et al, 2011; Tolar et al, 2011). The advantages of iPS cells over embryonic stem cells for regeneration therapy include the lack of ethical issues and, since iPS cells can be autologous, the elimination of immune rejection concerns. Another possibility is that iPS cells derived from revertant mosaicism (clinically evident as areas of normal skin in patients with heritable diseases such as epidermolysis bullosa) could be utilized to derive normal iPS cells to provide basically unlimited keratinocytes for grafting (Itoh et al, 2011; Uitto, 2011). Thus, iPS cells provide a novel approach to regenerative therapy for heritable skin diseases such as epidermolysis bullosa. A recent detailed review making a case for iPS cells for regenerative medicine is available (Lemaître et al, 2011).

2011- Isolation of single human hematopoietic stem cells capable of long-term multilineage engraftment

While the molecular regulation of specific hematopoietic stem cell properties such as long-term self-renewal is starting to be elucidated for murine HSCs (Sauvageau and Sauvageau, 2010), less is known regarding human hematopoietic stem cells. In a recent study, while CD49f identified hematopoietic stem cells with long-term multilineage potential, loss of CD49f identified transiently engrafting multipotent progenitors. In this study 28% of single Thy1+RholoCD49f+ cells had long term repopulating ability (Notta et al, 2011). The ability to quantitatively study subsets of hematopoietic stem cells has been a challenging task, involving many hematopoietic stem cell research groups. What we have learned from hematopoiesis will hopefully make the path to quantifying subgroups of epidermal stem cells a shorter one.

Prospects for the future

Surprisingly, the only tissue in which stem cells have been isolated to near purity and with in vivo proof of their long-term repopulating ability, is in the hematopoietic system. At present, the isolation of a pure population of epidermal stem cells has not been achieved. Populations of murine bone marrow cells have been enriched so that a purity of 1 in 3 to 1 in 7 cells is a primitive hematopoietic stem cell, and this year in human bone marrow 28% of single Thy1+RholoCD49f+ cells were reported to have long term repopulating ability (Yilmaz et al, 2006). As we move ahead in our understanding of epithelial stem cell biology, the same must be required for putative epidermal stem cell markers in epidermis. The field of hematopoiesis has reached a consensus decision to uphold an in vivo long-term competitive repopulation assay as the ‘gold standard’ for defining a stem cell (Purton and Scadden, 2007)(Chang et al, 2000). Interestingly, multipotent progenitors can be either long term repopulating cells or less primitive progenitors (Kondo et al, 1997; Akashi et al, 2000). While the need for such an assay in cutaneous biology is recognized (Strachan et al, 2008; Schneider et al, 2003; Charruyer et al, 2009)(Pouliot et al, 2005; Kaur et al, 2004), varying surrogate assays are used in practice, impeding the collaborative and effective advancement of cutaneous stem cell biology. Several of the most used surrogate assays have significant defects. For example, colony forming cells are commonly evaluated as a measure of the presence of stem cells. However, colony forming cells are definitely not synonymous with stem cells (see above). It is not clear whether long term repopulating primitive epidermal stem cells are a subset of or a different cell from the colony forming cell. Clonal analysis in vitro has the ability to measure self-renewal ability, but there is evidence that the most primitive stem cells may not have clonal capacity (hematopoietic stem cells with long-term bone marrow repopulating capacity have been shown to have minimal to no clonal capacity (Sutherland et al, 1990; Haylock et al, 1992; Budak et al, 2005).

Label retaining cells (in the hematopoietic system) are believed to identify one actively cycling subset of self-renewing stem cells but not the ‘dormant’ subset (van der Wath et al, 2009; Wilson et al, 2008). Since labeling in skin is only performed for a few days, if a similar situation exists in skin we are probably also only labeling a subset of epidermal stem cells. Thus, these surrogate assays have limitations and until we have a marker set for epidermal stem cells based on undisputed features of a stem cell (long term tissue repopulation (Strachan et al, 2008; Schneider et al, 2003; Charruyer et al, 2009)(Pouliot et al, 2005; Kaur et al, 2004) and/or self-renewal) it will be hard to define a hierarchy of progenitors and to provide a unique molecular signature for the most primitive epidermal precursor. Taking advantage of what appear to be ‘generic’ stem cell markers and profiling more primitive epidermal stem cells will hopefully aid and accelerate this process.

Progress in finding stem cell niches within the skin and epidermis has been steady and as more advanced techniques provide us with ever improved technology, we are moving towards understanding the locations and contributions of the different stem cell niches.

Aided by work in drosophila and in mammalian tissues we have also made steady progress in understanding the determinants of epidermal stem cell proliferation and differentiation. Although our understanding of the interactions between different regulatory pathways is limited there is extensive ongoing work in multiple laboratories.

A big challenge is to improve clinical therapy using epidermal stem cells, hair follicle derived multipotent cells and/or keratinocyte iPS cells. The new findings with iPS cells provide much hope for a rapid transition from basic science to the clinic.

Because it is a rapidly renewing tissue, similar to the gastrointestinal and hematopoietic systems, the epidermis is an attractive tissue in which to study stem cell biology. While our specialty is relatively small, keratinocytes now have the attention of the stem cell world, as an easily accessible population of cells, for many purposes. Our rapidly evolving understanding of epidermal stem cells has many potential uses that promise to translate into improved patient therapy.

Table 2

Evidence for an interfollicular stem cell (2001-present)

Reference	Conclusion/Interpretation	Study Aim	Technical approach	Result
Ghazizadeh & Taichman, 2001	Existence of multiplestem cells withrestricted lineages incutaneous epithelium	To determine thedistribution ofstem cells andtheir progeny inepidermis	In situ retroviralmediated gene transferto mark cutaneousepidermal stem cells(lacZ reporter gene)	Staining patternconsistent withmultiple stem cellpools
Braun, 2003	Interfollicular epidermisis maintained by astem cell compartmentdistinct from the hairfollicle stem cellcompartment	To determine thenumber andlocation of labelretaining cells inthe epidermis	Observation of labelretaining cells inwhole-mounts usinghistological analysisand immunolabeling oftissue sections	Label retaining cellsfrequently observedas individual, nonclusteredinterfollicular cells(Ki67 negative)
Ito, 2005	Follicular stem cells donot contribute toepidermis underhomeostaticconditions, but arerecruited after injury	To determinewhether bulgecells are requiredfor epidermalhomeostasis andrepair	Ablation of bulge cellsby a suicide geneencoding herpessimplex virus thymidinekinase using thekeratin 15 promoter andlineage mapping	Ablation of hairfollicle stem cellsleads to completeloss of hair folliclesbut survival of theepidermis
Levy, 2005(38)Levy, 2007	The interfollicular stemcell population isdistinct from thefollicular stem cellpopulation	To determinewhether stem cellsof the epidermisderive from thefollicle duringhomeostasis(2005) and woundhealing (2007)	Lineage analysis ofsonic hedgehog gene(ShhGFPcre; R26R)-expressing follicularcells	No labeled bulgederived cells wereobserved in theepidermis (2005).Follicular cellsparticipate in initialwound resurfacingand their progenypersist in the woundfor months
Langton, 2008	Hair follicle stem cellsare important but notnecessary for normalinterfollicular epidermishomeostasis or woundhealing	To determine therole of hair folliclestem-derived cellsin wound healing	Wound healinganalysis of Edaraddmutant mouse(developmental defectsin hair follicleformation)	Hair follicle-derivedstem cells participatein acute woundclosure. In theabsence of hairfollicles a larger areaof interfollicularepidermis is recruited

Adapted from Charruyer and Ghadially, What’s New in Dermatology, 2011.