Chapter 45. Epidermal Stem Cells

The cutaneous epithelium is a continually renewing tissue maintained in a dynamic equilibrium of proliferation in the basal layer and loss through terminal differentiation from the suprabasal layers. This process is orchestrated with great elegance by a hierarchy of stem cells, transient amplifying cells, and terminally differentiating cells. These populations of cells work together to maintain lifelong tissue function and to bring about tissue repair. This chapter focuses on the role of stem cells and their identification in the epidermis.

Proliferation in the cutaneous epithelium begins with the stem cells.1,2 Stem cells in this regard lack many characteristics of terminal differentiation, and have an intrinsically high proliferative potential relative to the other proliferating cells, but are generally capable of lifelong proliferation.3 Upon division, a stem cell produces off one daughter that remains a stem cell, and one daughter that goes on to produce a series of transit amplifying cells that serve to magnify or amplify the stem cell's division resulting in the production of many differentiated cells with minimal input from the stem cell. This hierarchical system that usually involves decreasing proliferative potential is illustrated in Fig. 45-1. Stem cells typically interact with their surroundings in a supportive, protective niche.1

Stem cells may be studied by the presence or absence of proteins on their surface that distinguish them from other proliferative cells.2 Such proteins may be internal, or more desirably, proteins on the cell surface that render the cells selective by various methods such as by magnetic bead separation or by fluorescence activated cell sorting (FACS).2 Stem cells may also be studied by cellular kinetic characteristics such as in slowly cycling label-retaining4 cells or through their patterns of mitotic activity.5 Stem cells can sometimes be identified and isolated according to their special physical properties such as cell size or buoyant density,6 or in conjunction with other properties such as in the so-called “side population” cells7 that have active Abcg2 transporters. Candidate stem cells identified or isolated by any of these methods may then be characterized by functional tools such as in vitro colony forming assays,8–10 or in an in vitro or in vivo skin reconstitution assay.2

Asymmetric Division

The hierarchical model for cell proliferation in the cutaneous epithelium implies some degree of cellular, genetic, or population asymmetry.3,11 The model of the stem cell hierarchy suggests a level of asymmetry that could be due to infrequent cell division or to chromosomal segregation.3 As the stem cell mechanism is thought to provide protection for the tissue as well as the cellular DNA, Cairns12,13 hypothesized that perhaps stem cells have a special mechanism for segregating their DNA and retaining an “immortal strand” at each division. Although there is some fairly convincing evidence that stem cells of the breast14 and intestinal epithelium15 may reserve an immortal DNA strand, recent investigation of the multipotential stem cells of the mouse hair follicles suggest that chromosome segregation does not occur.16,17 Moreover, the Tumbar laboratory developed a method to trace the proliferation history of hair follicle bulge keratinocytes and thus provided direct evidence in support of the infrequent division model for these particular stem cells.16

Lateral Migration

Cellular proliferation and terminal differentiation in the epidermis are usually thought to occur in columns. However, lateral migration is common during wound healing where a tongue of proliferating epithelium migrates over denuded dermis and reestablishes an epithelium complete with vertical proliferative units and terminal differentiation.18 In addition, Brash and colleagues have suggested that following irradiation of skin with ultraviolet light, clonal patches, each with its own p53 mutation, might reflect epidermal cell proliferation and differentiation beyond the confines of single proliferative units.19

Identification of Stem Cells in the Cutaneous Epithelium

Slowly cycling epidermal stem cells were first identified by of their cell kinetic behavior in the context of the epidermal proliferative units. Hence, Mackenzie identified mitotic basal keratinocytes beneath the edges of the hexagonal squames.20 In vertical cross sections of skin, Christophers21 had found that some basal cells in the shape of a hand mirror stained with a fluorescent dye characteristic of suprabasal cells. These studies were quantified by Potten in 197422 who called these units of structure epidermal proliferative units (EPUs). He focused on the central basal cells, noting that they were rarely mitotic and also rarely incorporated [3H]thymidine administered as a single pulse, and conjectured that the quiescent central cells might have stem cell activity whereas the peripheral cells may have transit amplifying cell activity. The next major advance in understanding the function of the EPU came with the identification of slowly cycling label-retaining cells in the center of the EPUs. Bickenbach4 and later Bickenbach and Mackenzie,23 Morris,24 and Potten25 found that administration of [3H]thymidine continuously for 3 days followed by a 4–8 week chase, could identify these central cells in light microscopic autoradiographs. Further characterization of the central stem cells and peripheral transit amplifying cells has been performed by a variety of in vivo and in vitro techniques.2 The presence of EPUs in some areas of thin human skin has also been noted, and may be as large as 2 millimeters in diameter.20 The EPU concept in skin is illustrated in Fig. 45-2.

The distribution of stem cells within the epidermis has been a subject of debate. Hence, Lavker and Sun26 found that mitotic cells and cells that were rarely mitotic (putative stem cells) were located respectively in the upper and lower aspects of the rete ridges. These investigators postulated that the deeper cells were physically more protected than the superficial cells. In contrast, further studies have demonstrated that cells with stem cell characteristics in human epidermis can vary depending upon the site.27 Ghazizadeh and Taichman28 used retrovirally marked human epidermal keratinocytes followed by grafting onto athymic nude mice to visualize proliferative units in human skin. These studies found presumptive stem cells to be distributed throughout the epithelium.

Markers and Selectable Determinants

Compared with the plethora of markers and selectable determinants for various hair follicle stem cells, few such markers have been identified in the epidermis. Notable exceptions are β-1 integrin,10 CD71 (the transferrin receptor),29 and LRIG1 (Fig. 45-3).30 β-1 integrin is a cell adhesion molecule and can be used as a selectable determinant to enrich for keratinocytes that have a high proliferative potential in vitro and that can reconstitute a graft. Cells staining brightly with a fluorescently labeled antibody to β-1 integrin keratinocytes are found in human epidermis situated at the bottom of the rete ridges or atop the dermal papillae depending on the location of the skin. CD71 is expressed on all proliferating cells and can be used in conjunction with α-6 integrin (the external component of the hemidesmosomes present on epidermal basal cells) to enrich for a population that reacts with a fluorescently labeled antibody to α-6 integrin, but does not bind to fluorescently labeled CD71. This is enriched for epidermal keratinocytes that have in vitro and in vivo properties of stem cells. In the mouse, cells with this phenotype are located in the hair follicle bulge. LRIG1 is a marker of human interfollicular stem cells and helps to maintain stem cell quiescence.30 In the mouse, LRIG1 immunoreactive cells are found in the hair follicle junctional zone between the sebaceous glands and the infundibulum.31

Alternative Mechanisms for Regulating Epidermal Proliferation

The stem cell concepts presented above have not gone unchallenged. Clayton et al have studied clonal expansion in the mouse tail epidermis together with mathematical modeling.32 These investigators conclude that the presence of stem cell/transit amplifying cell model in the tails is incompatible with a long-lived stem cell population and a short-lived transit amplifying cell population. Moreover, this group infers that clones of two basal cells in the tail can adopt any of the three fates: (1) both cells can remain proliferative, (2) both can differentiate and exit the cycle, or (3) that one cell can remain proliferative and the other differentiates. Thus, epidermis from different sites may have different mechanisms for proliferation and terminal differentiation. At the present time, there are no reports of this model being applied to mouse dorsum or human epidermis from any site.

Relationships among Various Epithelial Stem Cell Compartments

Cellular kinetic and labeling data suggest that there are multiple stem and progenitor compartments within the cutaneous epithelium. Under homeostatic conditions, these compartments are stable over intervals with essentially no interactions.33 Hence, there are multiple proliferative units within the hair follicles: the infundibulum of the follicle, the bulge, the upper isthmus, and the sebaceous gland. The progeny of these follicular and sebaceous proliferative units all appear to be able to contribute to the repair of wounded epidermis.

Regulation of Epidermal Stem and Transit Amplifying Cells

Identification and functional characterization of molecules regulating epidermal stem cells and transit amplifying cells is currently a subject of intense investigation.34–37 Mediators under study comprise complex pathways often shared by embryonic, morphogenetic, and adult homeostatic and repair processes. Regulatory “switches”34 include (1) stimuli that direct progenitor cells toward a particular type of terminal differentiation, (2) molecules and pathways characteristic of the niche and stem cell homeostasis, (3) molecules that differentially alter stem cell and transit amplifying cell proliferation, and (4) positive and negative regulators involved in commitment to terminal differentiation. Examples of such regulatory molecules are given in Table 45-1.

Skin Diseases Arising from Proliferative Dysfunction

Skin cancer is thought to arise from aberrant proliferation of keratinocyte stem cells.3,38 In this regard, a stem cell is a candidate for a tumor-initiating cell because of its long-term persistence in the tissue and because of its inherently high proliferative potential. The relationship between the epidermal stem cell and the so-called cancer stem cell is not known at the present time; however, current thinking is that the tissue stem cells, when corrupted, may become cancer stem cells that retain such stem cell properties as long-term self renewal, an ability to cast off transit amplifying cells, and production of terminally differentiated cells. Although stem cells are well protected against carcinogen-induced damage by virtue of being a rare and well-protected population, unlike the relatively short-lived transit amplifying cells, stem cells persist to endure the multiple mutations that lead to malignancy.

Psoriasis is thought to be an example of hyperproliferation of transit amplifying cells, which among inflammatory and dermal changes, is characterized by increased numbers of β-1 integrin dim cells in the suprabasal layers.39 Additionally, markers of proliferation such as Ki67 and C-myc are upregulated.

Epidermal stem cells can be cultivated and expanded in tissue culture.8,40 This has led to an application of epidermal cells in cell therapy of burn victims. Hence, from a small biopsy from nonaffected skin, skin cells including stem cells can be expanded ex vivo and then reapplied to affected skin. The transplanted tissue “takes” to debrided skin regions and ultimately forms a new epithelium with the capacity to persist for many years. This application is now standard therapy in many burn units.40 In the transplanted epithelium, it is noteworthy that epithelial appendages such as hair follicles, sebaceous glands, or eccrine glands are absent because techniques for in vitro morphogenesis of these tissues have not yet been developed. The lack of skin appendages in the grafted epidermis results in fragility as well as dryness due to lack of sebaceous glands, and poor thermoregulation due to lack of eccrine glands in the transplanted epithelium. These problems highlight the urgent need to develop in vitro methods to recapitulate embryonic morphogenesis of the cutaneous appendages. Ex vivo expanded epidermal keratinocytes are also used for treating ulcers and other chronic wounds.41

Epidermal Stem Cells and Gene Therapy

The accessibility of skin makes it an attractive target for gene therapy.42 Here, one goal is to correct mutant genes in the stem cells ex vivo and transplant resulting normalized epithelium to patients.42 Another goal is to correct defective expression of certain secreted proteins in skin stem cells and to apply the corrected epithelium to patients.43 Although the use of epidermal cells including stem cells was proposed more than a decade ago, these applications still have not been developed for clinical practice. It is possible that the predilection of epidermis to develop into vertical proliferative units rather than to expand laterally precludes these applications. However, the field of gene therapy is still considered to be in its infancy, and identification and targeting of multipotential skin stem cells, as well identification of factors resulting in the lateral migration of epithelium during wound healing will increase therapeutic applications. Additionally, other related means of manipulating the keratinocytes such as siRNA show great promise.44

In this chapter, I have discussed general properties of epidermal stem cells and some of the techniques for studying them. In this regard, keratinocyte stem cells together with transit amplifying cells and terminally differentiating cells play a role in the normal turnover and repair of the epidermis. Additional studies, perhaps with novel approaches are needed for better identification of epithelial stem and transit amplifying cells. Especially, more investigation is needed for an understanding of the molecular “switches” that regulate stem cell and transit amplifying cell homeostases, fate determination, and repair mechanisms. Moreover, there is a great need for understanding stem cell targeting as well as the lateral migration in wound healing, as these are key questions in making cell and gene therapy a reality. Finally, it must be understood that there will never be a “pure” population of stem cells. Rather, each new marker or selectable determinant discovered, and each new functional assay developed is essential for ultimately designing new treatments for skin diseases and skin cancer.