The skin is the first line of defense to protect the body from dehydration, injury, and infection. To meet these needs, the skin has evolved an elaborate differentiation process that results in a tough, water-impermeable outer covering that is constantly renewable. Mammalian skin consists of both dermal and epidermal components; this discussion will be restricted to the epidermal cells, referred to as keratinocytes. The mammalian epidermis is a stratified tissue, anchored to a basement membrane (Fig. 1A).

Fig. 1. (A) Diagrammatic representation of skin epithelial histology. Cells of the basal layer attach to an underlying basement membrane. Basal cells are mitotically active, but they lose this potential when they detach from the basement membrane and embark on the outward trek toward the skin surface. As basal cells enter the spinous layer, they strengthen their cytoskeletal and intercellular connections, gaining resilience to mechanical stress. Once this task is completed, the cells enter the granular layer, where they produce the epidermal barrier.
(B) Diagram of the epidermal proliferative unit. A putative slow-cycling epidermal stem cell occasionally divides, giving rise to a stem cell daughter and a transiently amplifying daughter. The transiently amplifying cell divides two to four times, and these progeny then leave the basal layer and execute a program of terminal differentiation.

The layer of cells directly contacting the basement membrane, termed the basal layer, contains proliferating cells. Like all keratinocytes, cells of the basal layer possess a network of 10-nm keratin intermediate filaments (IFs), but they are otherwise relatively undifferentiated. As the population of basal cells expands because of division, some cells detach from the basement membrane and begin to move outward toward the skin surface. The first change to occur is a strengthening of the IF network to increase the tensile strength of each cell. Cells achieve this by synthesizing large numbers of new sets of keratins, which assemble into IFs that aggregate into more resilient bundles or cables of IFs. IF cables anchor to cell-cell junctions called desmosomes, thus distributing force not over individual cells but over the entire tissue. As the suprabasal cells, now connected by desmosomes, move in tandem toward the skin surface, they deposit and enzymatically cross-link proteins beneath the plasma membrane to form the cornified envelope. These cells also make lamellar granules filled with lipids, which are extruded onto the cornified envelope scaffold, providing a water-impermeable seal that prevents the unregulated escape of fluids. After production of all materials is complete, the cells cease transcriptional and metabolic activity and undergo a programmed cell death that shares some similarities with apoptosis. The cells (squames) that are sloughed from the skin surface consist largely of dead protein-aceous sacs of IF cables; these cell remnants are continually replaced by inner cells moving outward.
In mouse skin, as measured by autoradiography, the entire differentiation process from basal layer to squame takes 10-14 days. Human epidermis turns over more slowly; however, the proliferative reserve of human skin epithelial stem cells, which supply sufficient progeny to maintain 1-2 m² of skin for decades, must be enormous.
An early observation in the field of skin biology was that epidermal keratinocytes could be grown in culture. As opposed to many other cell types that require transformation to be cultured effectively, epithelial cells taken directly from the skin can be passaged for many generations when cultured in the presence of a fibroblast feeder layer. When grown in the presence of an epidermal growth factor (EGF) receptor ligand such as EGF or transforming growth factor α (TGFα), human keratinocytes can be expanded by a factor of 10¹⁶.

Where Are the Skin Epithelial Stem Cells Located?
On initial histological evaluation of mammalian skin, there is no obvious morphologically distinct region, or niche, of the basal layer where stem cells might be located. It has been known from the 1970s that the epidermis is organized into columns of maturing cell layers ~10 cells wide (see Fig. 1B). It was initially hypothesized that the entire basal layer consisted of stem cells, then later that the Langerhans cells were stem cells. Later studies suggested that stem cells might comprise 2-7% of basal layer cells. One method of retrospectively demonstrating the presence of stem cells in epidermal cultures is to label the population of cells and then use them to reconstitute epidermal tissue in vivo. Thus, when epidermal cultures expressing the β-galactosidase reporter gene were grafted onto a mouse, the reconstituted skin exhibited clonal columns of β-galactosidase-expressing epidermal cells in the host animal (Fig. 1B). The size of the columns over the 12-week study period suggested that as many as 10-12% of basal layer cells might be "stem cells" capable of generating a single maturing column of cells.
Another method of identifying tissue stem cells makes use of their slow cycling nature. In a pulse-chase experiment, all dividing cells of a tissue incorporate nucleotide analogs such as bromodeoxyuridine (BrdUrd) or tritiated [³H]thymidine into newly synthesized DNAs. When the label is chased, only those cells that divide rarely and still reside within the tissue over time will retain their label. In oral epithelium, so-called label-retaining cells, or LRCs, are located in discrete regions of tongue and palatal papillae; in murine ear epidermis, LRCs reside in the basal layer, near the periphery of differentiating cell columns. Therefore, a model of skin epithelial maintenance emerged in the 1980s in which the periodic division of slow-cycling stem cells in the basal layer gives rise to transiently amplifying cells that populate most of the basal layer, dividing two or three times and then moving upward while differentiating into mature skin cells (Fig. 1B).
In the 1990s, researchers discovered that the majority of LRCs in the skin reside in the "bulge" region of the hair follicle, with only a small fraction of LRCs in the basal layer of interfollicular epidermis. Thus, the most clonogenic cells and the cells with highest label-retaining capacity in the mammalian haired epidermis occur in the hair follicle, in or near the bulge region.

Functional Characteristics of Skin Epithelial Stem Cells
Locating the putative epidermal stem cells represents a major advance in the field, allowing scientists to move forward with respect to the biochemical and functional characteristics of this important population of cells. Other stem cell fields, such as the hematopoietic system, are replete with cell surface markers that identify nearly every cell type, starting with stem cells and extending through the most differentiated forms of the progeny types. Specific markers of epidermal stem cells, however, are not yet known. Although these cells can be identified either in vivo by label retention or in vitro by clonogenicity, neither method of identification presently allows easy isolation of stem cells for analysis. Therefore, there is a strong need for specific epidermal stem cell markers.
Even in the absence of cell surface markers useful for isolation of stem cells, skin biologists have made advances in understanding some of the molecules important in conversion from stem cell to transit-amplifying cell. One such example is the protooncogene c-myc, a transcriptional regulator of proliferation in a large variety of cell types, including skin keratinocytes. Interestingly, overexpression of c-myc in transgenic mouse skin results in what appears to be depletion of the multipotent skin stem cells of the bulge and impaired wound healing. Surprisingly, increasing c-myc expression also seems to cause a cell fate change from hair follicle progenitor cells to sebum-producing cells, suggesting that c-myc levels may influence not only the decision of stem cell daughters to become transit-amplifying cells, but also the decision of which lineage to adopt.
Another factor associated with stem cells and/or their conversion to transit-amplifying cells is the transcription factor p63, a homologue of p53. The studies suggest that p63 and c-myc are both important regulators of skin keratinocyte function. A major issue still unresolved for both these factors is the extent to which they govern stem cell maintenance versus the production of transit-amplifying progeny.

The Multipotency of Skin Epithelial Stem Cells: What Is the Relationship Between Interfollicular and Bulge Stem Cells?
Substantial evidence supports the idea that stem cells in the interfollicular epidermis are less potent than bulge stem cells, leading to speculation that they are progeny, perhaps unipotent progeny, of multipotent bulge cells. In contrast to bulge cells, interfollicular stem cells do not have a clearly defined niche. There are more slow-cycling stem cells in the bulge than in the interfollicular epidermis, and the longevity of label retention in bulge cells is longer than that in interfollicular cells. Cells cultured from the bulge also have a higher clonogenic potential than interfollicular cells. Superficial burns that destroy the interfollicular epidermis but leave intact the hair follicles do not require skin grafting, whereas deeper burns in which the hair follicles are destroyed cannot regrow epithelium except from the edges.
The multipotency of bulge cells is demonstrated by the fact that these special cells can give rise to all lineages of skin epithelia, including interfollicular epidermis (Fig. 1B). Cells isolated from dissected bulge regions are capable of differentiating into stratified epidermis when grown to confluence in culture and transplanted onto athymic mice. The label-retaining property of slow-cycling stem cells has been exploited to evaluate bulge cell contribution to multiple epidermal lineages. Cells that retain label after an 8-week chase, which reside exclusively in the bulge, contribute to all of the layers of the hair follicle. Pulse labeling with two different nucleotides, timed precisely (based on cell cycle length) to label cells of the infundibulum region of the hair follicle, demonstrated an efflux of hair follicle cells out into interfollicular epidermis in both normal and wounded states. In another assay, the multipotency of bulge stem cells was demonstrated by transplantation of β-galactosidase-expressing transgenic whisker bulge cells into the bulge region of follicles from an unlabeled recipient mouse. Over time, β-galactosidase-expressing cells from transplanted bulges populated all of the epithelial compartments of the resulting chimeric follicles, including the sebaceous gland and the infundibular region above the bulge that is thought to be most similar to interfollicular epidermis.
Although the evidence supporting multipotency of the bulge cells is compelling, the characteristics of stem cells outside this niche are less certain. Are interfollicular epidermal cells unipotent or multipotent? To what extent, if any, do they differ from bulge stem cells? The answers to these questions await more extensive knowledge about the molecular characteristics of bulge cells.

Clinical Applications of Skin Epithelial Stem Cells: Grafting of Cultured Keratinocytes
Basic research into stem cell biology is partly oriented toward the eventual possibility of harvesting stem cells from a patient, modifying or expanding them, and reimplanting them to treat disease. Skin keratinocytes have proved useful for this already, because of their accessibility and ability to be cultured. The most prominent clinical use of cultured keratinocytes is in creating confluent epithelial sheets that can be gently removed from the culture dish and applied to reconstitute the epithelial portion of burns, chronic wounds, and ulcers. The advantage of this method is the use of the patient's own skin, which represents the optimal long-term repopulation strategy. Today, the most commonly used skin grafting technique employs a different approach, the use of split-thickness grafts taken from unaffected skin. This method is effective, but it is limited by the available surface area of unaffected skin and creates some degree of additional injury. The use of cultured keratinocytes allows a much greater surface to be covered and requires a smaller area of unaffected skin from which to harvest the keratinocytes for culture. At present, the use of cultured keratinocytes is limited by the length of time needed to grow the epithelial sheets in vitro, during which time the patient is susceptible to infection. The epithelial sheets are also extremely fragile and do not adhere well to some burn surfaces. Under development are skin substitutes that could function as dermal equivalents to hold the expanding keratinocytes, improve adhesiveness to the burn wound, and form a temporary wound cover to reduce infection rates. If subconfluent keratinocyte cultures could be implanted into the dermal equivalent, then this graft could be applied very early after the burn injury, with the epithelial cover maturing while the dermal equivalent functions as a temporary dressing, obviating the need for two surgeries.
Grafting of cultured skin epithelial stem cells has other potential applications besides replacing burned skin; in particular, it is exciting to consider the possibility of using the cultured keratinocytes as delivery instruments for gene therapy. Two groups have devised methods to use cultured human keratinocytes to correct inborn metabolic skin diseases. Keratinocytes were harvested from patients with recessive dystrophic epidermolysis bullosa, and the genetic defect was corrected either by genomic integration of the correct sequence using a bacteriophage integrase or by transgene expression using a lentivirus. The repaired keratinocytes were expanded in culture and grafted onto nude mice to produce healthy epithelia in which the defect was corrected. Although neither study included grafting back onto the original human being suffering from skin disease, these efforts represent a major advance toward the possibility of manipulating stem cells to treat human disease.

The search for the biochemical regulators of skin epithelial stem cell self-renewal and production of daughter cells that will populate one or several lineages of the epidermis is ongoing. The field has advanced significantly over the past three decades, especially with respect to the presence and location of discrete stem cell compartments. Some initial work has implicated the cell surface markers that, although not specific, may be useful to enrich populations of cells for stem cells to allow characterization. C-myc and p63 have also been identified as regulators of stem cell fate. In addition, recent years have experienced a flurry of reports of transcriptional regulation in stem cell maintenance and/or lineage determination. Despite these advances, scientists are not yet able to reliably isolate stem cells from skin epithelium to exhaustively study their transcriptional and functional characteristics. Eventually, when techniques exist to find the answers to these questions, doctors may be able to not only use skin epithelial stem cells to grow new skin to treat burns, but also to treat genetic diseases of skin and possibly even non-skin origin. We do not yet know whether it might be possible in the future to genetically engineer keratinocytes to inducibly secrete peptide hormones, such as insulin as a treatment for diabetes, or growth hormone as a treatment for growth hormone deficiency. Nor do we know whether keratinocyte stem cells possess sufficient plasticity to be differentiated into nonkeratinocyte cell types to correct defects of other tissues. The studies of the past three decades indicate that the future of skin stem cell research holds great promise.