Introduction

Skin is the largest organ of the human body. It provides the first line of defense against infectious agents, and prevents dehydration and injury. Skin has a number of appendages, such as hair, sebaceous and sweat glands, that also aid in the protection of inner organs from environmental assaults. Therefore, it is of extreme importance to maintain a healthy skin, which could be achieved through replacing damaged skin cells.

The cartoon shown in Figure 1 depicts the structure of the skin. Shown, are two layers: the dermis or inner layer that originates from mesodermal embryonic cells, and the epidermis or outer layer that are formed from ectodermal embryonic cells. These two layers are separated from each other by a basement membrane. Epidermis is a stratified tissue composed of cells called keratinocytes. The layer of keratinocytes that is closest to the basement membrane is called basal, and contains a number of constantly proliferating cells. It is these cells that migrate to the surface and form a protective layer, maintaining the outer covering of epidermis intact (2).

Epidermal stem cells are located in the basal layer of the epidermis. These cells give rise to transient amplifying cells that migrate to the surface and form a protective layer.

Transient amplifying cells

Several studies have been done to identify the location of epidermal stem cells. One of the methods was based on the fact that if they a cell is indeed a stem cell, it should have prolonged doubling time. The slow-cycling attribute is particularly important with regards to biology because it conserves the cells' proliferative potential. In addition minimal DNA replication would lead to reduced mutations from insults such as UV radiation. Pulse-chase experiments were conducted that labels the epidermal cells so that they can be tracked. The findings, depicted in Figure 2, showed that the cells that rarely divide will retain the label. These cells are the source of transient amplifying cells (TA cells). The TA cells divide three to five times before they terminally differentiate. The main function of TA cells is to increase the number of differentiating cells produced by one division of the stem cell. Transient amplifying cells allow the epidermal stem cells to remain quiescent in order to minimize the occurrence of mutations (3, 5). Thus, TA cells are descendents of stem cells in the epidermis and are responsible for the dynamic tissue renewal under normal conditions as well as tissue repair after wounding (4, 6).

Epidermal stem cells undergo mitosis, producing two daughter cells. One daughter cell retain the properties of the `mother' stem cell while the other daughter cell differentiate into a TA (Transient Amplifying)- cell. The TA cells are capable of generating large numbers of specialized differentiated cells within the epidermis. The entire process is repeated, while maintaining the integrity of the original stem cells, which are the source of skin renewal.

Location

Morasso and Tomic-Canic (2) indicate that there are three main locations in the adult skin where epidermal stem cells can be found; the bulge region of the hair follicle, the interfollicular epidermis (IFE), and the sebaceous gland (Figure 3). Further investigation showed that the bulge region contains cells that have the ability to give rise to the stem cells in the IFE region and sebaceous gland, indicating that basal (bulge region) stem cells are more primitive than their counterparts in the other two areas (2). Indeed, basal stem cells of the adult hair follicle can participate in tissue repair by responding to signals to regenerate the epidermis, hair follicles, and sebaceous glands (7).

Epidermal stem cells are shown the bulge region of the hair follicle, the interfollicular epidermis, and the sebaceous gland. However, the bulge region is thought to have stem cells that are more primitive than the other two areas.

Markers of Epidermal Stem Cells:

To aid in the identification of epidermal stem cell, ongoing research studies are in the process characterizing the signature of these cells. Specific markers identified for epidermal stem cells are shown in Table 1.

Table 1: Molecules associated with Epidermal Stem Cells.

EpSC: Epiderman Stem Cells

Intracellular Signaling in Epidermal Stem Cells

Wnt/Wingless signaling pathway directs cell fate in a variety of cell types and has an integral role in organogenesis. At the core of the pathway is the stability of ß-catenin, an essential molecule in Wnt/wingless signaling. Upon Wnt signaling, ß-catenin accumulates in the cytoplasm and is transported to the nucleus, where it interacts with members of the LEF/TCF family of transcription factors to activate gene expression. Mutation of ß-catenin, among other factors, results in the accumulation of cytoplasmic and nuclear ß-catenin, and constitutive signaling with concomitant gene activation. Such mutations have been linked to tumorigenesis, and are frequently observed in human tumors. For instance, in human skin tumors, pilomatricomas, ß-catenin is mutated in a high percentage of the analyzed cases (24).

THE FOLLOWING EXPANDS ON CONCEPTS DISCUSSED ABOVE:

Properties of Epidermal stem cells:

Self renewal

Multipotency and the ability to self-renew are general characteristic of stem cells. When transplanted into an athymic mouse, stem cells from the bulge region of the hair follicle are able to reconstitute epidermis, in the case of the appropriate environment (1). In addition, these keratinocytes are also able to form hair follicles and sebaceous glands (17). Moreover, cultured epidermal stem cells can give rise to "increasing number of holoclones" in vitro, demonstrating self-renewal ability of these cells. Also, epidermal stem cells are able to maintain multipotency and self-renewal ability in vitro longer than many stem cells of different origin (14).

Asymmetric Division

The ability to divide asymmetrically is a hallmark of stem cells since this type of division could preserve the `stem' functions, while reducing mutation. The rare division of stem cells give rise to, on average, one stem cell and one transient amplifying cell, which has a limited proliferative potential. Upon exhaustion of their proliferative potential, the rapidly proliferating TA cells undergo terminal differentiation. Blanpain et al (16) demonstrated the presence of two multipotent subsets of cells in the bulge region of the hair follicle; basal and suprabasal. The first maintains its attachment to basal lamina and another is detached from the extracellular matrix. Presence of the suprabasal population is detected during anagen phase, or active phase of hair follicle growth cycle. Suprabasal cells have altered their patterns of gene expression, and have entered an early "commitment phase" (17). As differentiation progresses, the suprbasal cells detach from basal lamina and rapidly proliferate before terminal differentiation. At this stage they are called transient amplifying cells.

The ability of epidermal stem cells either to remain in quiescent state or differentiate is dictated by the microenvironment of their niche. Bulge region is the "deepest and most protected place" in the epithelial layer, and as such is an ideal place for epidermal stem cells niche (14). Two factors, FGF18 and BMP6, present in this region have been shown to maintain cell quiescence (17). When dermal papilla, cells of mesenchymal origin contact bulge region, some of the epidermal stem cells differentiate giving rise to the hair follicle (14). Keratinocytes that reside in the bulge region interact with other cells, such as melanocytes, Merkel cells and arrector pili muscle cells. These interactions might play a role in maintaining a niche where the epidermal stem cells reside (18). Moreover, when engrafted, the bulge cells can assemble a new niche (17).

Multipotency

Epidermal stem cells and hair follicle stem cells were previously thought to be unipotent, generating only keratinocytes. The notion is that the lineage potential of adult stem cells is restricted to the tissue of origin. However, this has been recently challenged in studies showing epidermal stem cells generating cells of different lineages. In fact, skin-derived precursor cells during embryonic development persist in the adult thereby exhibiting primitive self-renewal properties (23). Recent studies have shown that dermal cells express markers of neurons, glia, smooth muscle cells, and adipocytes when grown in defined culture conditions (25). Thus, plasticity is another factor that is characteristic of epidermal stem cells.

Therapeutic Potential of Epidermal Stem Cells:

Wound repair

Epidermal stem cells play a central role in homeostasis and wound repair, and represent a cellular sources of tumor initiation and as a vehicle for gene therapy. These stem cells also have the potential for treating burn victims. Figure 4 demonstrate hair follicle epidermal stem cells that have been used in preparing skin equivalents, forming epithelium in deep burn wounds after implantation (22). These experiments show hope of being able to culture autologous epithelial grafts in vitro within a short time for implantation into patients. As they show multipotency to differentiate into almost all epithelial cell types, hair follicle stem cells can be used in preparing composite skin substitutes. Li et al (24) demonstrated that epidermal stem cells can regenerate a fully stratified epidermis by in vitro methods. Other studies found that the progenies of epidermal stem cells (transient amplifying and differentiating cells) have regenerative capacity as well (26).

Gene Therapy

Epidermal stem cells also hold promise for gene therapy because of their self- renewal ability. If they can be transduced with the vehicle containing the gene of interest, theoretically they should be able to maintain the gene replacement throughout the individual's life (25). Epidermolysis bullosa is an inherited skin disease that could be cured by this research. Researchers found that epidermal stem cells could be stably transduced with replication-defective retroviral vectors, and that this caused full phenotypic correction of the adhesion properties of JEB keratinocytes in rats (26). Other researchers used cultured human keratinocytes to correct this inborn metabolic skin disease in nude mice. They found two methods that were effective in correcting the genetic defect in the nude mice. They first harvested keratinocytes from patients with epidermolysis bullosa. Then correction was obtained either by genomic integration of the correct sequence using a bacteriophage integrase, or by transgene expression using a lentivirus (1). Other research has been done that shows effective gene transfer. This includes a protocol that yields consistently high retroviral gene transfer on a substrate of recombinant fibronectin (27). Cell-based therapy is another potential clinical benefit. Genetically engineered epidermal stem cells can be injected into the body and secrete proteins in the bloodstream for the lifetime of the organism. Researchers found that they increase the restoration of blood flow when injected into an ischemic murine hindlimb (28).

Hair Restoration

Epidermal stem cells can also form hair follicles and have the potential to treat baldness. Researchers at the Howard Hughes Medical Institute isolated murine epidermal stem cells, and showed that a single epidermal stem cell can differentiate into skin, hair and oil glands. Elaine Fuchs and colleagues took stem cells from normal mice, and grafted them on the backs of hairless mice. They were able to show the generation of normal skin with hair (16)

Tumorgenic Potential of Epidermal Stem Cells

The location of the epidermis and its constant exposure to ultraviolet radiation of the sun makes epidermal stem cells an important focal point for research on cancer. Stem cells are quiescent, rarely divide, and thus run the risk of DNA replication errors and oncogenic mutations, which can be the source for melanoma and cancer. Researchers at the University of Pennsylvania School of Medicine found a rare group of genes that can both determine and restrict the fate of the stem cell, and maybe involved in tumor development. Pax3, a pangene, causes the switch that leads to the formation of melanocytes while prevent differentiation. It appears that the targets of skin tumor initiation are stem cells found in the hair follicles, and the interfollicular epidermis (29). Other researchers devised a two-step model for generating a tumor in epidermal stem cells. These steps are initiation and promotion, and they showed that the initiation with benz(o)pyrene results in genetic damage, while the promoter stimulates the damaged cell to proliferate, leading to cancer (30). Further research into the relationships between these cells and cancer could lead to great discoveries that could benefit many patients.

Conclusion

Epidermal stem cells are necessary for the maintenance of the skin. They continually self- renew and differentiate into keratinocytes in order for the skin to protect against insults. Epidermal stem cells have been found to be located in the interfollicular epidermis, the bulge region of the hair follicle, and sebaceous gland. These cells maintain "stemness" by asymmetrical division. Transient amplifying (TA) cells derive from the epidermal stem cell and allow the stem cell to remain quiescent by producing differentiated cells and terminally differentiating approximately after three to five cycles. Epidermal stem cells continuously undergo self-renew to maintain the integrity of the epidermis. Ongoing research studies are in the process of identifying markers of these stem cells, although a specific marker has not been identified.

Epidermal stem cells have been demonstrated to be useful for therapeutic purposes. Due to their plasticity, it may be possible these stem cells have the ability to treat injured tissue throughout the body. Epidermal stem cells show great promise in treatment of burn patients, which is supported by results showing the ability of these cells of the hair follicle to regenerate epithelium of deep burn wounds. These cells would be excellent for gene therapy due to their self - renewing capacity, which has been demonstrated in patients suffering from epidermolysis bullosa. Understanding these stem cells is important for develop an understanding of cancer. Also, these cells may be useful in the treatment of baldness. It is important for scientists to conduct further research to fully understand epidermal stem cells in order to use these cells as treatment.

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Acknowledgements

This review was prepared by the following graduate students in the Stem Cell Biology Class, Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey:

Diane Motto, Irina Ogan, Shobana Shanmugasundaram, Justin Sweet, Tiffany Toliver

Teaching Assistant: Marianne Castillo

The review was edited by two stem cell biologists.