1. Keratinocytes

There are two approaches to commit ES cells and adult stem cells (of non-epidermal origin) to the keratinocyte lineage in vitro. One approach would be to expose the cells to a cocktail of exogenous cytokines, growth factors, chemicals, and extracellular matrix (ECM) substrata over a prolonged duration of in vitro culture. Only a fraction of the stem cells would be expected to undergo commitment to the keratinocyte lineage, because many of these cytokines, growth factors, chemicals, and ECM substrata would exert non-specific pleitropic effects on stem cell differentiation into multiple lineages. At best, the cocktail combination of various cytokines, growth factors, chemicals, and ECM substrata can be optimized by trial and error, to maximize the proportion of stem cells committing to the keratinocyte lineage, while at the same time yielding a large number of other undesired lineages. Hence, extensive selection/purification and proliferation of the commited keratinocyte progenitors is likely to be required.

By using such an approach, Coraux et al.54 managed to achieve commitment and subsequent differentiation of murine ES cells into the keratinocyte lineage, in the presence of a cocktail combination of bone morphogenetic protein-4 (BMP-4), ascorbate, and ECM derived from human normal fibroblasts (HNFs) and murine NIH-3T3 fibroblasts. Nevertheless, it must be noted that the study of Coraux et al.54 also reported a high degree (approximately 80%) of non-specific differentiation into multiple uncharacterized lineages, and no attempt was made to purify differentiated keratinocytes or keratinocyte progenitors from the mixture of lineages derived from murine ES cells. Bagutti et al.61 reported that coculture with human dermal fibroblasts (HDFs) as well as HDF-conditioned media could induce beta integrin- deficient murine ES cells to commit and differentiate into the keratinocyte lineage. However, as with the study of Coraux et al.,54 the keratinocytes were interspersed with differentiated cells of other lineages. Recently, differentiation of human ES cells into the keratinocyte lineage was also reported by Green et al.62 However, this study was based on in vivo teratoma formation within a SCID mouse model, and to date, there are no parallel in vitro studies that have been reported.

With adult stem cells of non-epidermal origin, there are also few studies 63, 64 which have successfully achieved re-commitment and trans-differentiation to the keratinocyte lineage. Even so, these studies were based primarily on the transplantation of undifferentiated stem cells in vivo, with the observed trans-differentiation occurring sporadically and at extremely low frequencies. Moreover, the validity of the experimental data may be clouded by controversy over the artifact of stem cell fusion in vivo.65 To date, there are no parallel in vitro studies that have achieved recommitment and trans-differentiation of non-epidermal adult stem cells to the keratinocyte lineage. It can therefore be surmised that the use of exogenous cytokines, growth factors, chemicals, and ECM substrata to induce ES cell and nonepidermal adult stem cell commitment to the keratinocyte lineage is a relatively inefficient, time-consuming, and labor-intensive process that would require extensive selection and purification of the committed keratinocyte progenitors. Hence, it would be technically challenging to apply this to the clinical situation.

The other approach for inducing ES cell and non-epidermal adult stem cell commitment to the keratinocyte lineage is through genetic modulation. This may be achieved by transfecting stem cells with recombinant DNA constructs encoding for the expression of signaling proteins that promote commitment to the keratinocyte lineage. Of particular interest are the Lef-1/Tcf family of Wnt regulated transcription factors that act in concert with b-catenin,66, 67 c-myc which is a downstream target of the Wnt-signaling pathway,68, 69 and the transactivation domain containing isoform of transcription factor p63 (Tap63).70, 71 Interestingly, the transcription factor GATA-3, which is well known to be a key regulator of T-cell lineage determination, has also been shown to be essential for stem cell lineage determination in skin, where it is expressed at the onset of epidermal stratification and Inner Root Sheath (IRS) specification in follicles.72 Recombinant overexpression of p6373 and c-Myc74 has been reported to promote commitment and differentiation to the keratinocyte lineage.

The disadvantage of directing differentiation through genetic modulation is the potential risks associated with utilizing recombinant DNA technology in human clinical therapy. For example, the overexpression of any one particular protein within transfected stem cells would certainly have unpredictable physiological effects upon transplantation in vivo. This problem may be overcome by placing the recombinant expression of the particular protein under the control of switchable promoters, several of which have been developed for expression in eukaryotic systems. Such switchable promoters could be responsive to exogenous chemicals,75 heat shock,76 or even light.77 Genetically modified stem cells may also run the risk of becoming malignant within the transplanted recipient. Moreover, there are overriding safety concerns with regard to the use of recombinant viral based vectors in the genetic manipulation of stem cells.78 It remains uncertain as to whether legislation would ultimately permit the use of genetically modified stem cells for human clinical therapy. At present, the potential detrimental effects of transplanting genetically modified stem cells in vivo are not well studied. More research needs to be carried out on animal models to address the safety aspects of such an approach.

More recently, there is emerging evidence that some transcription factors (which are commonly thought of as cytosolic proteins) have the ability to function as paracrine cell to cell signaling molecules.79 This is based on intercellular transfer of transcription factors through atypical secretion and internalization pathways.79 Hence, there is an exciting possibility that transcription factors implicated in commitment to the keratinocyte lineage may in the future be genetically engineered to incorporate domains that enable them to participate in novel paracrine signaling mechanisms. This in turn would have tremendous potential for inducing the commitment of ES cells and non-epidermal adult stem cells to the keratinocyte lineage.

Skin appendages, including hair follicles, sebaceous glands and sweat glands, are linked to the epidermis but project deep into the dermal layer. The skin epidermis and its appendages provide a protective barrier that is impermeable to harmful microbes and also prevents dehydration. To perform their functions while being confronted with the physicochemical traumas of the environment, these tissues undergo continual rejuvenation through homeostasis, and, in addition, they must be primed to undergo wound repair in response to injury. The skins elixir for maintaining tissue homeostasis, regenerating hair, and repairing the epidermis after injury is its stem cells.

The hair follicle is composed of an outer root sheath that is contiguous with the epidermis, an inner root sheath and the hair shaft. The matrix surrounding the dermal papilla, in the hair root, contains actively dividing, relatively undifferentiated cells and is therefore a pocket of MSCs that are essential for follicle formation. The lower segment of each hair follicle cycles through periods of active growth (anagen), destruction (catagen) and quiescence (telogen).80 A specialized region of the outer root sheath of the hair follicle, known as the bulge, is located below the sebaceous gland, which is also the attachment site of the arrector pili muscle, receiving inputs from sensory nerve endings and blood vessels. Furthermore, the hair follicle bulge is a reservoir of slow-cycling multipotent stem cells.81, 82 Subsets of these follicle-derived multipotent stem cells can be activated and migrate out of hair follicles to the site of a wound to repair the damaged epithelium; however, they contribute little to the intact epidermis. These hair follicle stem cells can also contribute to the growth of follicles themselves and the sebaceous gland. For example, in the absence of hair follicle stem cells, hair follicle and sebaceous gland morphogenesis is blocked, and epidermal wound repair is compromised.83 In addition to containing follicle epidermal stem cells, the bulge contains melanocyte stem cells.84 Recent studies show that nestin, a marker for neural progenitor cells, is selectively expressed in cells of the hair follicle bulge and that these stem cells can differentiate into neurons,85 glia, keratinocytes, smooth muscle cells, melanocytes and even blood vessels.86, 87 Examination of close developmental and anatomical parallels between epithelial tissue and dermal tissue in skin and hair follicles has revealed dermal tissue to have stem cells. Paus et al. indicated that hair follicle dermal sheath cells might represent a source of dermal stem cells that not only incorporate into the hair-supporting papilla, low down in the follicle, but also move up and out from the follicle dermal sheath into the dermis of adjoining skin.88 Hair follicle dermal sheath cells taken from the human scalp can form new dermal papilla, induce the formation of hair follicles, and produce hair shafts when transplanted onto skin.89 There is also a clear transition from dermal sheath to dermal papilla cells.90 When the follicle dermal cells are implanted into skin wounds, they can be incorporated into the new dermis in a manner similar to that of skin wound-healing fibroblasts.91 However, these cell populations still lack specific markers for purifying and distinguishing the stem cells from their progeny. Furthermore, of prime importance is improving our understanding of the relation between bulge cells and interfollicular epidermal stem cells and between bulge cells and other stem cells inhabiting the skin and the mechanisms of hair growth.

Recently, cell replacement therapy has offered a novel and powerful medical technology for skin repair and regeneration: a new population of stem cell, called a neural crest stem cell, from adult hair follicles, was discovered to have the ability to differentiate in vitro to keratinocytes, neurons, cartilage/bone cells, smooth muscle cells, melanocytes, glial cells, and adipocytes.9296 In mammalian skin, skin-derived neural progenitors were isolated and expanded from the dermis of rodent skin and adult human scalp and could differentiate into both neural and mesodermal progeny.97, 98 Skin-derived neural progenitor cells were isolated based on the sphere formation of floating cells after 37 days of culture in uncoated flasks with epidermal growth factor and fibroblast growth factor, and characterized by the production of nestin and fibronectin, markers of neural precursors. In addition, skin-derived neural progenitor cells were identified as neural crest derived by the use of Wnt1 promoter driving LacZ expression in the mouse. Some of the LacZ-positive cells were found in the skin of the face, as well as in the dermis and dermal papilla of murine whisker.99 These skin derived neural crest cells have already shown promising results in regenerative medicine such as the promotion of regenerative axonal growth after transplantation into injured adult mouse sciatic nerves 95 or spinal cord repair,100 resulting in the recovery of peripheral nerve function. This new study marks an important first step in the development of real stem-cell-based therapies and skin tissue regeneration.

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Stem Cells for Skin Tissue Engineering and Wound Healing