Although the present study focuses predominantly on keratino

Although the present study focuses predominantly on keratinocytes, it has been known for many years that fibroblasts play an important role in wound healing and in remodeling the extracellular matrix. Thus, an ideal bioengineered graft will always need to incorporate fibroblasts as well as keratinocytes. The combination of keratinocytes and fibroblasts with a keratinocyte/fibroblast ratio of 1:9 in a spray device was shown clinically to be very effective in promoting wound closure (Goedkoop et al., 2010; Kirsner et al., 2012). However, the use of such growth-arrested corticotropin releasing factor still requires time to stimulate wound coverage, whereas keratinocytes and fibroblasts grown in a fibrin scaffold can be grafted instantly. Here, we have shown that fetal cells in such a combination grow well for at least 8 weeks in a human-to-mouse skin graft, suggesting that they are a viable option for covering open wounds. The improved method of preparing fibrin gels directly from whole plasma will be useful for constructing bioengineered skin equivalents to provide a more cost-effective and clinically suitable product. Fetal cells have also been shown to adapt well to various biocompatible materials with high survival rates (Montjovent et al., 2005; De Buys Roessingh et al., 2006), favoring their use in tissue engineering. De Buys Roessingh et al. (2006) reported that fetal fibroblasts are also resistant to various environmental stresses and low oxygen conditions, suggesting that they are likely to survive when grafted into hostile wound environments. There is much discussion about the potential of induced pluripotent stem cells (iPSCs) as an autologous cell source to generate large numbers of tissue cells for therapeutic applications, including the generation of keratinocytes (Guenou et al., 2009). When compared with the handling methods required for iPSCs and embryonic and mesenchymal stem cells, the isolation and cell-culture procedures used for fetal skin cells are technically less demanding. Expansion and maintenance of iPSCs in an undifferentiated state and during subsequent reprogramming require the addition of many specific growth factors, presenting a financial obstacle against upscaling of stem cell cultures for clinical applications. Unlike stem cells, fetal keratinocytes are already programmed for efficient, full epidermal differentiation, and also have high expansion potential and low immunogenicity. Although it is speculative at this point, another possible benefit of using immature cells such as those described here for grafting is that it may be possible to reinitiate appendage formation from fetal cells. The absence of appendages such as hair follicles and sweat glands is one of the most difficult consequences of large-scale grafting. Holbrook et al. (1993) and Holbrook and Minami (1991) reported that fetal skin tissue from a critical window of time (9–12 weeks gestation) could initiate follicle morphogenesis in vitro. In addition, primary cultures of mouse fetal and neonatal skin cells containing both epidermal and dermal cells will reform skin, complete with hair follicles, if transplanted into subcutaneous sites in the mouse (Yuspa et al., 1970; Worst et al., 1982). The factors that control sweat-gland development are even less understood, but recent publications have begun to address the nature of sweat-gland stem cells (Lu et al., 2012) and the interaction between progenitor cells and extracellular matrix in generating sebaceous glands (Horsley et al., 2006). Human-to-mouse culture grafts using cultured fetal cells as described here will be useful for studies on wound healing and diseases. Wound healing in adults usually results in scarring, which can cause functional restrictions in movement as well as negative physical and psychological effects on the patient. Formation of hypertrophic scars and keloids is also a burden and is difficult to treat medically. The developing fetus has a remarkable ability to heal skin wounds by regenerating normal epidermis and dermis with restoration of skin architecture, strength, and function in the absence of any scar formation (Bullard et al., 2003).