thymidine phosphorylase inhibitor br Results br Discussion H

Results
Discussion Human fetal dorsal skin, from which the fetal keratinocytes were cultured for this study, was analyzed histologically to correlate morphologic changes of the skin to biochemical changes in structural proteins during development. K18, which along with K8 is typical of simple epithelia and early embryonic stages (Moll et al., 1982), and K19, which is expressed in adult mixed epithelial regions and possibly is a stem cell niche indicator (Stasiak et al., 1989), were still expressed in the basal epidermal layer and periderm of fetal skin up until 22 weeks. K17, which is typically expressed by “activated” keratinocytes, was present in fetal epidermis but reduced with increasing age. If fetal thymidine phosphorylase inhibitor are ultimately to be used for clinical applications, quality-control measures will be needed to ensure that the cells being propagated retain their defined state. Thus, it is significant that the fetal phenotype persists in tissue culture, as shown by the retention of fetal keratin expression in culture. Fetal keratinocyte cultures can therefore be distinguished from their adult counterparts by expression of K18 and K19. We have shown that fetal keratinocytes can be stably and successfully cultured in vitro while maintaining their normal phenotype and karyotype. No slowing of growth was observed until cells were beyond 20 pds, about twice as many cell divisions as observed for similar adult cells. This significantly higher proliferative potential suggests that fetal cells can provide a long-lived (and thus more economical and more accessible to a greater number of patients) cell source for tissue-regeneration applications. This will facilitate exhaustive characterization of a single fetal keratinocyte bank prior to clinical use. By using the isolation and culture techniques described here, one can induce a 4 cm2 sample of fetal skin to generate sufficient cells to expand to an area of 16 m2 within 1 week of recovering live cells from a frozen cell bank (Figure S5B). Here, we cryopreserved cells using a progressive freezing method and achieved a recovery of >80% even after 3 years of liquid nitrogen storage, showing that these cells are robust in tissue culture. We have achieved this efficiency using serum-free culture without mouse-derived fibroblast feeder cells; therefore, the process should be easily and quickly adapted to meet GMP culture requirements. With this yield and efficiency, additional steps to enrich for stem cells may be unnecessary—anything that reduces handling will increase cell viability and thus further increase cell yield. In spite of the developmental immaturity of the starting material, second-trimester fetal keratinocytes are clearly capable of achieving fundamentally normal adult-type differentiation in vitro, as they can form a stratified epidermal structure in an organotypical culture system that expresses major structural proteins of adult epidermis. Proof of principle was established in a preclinical human-to-mouse model using immune-deficient mice (Del Rio et al., 2002) optimized for grafting cultured human fetal keratinocytes and fibroblasts. The successful engraftment and stable skin regeneration achieved using cultured fetal skin cells show that these cells can generate mature, differentiated epidermis in vivo. The biggest obstacle to skin grafting using anything other than the patient’s own cells is immune rejection. The data presented here reveal low MHC I expression and no MHC II expression in the fetal skin cells. The fetal cells were also shown to elicit no proliferative response in naive T cells. Coculture with fetal keratinocytes or fetal fibroblasts even led to suppression of T cell proliferation. This may be due to production of factors with immunosuppressive activity (Kehrl et al., 1986; Lúdvíksson et al., 2000; Taylor et al., 2006) or other mechanisms that operate in the state of mutual immune tolerance that exists between the fetus and mother during pregnancy (Munn et al., 1998; Meisel et al., 2004; Hunt et al., 2005). The effect of fetal skin cells on regulatory T cells (Treg), which are capable of modulating tolerance in the immune response (Sakaguchi et al., 2001), may also play a role. In a previous study, fetal liver mesenchymal stem cells were shown to exhibit various levels of inhibitory immune effects (Götherström et al., 2004). In a related study, Zuliani et al. (2013) recently reported evidence of suppression of peripheral blood mononuclear cell (PBMC) proliferation in a sample of fetal keratinocytes, although they made no comparison with adult cultures. These authors suggested a role for indoleamine 2,3 dioxygenase (IDO) in the immunosuppressive effects. The in vitro data presented here suggest that fetal keratinocytes may have an “immunological advantage” that could be of significant benefit in future clinical applications of these cells.