br Results We investigated the identity

Results We investigated the identity of human NPCs extracted from the foreskin dermis through fluorescence-activated cell sorting (FACS)-based isolation of cell subpopulations and subsequent characterization of the sorted myd88 pathway (Figure 1). Immunofluorescent and flow-cytometry analyses of sphere cultures showed the existence of a rare p75NTR (also known as Gp80-LNGFR, CD271, and TNFRSF16)-positive cell subpopulation (2.9% ± 1.7% of total; n = 24) in primary dermal sphere cultures that was Nestin+ and CD34− and presented a characteristic morphology (i.e., a single process extending from the soma; Figure 1B; Movie S1 available online). Intriguingly, 74.6% ± 2.9% of p75NTR+ cells coexpressed SOX2 by immunofluorescence analyses (Movie S2), indicating that p75NTR+ cells might be more precursor-like. Isolation of p75NTR+CD34− cells enabled a significant enrichment of precursors committed to the neural lineage in vitro, as assessed by an average 22.7-fold increase (18.2% versus 0.8% of total cells; n = 6) in their neural differentiation capacity when compared with p75NTR-CD34− cells (Figure 1C). To determine whether p75NTR+ cells exhibit the same differentiation potential in ovo, we sorted, expanded, and injected human cells into the neural crest migratory stream (Hamburger-Hamilton stage 17 [HH17] chicken embryos, hindlimb-level somites; Figure 2A). Cross-sections of HH26 embryos, immunolabeled with anti-human nuclei (anti-HuNu) for the detection of transplanted cells, showed that the human p75NTR+ cells survived; migrated to the neural crest, dorsal root ganglia (DRG), and skin; and differentiated into βIII tubulin+ cells (Figures 2B–2F). Since βIII tubulin was recently reported to mark human melanocyte lineage cells, as well as peripheral neurons in the dermis (Locher et al., 2013), we confirmed the peripheral neuronal phenotype of HuNu+ cells by showing coexpression of neurofilament 200 (NF200; Figures 2G–2I). These markers were not present in the original dermal cultures (Figure S1). In contrast, p75NTR− cells were hardly detected at the HH26 stage, if at all, and most of the surviving cells did not express any of the aforementioned markers (Figures 2J and 2K; data not shown). A quantification of transplant-derived HuNu+ cells showed that, as expected, p75NTR+ NPCs showed increased survival, migration to peripheral tissue, and neural differentiation when compared with both unsorted and p75NTR− cell fractions. On average, 10.8-fold more p75NTR+ cells survived, migrated to peripheral tissue, and differentiated into peripheral neurons than p75NTR− cells (Figure 2K). These results confirmed the neurogenic fate of dermis-derived p75NTR+ cells in vivo.
Discussion This study provides experimental evidence that primary dermospheres obtained from human skin contain a mixture of SOX2+ multipotent precursors and more committed cells, with SOX2+ cells being derived from either Schwann or perivascular cells. It also suggests that SOX2 expression levels are tightly regulated in dermal precursors and determine the ability of these cells to generate neural progeny. Embryonic Schwann cell markers such as CDH2 and CDH19 are known to be sharply downregulated in Schwann embryonic development at the iSC stage (Takahashi and Osumi, 2005; Wanner et al., 2006), and thus upregulation of these mRNAs in adult Schwann cells is unexpected (Mirsky et al., 2002). It is tempting to speculate that their appearance in the double-positive cells suggests a resident precursor cell status; alternatively, it may reflect Schwann cell dedifferentiation as a result of dermal disaggregation procedures (Jessen and Mirsky, 2008; Parrinello et al., 2010). Of note, recent murine data demonstrated that cellular dedifferentiation plays a major part in SOX2 upregulation in these cells (Johnston et al., 2013). The extent to which cellular dedifferentiation is related to the appearance of human dermis-derived NPCs in sphere culture and beyond remains to be determined.