(151a) From Skin to Nervous System – Epidermal Neural Crest Stem Cells, a Novel Autologous Candidate for Therapeutic Applications

Neural crest (NC) cells play a central role in forming the peripheral nervous system, the craniofacial skeleton, and the pigmentation of the skin during development due to their broad multilineage differentiation potential into neurons (Neu), Schwann cells (SC), melanocytes (MC), and smooth muscle cells (SMC). Recently, we identified an easily accessible source of pluripotent NC stem cells from human inter‐follicular keratinocyte (KC) cultures (KC‐NC) isolated from glabrous neonatal foreskin. Using small molecules FGF2 and IGF1 as well as inhibition of Tgf-β1 we are able to maintain SOX10+/FOXD3+ KC-NC in vitro (Fig. 1a). Genome wide transcriptomic analysis, as well as single cell RNA-seq provided evidence for the upregulation of NC specific genes and differentiation trajectories (Fig. 1b). The clonal multipotentency of the diverse KC-NC population was verified with in vitro clonal assays, further differentiated into NC specific lineages (Neu, SC, MC, SMC, Fig. 1c). Each differentiation was successfully subjected to functional assays involving electrophysiological tests for neurons, L-Dopa assays for MC and gel compaction assays for SMCs. In ovo transplantation further verified the multipotency of KC-NC with GFP+ KC-NC migrating into a variety of areas in the transplanted embryos, including axonal alignment with Tuj1+ nerve axons. Transitioning onto the more clinically relevant model of KC-NC from adult skin, we showed that, apart from slightly longer doubling times, adult KC-NC showed no other signs of cellular senescence, maintained their multipotency in vitro and migrated along stereotypical pathways and contributed to multiple NC derivatives in ovo (Fig. 1d), making them a promising candidate for autologous cell transplantation therapeutic strategies. In order to test the in vivo functionality of our KC-NC we adopted a CNS myelin deficient mouse model (shi/shi) and observed migration of KC-NC out of the lesion site in the corpus callosum and proliferation after the first weeks of transplantation (Fig. 1e). Taken together our work suggests that Epidermal Neural Crest Stem cells are a promising source of autologous transplantation to be tested for a variety of nervous system degenerative disorders.