(528a) From Skin to Nervous System : Experimental and Bioinformatics Approaches Investigating Signaling in Neural Crest Stem Cells from Interfollicular Human Epidermis
Neural Crest cells (NC) play a central role in forming the peripheral nervous system, the craniofacial skeleton and pigmentation of the skin during development due to their broad multilineage differentiation potential into neurons, Schwann cells, melanocytes, and mesenchymal stem cells. Recently, we identified an easily accessible source of pluripotent neural crest stem cells from human inter-follicular keratinocyte (KC) cultures. Here, we investigate the importance of two growth factor, FGF2 and IGF1 in the induction of KC to NC, with respect, to the expression of potent NC markers Sox10 and FoxD3 and proliferation potential. Our approach contains both experimental and high-throughput genomic sequencing tools in order to shed light to the gene regulatory networks and pathways that govern our system. Using chemical inhibition and shRNA knockdown strategies, we uncovered that the downstream regulatory pathways AKT/PI3K, MEK/ERK and cJun are critical in Sox10 and FoxD3 regulation in our system. In addition, 10X single cell RNA-sequencing is employed to analyze the different stages of the KC to NC induction over time. After identifying clusters with discrete genetic profiles, such as KC expressing epidermal markers or pluripotent NC (towards the later days of induction), we compare our findings with genetic profiles from human embryonic stem cell or induced pluripotent stem cell-derived NC. This high-throughput RNA-seq analysis provides insights into the genes and pathways that play a critical role in our KC-NC system. In summary, our study provides a better understanding of the role of FGF2 and IGF1 on the induction of NC from KC cultures and sheds light on the pathways through which these growth factors regulate key NC transcription factors Sox10 and FoxD3, which can prove pivotal in sustaining the pluripotent phenotype of NC, rendering them a potent stem cell source for applications in cell therapies.