(770d) The Role of FGF2 in Reprogramming of Epidermal Keratinocytes Toward Neural Crest Fat

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 differentiation diversity these cells have been a focal point of research for disease therapeutics via cell transplantation. However, an easily accessible autologous cells source for therapeutic cell transplantation remains one of the main challenges facing the field. Recently our lab discovered that human epidermal inter-follicular keratinocytes (KC) can turn into neural crest (NC) stem cells without ectopic expression of transcription factors or reprogramming to the pluripotent state. Here we studied the role of FGF2 in this novel phenomenon.

Keratinocytes were isolated from glabrous foreskin of 1-3 day old neonates and exposed to KC-NC induction medium containing FGF2 growth factor. FGF receptor knockdown through lentiviral transfection was used, as well as chemical inhibitors for various signaling pathways. Clonal density experiments were performed using single keratinocyte cells to create a pure KC population that can give rise to NC. NC were further differentiated into Schwann cell, peripheral neuron, melanocyte and smooth muscle cell lineages. Gene expression was assessed with RNA-Seq and validated through quantitative real time RT-PCR. On protein level immunocytochemistry was used in order to detect markers in NC and differentiated lineages.

Our results show that FGF2 is necessary for KC-NC reprogramming (Fig:1A), as evidenced by use of chemical inhibitors as well as gene knockdown using shRNA. We identified the FGF receptor that mediates the signaling as well as subsequent pathways including Akt. Ultimately, FGF signaling leads to expression of the NC-specific transcription factor, SOX10, which is necessary for NC reprogramming. The newly produced NC express NC genes including SOX10, Nestin, Pax3, FoxD3 (Fig:1B) and could be coaxed to differentiate into peripheral neurons, Schwann cells, melanocytes and smooth muscle cells as shown by molecular as well as functional assays.

In conclusion, we provide mechanistic insight into the process of keratinocyte reprogramming to neural crest stem cells. This work represents a paradigm shift in stem cell biology as it demonstrates the unusual plasticity of human adult KC that can turn into many different cells types without genetic modification or reprograming to the pluripotent state. The dearth of cell sources for treatment of neurogenic disorders, combined with the accessibility and growth potential of human epidermal cells suggest that the proposed work could have tremendous implications for the use of cell therapy for treatment of many debilitating diseases.