(3dw) From Skin to Nervous System : Epidermal Neural Crest Stem Cells, an Autologous, Multipotent Cell Source for Neurodegenerative Disorders

Neural crest (NC) cells play a central role in formation of the peripheral nervous system, craniofacial skeleton, and skin pigmentation 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 multipotent NC stem cells from human inter‐follicular keratinocyte (KC) cultures (termed 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)[Ref. 1]. Genome wide transcriptomic analysis, as well as single cell RNA-seq showed upregulation of NC-specific genes and cell differentiation trajectories indicative of the epidermal origin of these cells (Fig. 1b). Clonal analysis verified the clonal multipotency of KC-NC towards all NC specific lineages (Neu, SC, MC, SMC, Fig. 1c) with varying efficiencies. In addition to biochemical assays, the identify of each NC derivative was examined using functional assays, including electrophysiological tests for neurons, L-Dopa assays for MC and contractile force generation for SMC. Most notably, upon transplantation into chick embryos, KC-NC migrated along stereotypical pathways contributing to all NC-derivatives including neurons, Schwann cells, SMC and putative MC [Ref. 2]. Surprisingly, KC-NC could also be derived from aged adult donors (67-93 years old), maintained their multipotency in vitro and contributed to all NC derivatives in vitro as well as in ovo (Fig. 1d). In addition, CpG methylation analysis showed that the epigenetic age of KC-NC was significantly lower than that of KC and the chronological age of the donors [Ref. 3]. We are also examining the regenerative potential of OPCs by transplantation into the corpus callosum of the shiverer mouse model (shi/shi), an established model of demyelinating disease, using a hydrogel, conjugated with growth factors as a delivery mechanism. Our data so far show that OPCs migrated extensively and continued to proliferate a few weeks post-transplantation (Fig. 1e). Our current efforts focus on long-term transplantation experiments to determine the potential of these KC-NC for axonal myelination.

Given the accessibility of human skin and the high proliferation capacity of KC and KC-NC, these cells represent a potentially useful source of autologous multipotent stem cells for treatment of demyelinating diseases or spinal cord injuries. They could also be used to study diseases of the central or peripheral nervous system e.g. neurocristopathies, similar to hiPSC but without the need for reprogramming to the pluripotent state.

References

[1] Tseropoulos G., Andreadis S.T. et al., Derivation of neural crest stem cells from human epidermal keratinocytes requires FGF‐2, IGF‐1, and inhibition of TGF‐β1, Bioengineering & Translational Medicine 3 (3), 256-264, 2018

[2] Bajpai V.K., Tseropoulos G., Andreadis S.T. et al., Reprogramming postnatal human epidermal keratinocytes toward functional neural crest fates, Stem Cells 35 (5), 1402-1415, 2017

[3] Moghadasi Boroujeni S., Tseropoulos G., Andeadis S.T. et al. Neural crest stem cells from human epidermis of aged donors maintain their multipotency in vitro and in vivo, Scientific reports 9 (1), 1-12, 2019