Exploring the Myelinogenic Potential of Human Keratinocytes Derived Neural Crest Cells: Implications for Demyelinating Diseases

Introduction: During embryonic development, neural crest stem cells (NCSC) migrate laterally along the length of developing notochord and give rise to diverse cell types (e.g. peripheral neurons, Schwann cells, melanocytes and skeletal and connective tissue). In this work, we have shown, for the first time, that human epidermal stem cells (also called keratinocytes (KC)) derived from neonatal and adult skin can be coaxed to acquire functional NCSC fate under defined culture conditions without any transgene overexpression. We further demonstrated using an in-vivo chicken embryo model that human KC derived neural crest cells (KC-NCSC) can migrate and differentiate into functional neural crest derivatives in the developing embryo. Finally, we differentiated KC-NCSC towards myelin producing Schwann cells, which were successfully implanted in the mouse model of dysmyelination to examine their potential to myelinate neurons.

 Materials & Methods: Human neonatal foreskin KC and adult skin KC were isolated and KRT14+  KC were induced to undergo neural crest fate conversion under an NC induction medium (NCIM) treatment for 8-12 days. KC derived neural crest stem cells (KC-NCSC) were analysed transcriptionally (through global RNA sequencing (RNASeq) and qRT-PCR) as well as translationally (through flow cytometry, immunocytochemistry, and immunoblotting). To demonstrate that KC possess the ability to undergo NC transformation at single cell level, clonal cultures of KC were established and induced to acquire NC fate. For chicken embryo experiments, we implanted KC-NCSC into cranial portion of 10-13 somite stage chicken embryos and examined embryos 2 and 4 days afterwards. To coax KC-NCSC to undergo Schwann cell differentiation, KC-NCSC were cultured in differentiation media containing FGF2, CNTF, GDNF, and NRG1 for 15-20 days. Further, we injected KC-NCSC derived Schwann cells into a Shiverer/Rag2 (-/-) mouse model after modifying them with lentivirus carrying mCherry reporter to allow us monitor mCherry+ cells after explantation. Mice were sacrificed at day 7 day and day 30 intervals and were studied for myelination using immunohistochemistry.

 Results and Discussion: After isolation, KC grew as typical epithelial colonies and after 8-12 days of induction treatment small spindle shaped cells appeared to be delaminating from tighter KC colonies (Fig1A). These were termed as KC-NCSC as they were uniformly positive for NC genes i.e. SOX10, FOXD3, PAX3, KIT, NGFR, NES and lacked KRT14/KRT5 (KC markers) as evidenced by transcriptional and translational analyses (Fig1B, C and D). KC-NCSC induction upregulated key epithelial to mesenchymal transition (EMT) genes like SNAI1, SNAI2, TWIST, FOXC2, VIM, CDH2 and downregulated CDH1. Illumina based RNA sequencing analysis showed that in contrast to KC, KC-NCSC possess global transcriptional profile similar to native NC as demonstrated by ROC analysis and 3D MDS plot (Fig1E). Clones of KC-NCSC expressed characteristic NC genes confirming clonal NC potential of KC. Under appropriate differentiation conditions, KC-NCSC differentiated and matured along functional neuronal, Schwann cell, melanocyte and mesenchymal linages as confirmed by mRNA and protein analysis as well as functional tests for each lineage. Upon implantation in chicken embryos, KC-NCSC migrated extensively and differentiated into multiple NC derivatives including neurons, glia, myocytes and melanocytes as confirmed by immunohistochemistry (results summary in Table1G). KC-NCSC efficiently differentiated into Schwann cell phenotype as confirmed by immunocytochemistry for key schwann cell genes like MPZ (P0), Myelin basic protein (MBP) and PLP as well as upregualtion of these genes at the transcriptional level (Fig1F).

Conclusions: In summary, this work establishes KC as novel source of bonafide NCSC and KC-NCSC derived Schwann cells, which were successfully transplanted into murine dysmyelinating disease model for studying their myelinogenic potential. This study has significant implications for the use of cellular therapies for treatment of devastating demyelinating diseases like multiple sclerosis.