390556 Direct Reprogramming of Skin Derived Stem Cells into Functional Neural Crest Stem Cell Fate
Introduction: Neural crest stem cells (NCSC) are multipotent cells that arise during embryonic development at the junction of neural and non-neural ectoderm and migrate laterally along the length of developing notochord (Sauka-Spengler & Bronner-Fraser, 2008), in process, giving rise to diverse cell types including peripheral neurons, Schwann cells, melanocytes, corneal endothelium, cartilage, bone, smooth muscle etc. In the present work, we provide first evidence that skin stem cells (SSC) can be coaxed to acquire NCSC fate under chemically defined conditions (without any genetic modification of the SSC) and resulting NCSC efficiently differentiate into functional neural crest (NC) lineages i.e. neurons, smooth muscle cells (SMC), osteocytes, chondrocytes, adipocytes, and endothelium.
Materials & Methods: Human neonatal foreskin SSC and adult skin SSC were isolated by separating epidermis from the dermis by enzymatic treatment and grown either on 3T3-J2 murine fribroblasts feeder (Rheinwald & Green, 1975) as colonies or in SSC serum free medium as feeder-free culture. SSC specific promoter driven puromycin cassette was cloned into a lentiviral vector and SSC were transduced and puromycin selected to isolate bona fide SSC. To induce SSC into neural crest fate, SSC were cultured in type1 collagen coated dishes in presence NC induction medium (NCIM) for 8-12 days. SSC derived neural crest stem cells (SSC-NCSC) were analysed transcriptionally (through global RNA sequencing (RNASeq) and quantitative real time PCR (qRT-PCR) as well as translationally (through flow cytometry, immunocytochemistry, and immunoblotting). To demonstrate that SSC possess the ability to undergo NC transformation at single cell level, clonal cultures of SSC were established and induced to acquire NC fate by NCIM treatment. To establish the multipotentiality of SSC-NCSC, they were further induced to differentiate into neuronal, myogenic, osteogenic, chondrogenic and endothelium fate by lineage specific differentiation medium (DM)/condition and characterized molecularly as well as functionally.
Results and Discussion: SSC promoter-Puro selected homogenous population of SSC (Fig1.A) grew as epithelial colonies and after 8-12 days of induction treatment small spindle shaped cells appeared to be delaminating from SSC colonies (Fig1. A). These emigrating cells were termed as SSC-NCSC due to their similarity with NCSC in terms of transcriptional and translational profile (Fig. 1B, 1C). The key NC markers i.e. SOX10, PAX3, NES, FOXD3, MSX1, IRX3, B3GAT1 and, NGFR were upregulated in SSC-NCSC while SSC markers were downregulated (Fig. 1B, 1C). Global RNASeq analysis shows SSC-NCSC clustered close to human embryonic stem cell (hSSC) derived NCSC. SSC-NCSC differentiated into mesenchymal lineages i.e. osteocytes, chondrocytes and adipocytes as evidenced by RT-PCR analysis and functional stains von kossa, oil red O and alcian blue, respectively (Fig1D, non-induced controls are in inset). SSC-NCSC also differentiated towards functional SMC as they expressed ACTA2, CNN1 and MYH11 (Fig1E, ACTA2) and fibrin based vascular media engineered using SSC-NCSC derived SMC displayed contractility in response to receptor mediated and non-receptor mediated vasoagonists. Approximately, 23% SSC-NCSC expressed CD31 and could uptake LDL and formed tube on Matrigel suggestive of endothelial potential of SSC-NC (Fig.1E). Finally, SSC-NCSC were successfully differentiated towards peripheral neurons as suggested by immunostaining (TUBB3, TH, NeuN, Peripherin) (Fig1.F, TUBB3). Electrophysiological analysis showed that SSC-NCSC derived neurons could generate action potentials upon stimulation indicating their functional maturity (Fig1.F). SSC colony derived from single cell could also be induced into NC fate suggesting clonal transdifferentiation potential of SSC. In addition, adult SSC from (45 year old, male) also gave rise to SSC-NCSC suggesting limited effect of aging on NC potential of SSC.
Conclusions: This is first report of direct reprogramming of SSC into functional neural crest fate which has implications in stem cell biology, direct reprogramming and regenerative medicine.