(84f) 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.