Reprogramming postnatal human epidermal keratinocytes toward functional neural crest fates

Reprogramming postnatal human epidermal keratinocytes

toward functional neural crest fates

Vivek K. Bajpai

1,#

Laura Kerosuo

2,*

Georgios Tseropoulos

1,*

Kirstie A. Cummings

3,*

Xiaoyan Wang

Pedro Lei

Biao Liu

4,5

Song Liu

4,5

Gabriela Popescu

Marianne E.

Bronner

, and

Stelios T. Andreadis

1,6,7,§

Department of Chemical and Biological Engineering, University at Buffalo, Buffalo, NY 14260

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA

91125

Department of Biochemistry, Neuroscience Program, School of Medicine and Biomedical

Sciences, University at Buffalo, Buffalo, NY 14214

Center for Personalized Medicine, Buffalo, NY 14263

Dept. of Biostatistics and Bioinformatics Roswell Park Cancer Institute, Buffalo, NY 14263

Department of Biomedical Engineering, University at Buffalo, NY 14260

Center of Excellence in Bioinformatics and Life Sciences, Buffalo, NY 14263

Abstract

During development, neural crest cells are induced by signaling events at the neural plate border of

all vertebrate embryos. Initially arising within the central nervous system, neural crest cells

subsequently undergo an epithelial to mesenchymal transition to migrate into the periphery, where

they differentiate into diverse cell types. Here we provide evidence that postnatal human epidermal

keratinocytes, in response to FGF2 and IGF1 signals, can be reprogrammed toward a neural crest

fate. Genome-wide transcriptome analyses show that keratinocyte-derived neural crest cells are

similar to those derived from human embryonic stem cells. Moreover, they give rise

in vitro

and

vivo

to neural crest derivatives such as peripheral neurons, melanocytes, Schwann cells and

Address for all Correspondence: Stelios Andreadis, Ph.D., Professor, Bioengineering Laboratory, 908 Furnas Hall, Department of

Chemical and Biological Engineering, Department of Biomedical Engineering, and Center of Excellence in Bioinformatics and Life

Sciences University at Buffalo, The State University of New York, Amherst, NY 14260-4200, USA, Tel: (716) 645-1202, Fax: (716)

645-3822, sandread@buffalo.edu.

Current Address: Department of Chemical and Systems Biology, School of Medicine, Stanford University, Stanford, CA, 94305

These authors contributed equally to the work.

AUTHOR CONTRIBUTIONS

V.K.B. conceptualized and designed the study and performed the experiments. V.K.B., S.T.A.

designed experiments and performed data analysis and interpretation. L.K., M.E.B. performed chicken embryo experiments. K.A.C.,

G.P. did electrophysiological analysis. G.T., X.W., P.L. performed experiments. B.L., S.L., V.K.B. did RNA sequencing analysis. All

authors read the manuscript and provided their inputs. V.K.B., S.T.A., M.E.B. wrote the manuscript.

CONFLICT OF INTERESTS

None

ACCESSION NUMBERS

GEO: GSE72268

SUPPLEMENTARY INFORMATION

Supplemental figures and list of antibodies and primer pairs used in this study are provided as supplementary information.

HHS Public Access

Author manuscript

Stem Cells

. Author manuscript; available in PMC 2018 May 01.

Published in final edited form as:

Stem Cells

. 2017 May ; 35(5): 1402–1415. doi:10.1002/stem.2583.

Author Manuscript

mesenchymal cells (osteocytes, chondrocytes, adipocytes and smooth muscle). By demonstrating

that human KRT14+ keratinocytes can form neural crest cells, even from clones of single cells, our

results have important implications in stem cell biology and regenerative medicine.

Graphical abstract

In this study we demonstrate that human epidermal keratinocytes (KC) can be efficiently

reprogrammed to acquire neural crest (NC) fate in response to FGF2 and IGF1 signals. Clonal KC

gave rise to NC cells suggesting KC possess NC competence at the single cell level. KC derived

NC (KC-NC) differentiated into multiple NC derivatives both in vitro and in vivo and were

clonally multipotent. This study has significant implications for stem cell biology and regenerative

medicine.

Keywords

Neural crest; Epidermal keratinocytes; Neural plate border; Neural crest induction;

Reprogramming

INTRODUCTION

In vertebrate embryos, neural crest (NC) cells form important components of the peripheral

nervous system, craniofacial skeleton and pigmentation of the skin. Because of their diverse

developmental potential, there has been great interest in producing NC cells from human

embryonic stem cells (hESC)[

] and induced pluripotent stem cells (iPSC)[

]. In fact,

recently it has been shown that introduction of a single transcription factor, SOX10, plus

environmental factors was sufficient to reprogram fibroblasts toward a NC fate[

]. However,

cell reprogramming typically involves introduction of genes via lentiviral infection[

] with

variable transduction efficiency, thereby often requiring selection or sorting of the minority

transduced subpopulation. Moreover, virally-mediated genomic integration runs the risk of

mutations that may lead to tumorigenesis.

An alternative approach is to use growth factors to reprogram cells in order to recapitulate

the embryonic process of NC formation under conditions reflecting the endogenous state. In

the embryo, NC cells are induced at the neural plate border by neighboring tissues that

provide signals like WNT, FGF, BMP, and NOTCH[

–

]. These imbue the border region

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with the ability to form NC via an evolutionarily conserved gene regulatory network (GRN)

[

]. Newly formed neural crest cells express a characteristic set of transcription factors

including FOXD3, PAX3/7, SNAI2, TFAP2A, MSX1/2, c-MYC, and SOX9, which in turn

activate the transition to a migratory and multipotent population[

]. For example, the

combination of FGF2 and WNT activation or BMP inhibition is sufficient to commit naïve

embryonic ectoderm into NC cells [

Here we report that human inter-follicular epidermal keratinocytes (KC) - derived from non-

neural ectoderm - express several neural plate border genes. Notably, in response to FGF2

and IGF1, KC activate a NC program at the clonal level. NC cells derived from KC (KC-

NC) display a global gene expression profile similar to that of human embryonic stem cell

derived NC. Under differentiation conditions, clonal KC-NC give rise to functional NC

lineages including peripheral neurons, Schwann cells, melanocytes and mesenchymal stem

cell derivatives. Moreover, human KC-NC migrate and differentiate into NC derivatives

upon implantation into chicken embryos. Our results show that postnatal human epidermal

KC can be rapidly and easily reprogrammed into neural crest cells, demonstrating their

utility for regenerative medicine and stem cell biology.

MATERIALS & METHODS

Isolation of human inter-follicular epidermal keratinocytes

Human studies were performed as per institutional guidelines. Glabrous (lacking hair

follicles) foreskin from 1-3 day old neonates was procured from Women and Children

Hospital, Buffalo. Adult (45, 64 and 96 years old) skin (abdomen or thigh) tissues were

procured from the School of Medicine, University at Buffalo, SUNY. Keratinocytes (KC)

were isolated as described previously[

]. Briefly, skin samples were washed 3 times with

PBS, cut into pieces (~5mm×5mm), enzymatically digested with dispase I (Zen-Bio) for

15-20 hours at 4°C. Afterwards, epidermis was separated from dermis manually using fine

forceps. The epidermis was further treated with trypsin-EDTA (Life Technologies) for 10-15

min at 37°C, filtered through 70 μm cell strainer (BD Biosciences), centrifuged and plated

on a confluent monolayer of growth-arrested 3T3-J2 mouse fibroblast feeder cells in

keratinocyte growth medium consisting of a 3:1 mixture of DMEM (high glucose) and

Ham's F-12 medium (Life Technologies) supplemented with 10%(v/v) fetal bovine serum

(FBS, Gibco), 100 nM cholera toxin (Vibrio Cholerae, Type Inaba 569 B, Calbiochem), 5

μg/ml transferrin (Life Technologies), 0.4 μg/ml hydrocortisone (Sigma), 0.13 U/ml insulin

(Sigma), 1.4x10

−4

M adenine (Sigma), 2x10

−9

M triiodo-L-thyronine thyronine (Sigma), 1x

antibiotic-antimycotic (Life Technologies) and 10 ng/ml epidermal growth factor (EGF, BD

Biosciences). After 8–10 days of culture, 3T3-J2 feeder layer was detached using versene

leaving behind KC colonies. KC colonies were treated with Trypsin-EDTA and then rinsed

with serum-free and EGF-free keratinocyte growth medium. KC were further cultured in

EpiLife medium (Life Technologies) before using them for NC induction. Passage 1-3 KC

were used in all experiments.

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Induction of KC into neural crest stem cell fate

For induction into the NC fate, KC were cultured at a density of 10-15×10

cells/ cm

collagen type I coated dishes (10μg collagen type I per cm

; BD Biosciences) in the

presence of neural crest induction medium (NCIM) comprising of basal medium (EBM-2

medium; Lonza) plus 2% (v/v) FBS, 10 μg per ml heparin, 100 μg per ml ascorbic acid and

0.5 μg per ml hydrocortisone, 1x Gentamicin/Amphotericin-B supplemented with 10 ng/ml

fibroblast growth factor 2 (FGF2, BD Biosciences), 10 ng/ml Insulin like growth factor 1

(IGF1, BD Biosciences). Other induction factors and inhibitors tested were EGF, WNT1

(Life Technologies), Chir99021 (Tocris), NRG1 (Sigma), BMP4 (Gibco), and PD173074

(Cayman).

Neural crest induction of KC clones

To ensure each KC colony was derived from a single cell, we plated 0.5 cells per 100 μl KC

growth medium per well of a 96 well plate on 3T3-J2 feeder cells. Wells with more than one

colonies were excluded. Total 174 single cells KC clones from three different donors grew

and all the clones were used for NC induction treatment irrespective of their colony size.

Differentiation of KC-NC into NC derivatives

For peripheral neuronal differentiation, after 10 day of induction KC-NC were cultured on

poly-L ornithine (Sigma) (2 μg/cm

)/ laminin (Life Technology) (5 μg/cm

) coated dishes in

presence of NCIM plus 1 ng/ml of transforming growth factor-beta 1 (TGF-

1, Biolegend)

for 24 hours followed by one week culture in neuronal differentiation media (EBM2 basal

medium, 1% (v/v) FBS, 50 ng/ml brain derived neurotrophic factor (BDNF, Peprotech), 50

ng/ml glial derived nerve factor (GDNF, Peprotech), 200 ng/ml nerve growth factor (NGF,

Peprotech), 20 ng/ml neurotrophin 3 (NT3, Peprotech), 0.5μM N(6),2'-O-dibutyryladenosine

3':5' cyclic monophosphate (dbcAMP) (Sigma), 0.5x Glutamax, (Life Technology)).

For melanocyte differentiation, after 7 days of NC induction treatment, KC-NC were

cultured in melanocyte differentiation medium (EBM2 basal medium, 5% (v/v) FBS, 100 ng

per ml SCF (Life Technology), 200 nM Endothelin 3 (EDN3, Sigma), 50 ng/ml WNT1, 10

ng/ml FGF2, 5μg/ml Insulin, 1 pM cholera toxin, 10 nM 12-O-tetra-decanoylphorbol-13-

acetate (TPA, Sigma) and 10μM SB431542 (Cayman) for 5 weeks. For L-DOPA assay, KC-

NC derived melanocytes were fixed with 4% (w/v) paraformaldehyde for 20 min at room

temperature, washed three times with PBS and incubated with freshly prepared 5 mM L-

DOPA (Sigma) overnight at 37°C. After incubation, cells were post-fixed with 4%

paraformaldehyde for 20 min at room temperature, washed with PBS and visualized for

melanin pigment under bright field microscopy. Primary human melanocytes used as control

were isolated from foreskin tissues using previously described protocols [

] and cultured in

medium M254 (Thermo Fisher Scientific) supplemented with human melanocyte growth

supplement (HMGS) (Thermo Fisher Scientific).

For Schwann cell differentiation, after 7 days of induction treatment, KC-NC were plated on

poly-L ornithine/laminin coated dishes and cultured in Schwann cell differentiation medium

(EBM2 basal medium, 2% (v/v) FBS, 100ng/ml ciliary neurotrophic factor (Life

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Technologies), 100 ng/ml NRG1, 4 ng/ ml FGF2, 200μg/ ml ascorbic acid, 0.5x Glutamax,

10μM SB431542 for 5 weeks.

For mesenchymal differentiation, after 10 days of induction KC-NC were transferred to

mesenchymal growth medium (MGM) comprising of DMEM plus 10% (v/v) mesenchymal

stem cell qualified-FBS (MSC-FBS, Life Technologies) for 3 weeks. At this point, KC-NC

were analyzed for MSC immunophenotype and induced into mesenchymal lineages by

lineage specific differentiation medium[

]. Bone marrow derived mesenchymal stem cells

(Stem Cell technologies) were cultured in DMEM plus 10% (v/v) MSC-FBS and used as

positive control. Osteogenic, chondrogenic and adipogenic differentiation was induced and

functionally analyzed as described previously[

]. Smooth muscle cell (SMC)

differentiation was induced by MGM supplemented with 10 ng/ml TGF-

1 for one week.

Clonal multipotency of KC-NC

Single cell KC-NC were plated in 96 well plate by limiting dilution. Each well was

examined microscopically to ensure that only one cell gave rise to the clone. We derived 6

KC-NC clones per donor from 4 different neonatal donors (total 24 KC-NC clones). Each

clone was expanded in NCIM and split into 24 wells of a 96 well plate for differentiation

experiments. Six wells were used for differentiation into each of the following lineages:

smooth muscle cells (SMC), melanocytes (Mel), Schwann cells (SC) and neurons (N). Each

lineage was evaluated by immunostaining using lineage specific marker antibodies.

Gel compaction assay and vasoreactivity assay for testing KC-NC derived smooth muscle

cell function

Gel compaction assay measures the ability of SMC to generate force and compact fibrin

hydrogels. To this end, 1×10

KC-NC derived SMC or control human aortic smooth muscle

cells (ASMC, Life Technologies) were mixed with 0.8 ml of fibrinogen (Enzyme Research

Laboratories) that was polymerized by addition of 0.2 ml thrombin (Sigma) in a 24-well

plate at 37°C for 1 hr. The final concentration of fibrinogen and thrombin in the fibrin

hydrogel was 2.5mg/ml and 2.5 U/ml, respectively. The resulting KC-NC derived SMC

constructs were incubated in MGM supplemented with 2ng/ml TGF-

1. After 1 hr

incubation, the fibrin gels were released from the surface of the plate to enable gel

compaction, an indicator of SMC contractile function. At indicated times (0, 24, 48, 72, and

96 hr) images were acquired using a EC3 imaging system (UVP) and the area of each

hydrogel (A) was measured using Image J software and normalized to the initial hydrogel

area (A

). The results were plotted as the ratio A/A

and therefore, the smaller the area the

higher the contractile function of KC-NC derived SMC.

Vasoreactivity assay was performed as described previously[

]. Towards this end, fibrin

hydrogels (final concentration of fibrinogen and thrombin at 2.5 mg/ml and 2.5 U/ml,

respectively) containing 1×10

KC-NC derived SMC or control ASMC were polymerized

around a 6.0 mm diameter mandrel of poly(di-methyl siloxane). After 1 hr, hydrogels were

detached from the walls and incubated in a medium comprising of MGM plus 2 μg/ml

insulin, 2 ng/ml TGF-

1, 300 μM ascorbic acid phosphate (Alfa Aesar) and 2 mg/ml

amino-n-caproic acid (Sigma) for 2 weeks. After two weeks, compacted hydrogels were

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released from the mandrels and mounted on an isolated tissue bath containing Krebs–Ringer

solution at 37°C. Each hydrogel was mounted on two hooks through the lumen; one hook

was fixed and the other was connected to a force transducer. Hydrogels were equilibrated at

a basal tension of 1.0 g and constant length for approximately 1 hr. After equilibration,

vasocontractile agonists (endothelin-1 (20 nM) (Sigma), the thromboxane A2 mimetic

U46619 (1 μM, Sigma) or potassium chloride (KCl; 118 mM, Sigma) were added to the

tissue bath and isometric contractions were recorded using a PowerLab data acquisition unit

and analyzed with Chart5 software (ADInstruments). To determine the mechanical

properties (Young’s modulus (YM) and ultimate tensile stress (UTS)) of the hydrogels,

samples were incrementally stretched until they broke using the Instron 3343 (Instron).

Contractility and UTS/YM were normalized to the cross-sectional area of the hydrogels and

expressed in Pa and kPa, respectively.

Electrophysiological measurements

KC-NC derived neurons were differentiated on coverslips and transferred into a perfusion

chamber with a solution containing the following components (in mM): 140 NaCl, 4 KCl, 2

CaCl

2 MgCl

10 HEPES, 10 D-glucose, 10 sucrose (pH 7.4 with NaOH)) for

electrophysiological recordings. To measure action potentials, pipettes were fire-polished

and filled with an intracellular solution containing (in mM): 135 K-gluconate, 7.3 KCl, 10

phosphocreatine, 10 HEPES, 2 MgATP, 0.3 Na

GTP (pH 7.3 with KOH; 300 mOsm) with a

final resistance of 1-2 M

. Following the formation of a G

seal and establishment of

electrical access to the cell, action potentials were measured either by applying a very brief

(1-2 ms) but large (0.8 nA) current injection or by injecting current for a more prolonged

amount of time (200 ms) with each subsequent recording increasing by 50 pA until an action

potential was fired. To verify the involvement of voltage-gated Na

channels which are

required for action potential formation, tetrodotoxin (TTX, 1 μM) was applied and allowed

to diffuse before recording. To measure current through Na

and K

channels, voltage-clamp

was employed. Pipettes and recording solutions were identical to those used to measure

action potentials. Following formation of a G

seal, cells were electrically accessed through

the application of negative suction pressure. Pipette capacitance and series resistance

compensation were performed according to standard procedure. Cells were held at

hyperpolarized potentials (−90 mV) and stepped to various voltages (Δ10 mV; 200 ms)

following a step back to the hyperpolarized potential for a sufficient amount of time (5 s) to

allow for recovery from inactivation. Na

and K

channel currents were blocked using 1μm

TTX and 40mM TEA, respectively. Following acquisition of data from steps to −100 to +50

mV, traces were analyzed using pClamp (Molecular Devices) and displayed as current/

voltage plot.

Quantitative real time PCR

At the indicated times total RNA was isolated using RNeasy kit (Qiagen) as per

manufacturer’s instructions. First strand cDNA was synthesized from 1 μg of total RNA

using QuantiTect kit (Qiagen). To determine the kinetics of gene expression during

differentiation, quantitative real time PCR was performed on the Bio-Rad CFX96 Real-Time

PCR detection system with the SYBR Green mix (Bio-Rad) according to manufacturer’s

instructions. Primers information is given in supplementary table 1. KC (day 0, before start

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of induction treatment) were included as a calibrator in our qRT-PCR analysis. The level of

each mRNA was quantified using the ΔΔC

method and normalized with the expression

level of the housekeeping gene, 18sRNA. The specificity of each product was verified by

melt curve analysis and gel electrophoresis.

RNA sequencing

KC and KC-NC were characterized in terms of their global expression profile by next

generation RNA sequencing using Illumina platform. To this end, total RNA was isolated

from both KC (n=3) and the respective KC-NC (n=3) and quality control analysis was

performed by RNA gel and Agilent Bio-analyzer. Sequencing libraries were prepared as per

standard Illumina protocols and sequenced in pair-end (2x50 bp) rapid run mode. After

quality control of raw data using Illumina pipeline, RNA-Seq reads were mapped to the

human genome (hg19) using Tophat (version 2.0.11) with default settings. The mapping

quality was examined by RSeQC 2.3.9. Then the read counts for each gene in the accepted

hits are counted by the R package qRNASeq. For each gene, total reads within the exon

regions were counted and overlapped regions from different transcripts were united. The

differentially expressed genes between KC and KC-NC were detected by the R package

DESeq2, using a design formula considering patient variability. Hierarchical clustering and

3D multi-dimensional plot (3D MDS) were generated in R using hclust() and cmdscale

functions, respectively. Heat maps were generated using the ‘heatmap.plus’ function in R.

The KC-NC gene expression profile was compared to that of human embryonic stem cell

derived neural crest cell signatures reported in the literature [

] as well as KC, using

ROCR and verification libraries in R Bioconductor (v3.1.3). RNA sequencing data is

available on the NCBI Gene Expression Omnibus (GEO) and accessible through GEO

Series accession number GSE72268.

SOX10 promoter methylation assay

SOX10 CpG Island (Chr22: 38379093-38379964 (hg19), containing 82 CpG) whose

methylation status inversely correlates with SOX10 transcription [

] was examined by

MethylScreen technology[

], using the Epitect methyl II PCR assay (catalog#

EPHS109833-1A, Qiagen) as per manufacturer's instructions (EpiTect Methyl II PCR Assay

Handbook – Qiagen). Genomic DNA was isolated from KC and the corresponding KC-NC

(n=5 donors) using PureLink genomic DNA isolation kit (Life Technologies). One μg of

genomic DNA from both KC and the respective KC-NC was mock-digested (no enzyme) or

digested with methylation-dependent (digests methylated DNA) and methylation-sensitive

(digests unmethylated and partially methylated DNA) restriction enzymes (provided with

EpiTect Methyl II DNA Restriction Kit (Qiagen)) individually or together. Methylation

status of the target sequence was measured using quantitative real time PCR with target

sequence specific probes. The raw ∆CT values from all 4 restriction digestion groups were

plugged in the data analysis spreadsheet (

http://www.sabiosciences.com/

dna_methylation_data_analysis.php

), which automatically calculates the relative amount of

methylated and unmethylated DNA fractions.

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Immunocytochemistry and flow cytometry

Cells were fixed (4% (w/v) paraformaldehyde), permeabilized (PBS with 0.1% (v/v) triton

X-100, Sigma), blocked (5% (v/v) normal goat serum in PBS, Life Tech or BlokHen

solution (Aves labs)) and incubated with primary antibodies (Supplementary Table 2)

followed by appropriate secondary antibodies conjugated with Alexa 488 or Alexa 594.

Hoechst 33342 (Thermo Fisher Scientific) was used for nuclear staining. Cells that were

incubated with only secondary antibody served as controls. For flow cytometry detection of

KRT14, SOX10, NES and PMEL, cells were fixed with 4% (w/v) paraformaldehyde,

permeabilized with 0.1% (v/v) triton X-100, and incubated with the respective primary

antibodies (Supplementary Table 2). Alexa 488 or Alexa 647-R conjugated secondary

antibodies were used. For all surface proteins, cells were detached with 5mM EDTA and

stained with appropriate primary and secondary antibodies. Cells were run in a FACS

Calibur (BD) or CytoFLEX (Beckman Coulter) flow cytometer and the data were analyzed

using CellQuest software (BD) or FlowJo (FlowJo LLC), respectively.

Imaging and Image analysis

Images were acquired using a Zeiss Axio Observer Z1 inverted microscope with an ORCA-

ER CCD camera (Hamamatsu, Japan). Fluorescence intensity quantification was performed

using NIH ImageJ as described previously[

]. Briefly, cells from all time points were

stained in one batch and images were acquired using fixed exposure time for each

fluorescent secondary antibody. Corrected total cell fluorescence intensity (CTCF) was

calculated as per following formula:

In ovo

transplantations

To this end, KC and the respective KC-NC were transduced with lentivirus containing

minimal CMV promoter driven EGFP reporter. Approximately 50-60% cells were

transduced by the lentivirus as assessed by examining EGFP+ cells under fluorescence. KC-

NC (n=8) or KC (n=3) were trypsinized and

≈

30-60 cells per embryo were transplanted

ovo

onto the head mesenchyme to join the migrating neural crest cells of 10-13 somite stage

chick embryos (Fig. 5B). The eggs were sealed, kept humidified by adding Ringer’s

balanced salt solution once a day, and fixed 2-3 days later in 4% (v/v) paraformaldehyde

overnight at 4°C and washed 2x with PBS. Finally, the embryos were embedded in 15%

(w/v) sucrose / 30% (w/v) gelatin in PBS and cryosectioned (12μm sections), and de-

gelatinized by incubation in 42°C PBS for 1.5h prior to incubation in blocking buffer (5%

(v/v) donkey, 5% (v/v) goat serum, 1% (v/v) DMSO in PBS-0.1% (v/v) tween 20) and

mounting. First, the slides were screened under the microscope to look for EGFP+ cells and

the appropriate slides were marked. The EGFP+ cells were distinguished from the highly

autofluorescent blood cells found abundantly in the capillaries in the mesenchyme by

checking their fluorescence additionally on the red and blue channels, which makes the

blood cells to look white in the images (see Fig. 5C). Depending on the location of the

EGFP+ cells, the marked sections were decoverslipped (PBST treatment for 1-2 days) and

stained with antibodies accordingly. The following primary antibodies were used (overnight,

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4°C): BLBP (ABN14 Millipore, 1:200), SMA (A5228 Sigma, 1:1000), Tuj-1 (Covance

MMS-435P, 1:400), GFAP (SMI22 Sternberger Monoclonals, Covance 1:800) and human

nuclear antibody (MAB 1281 Millipore, 1:100). Subsequently the following Alexa Fluor

conjugated secondary antibodies (overnight, 4°C were used: 488 donkey anti-rabbit, 647

donkey anti-rabbit, 568 goat anti-mouse IGg2a, 633 goat anti-mouse IGg2a (Molecular

Probes).

Clonogenic assay and population doubling

Clonogenic assay was performed as described previously[

]. Briefly, KC-NC were seeded

(10 cells/cm

) in a 100 mm culture dish and cultured for one week in NCIM. Afterwards,

plates were fixed with a solution of methanol and acetic acid (3:1 v/v), stained with trypan

blue and photographed. Images were analyzed using ImageJ to determine the area and

effective diameter of each clone. For proliferation studies, 40,000 KC-NC were seeded in

culture plates in triplicates or quadruplicates and grown in NCIM. Every 3 days, the cells

were counted and the doubling time and cumulative cell numbers were calculated assuming

geometric growth.

Immunoblotting

Immunoblotting on KC and the respective KC-NC protein lysate for CDH1 (E-cadherin) and

CDH2 (N-cadherin) was done as described previously[

Statistical analysis

Experiments were performed with KC from at least 3 human donors (range: 3–30 human

donors) and each experiment was repeated at least three times. Data were expressed as mean

± standard deviation except electrophysiological data, which is mean ± SEM. Comparison

among groups were performed using one-way ANOVA followed by post-hoc analysis using

Tukey’s HSD test or two-tailed t-test. Wilcoxon rank-sum tests were performed for paired

comparisons. Statistical significance was defined as p < 0.05.

RESULTS

Human Keratinocytes (KC) display neural plate border characteristics

An adult neural crest population has been reported to be present in hair follicle’s bulge

region[

]. In addition, skin derived precursors (SKPs) having similar characteristics as

neural crest cells have been derived from mouse[

] and human dermis[

]. To avoid

possible contamination from bulge neural crest, we isolated human interfollicular[

]

epidermal KC from glabrous (lacking hair follicles) foreskins (1-3 day old neonates).

Furthermore, we manually separated epidermis from dermis after enzymatic tissue digestion

to avoid the presence of dermal SKPs in our cultures. KC on 3T3-J2 feeder layer grow as

distinct compact colonies of cuboidal cells. Versene treatment and multiple subsequent PBS

washes removed the 3T3-J2 cells and any melanocytes that might be present on top of the

feeder cells (Fig.S1A). Indeed, we confirmed the purity of our KC population by performing

immunocytochemistry and flow cytometry analysis for melanocyte markers (n=3 donors).

As shown by flow cytometry and immunostaining, primary human foreskin derived

melanocytes express the melanocyte marker PMEL (also known as SILV or HMB45) and

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lack KRT14 (a KC marker; Fig.S1B,C). In contrast, KC lack PMEL (and MITF) and express

KRT14 (Fig.S1B,C). Finally, single cell derived clonal cultures of KC were established to

ensure that the starting cell population in our experiments is bona fide KC.

All experiments were done with KC from at least three human donors (range: 3-30 donors)

and each experiment was repeated at least three times. Passage 1 to 3 KC were used for all

experiments. Human foreskin KC expressed type I intermediate filament KRT14 (99.6%

cells), TP63 (97.9%), ITGB1 (99.7%) with variable immunofluorescence intensity (n=3

donors; Fig.S2A), indicative of basal/transit amplifying KC. When examined for the

presence of genes at the embryonic neural plate border, we found that KC expressed several

neural crest and neural plate border markers

(MYC, SOX9, SNAI2 MSX2, IRX2, DLX3,

TFAP2A , and KLF4)

when compared with human dermal fibroblasts that were of

mesodermal origin (n=3 donors) from the same donor and used as a control (Fig.S2B).

Immunostaining revealed that 81.8% of KRT14+ KC were positive for SNAI2, 60% were

MYC+ and 96.7% were TFAP2A+ (n=3 donors; Fig.S2A). We further examined adult

human KC for the expression of neural plate (NP) border markers. To this end, we isolated

abdominal or thigh skin KC from three human donors (45, 64 and 96 years old). qRT-PCR

for

MYC, SOX9, SNAI2 MSX2, IRX2, DLX3, TFAP2A

and

KLF4

(Fig.S3A) and

immunostaining for TFAP2A, SNAI2 and KLF4 (Fig.S3B) showed that adult KC express

NP border markers, albeit at lower level compared to neonatal KC. The enrichment of

several neural plate border markers in KC led to the intriguing possibility that they might be

reprogrammed to a NC fate under appropriate environmental conditions.

Reprogramming of KC into NC cells

To identify factors that may induce NC fate in human KC, we tested signaling pathways

previously implicated in neural crest formation[

–

] using FGF2, WNT1, WNT activator

Chir99021, EGF, IGF1, BMP4, NRG1 and combinations thereof[

]. We found that FGF2

significantly promoted NC fate as evidenced by SOX10 and NES (Nestin) dual positive KC-

NC cells (1,313±394 cells out of 3,000 plated cells; p=1.08x10

−8

, n=3 donors) after 6 days

of induction treatment (Fig.1B). NES is an intermediate filament protein that is expressed

both in CNS progenitors and neural crest stem cells [

]. This induction was blocked by

FGFR chemical inhibitor PD173074, yielding a drastic decrease in SOX10+NES+ dual

positive cells (p=0.0013, n=3 donors; Fig.1C). The combination of FGF2 and IGF1 further

potentiated the induction process as shown by significant increase in SOX10+NES+ cells

compared to FGF2 alone (p=2.39x10

−6

, n=3 donors; Fig.1D). It also led to significant

increase in expression of neural crest genes (

SOX10, NES, PAX3, FOXD3, NGFR

and

B3GAT1

), EMT genes (

TWIST1

) and the cell proliferation marker,

KI67

(n=3 donors;

Fig.S2C). As NC migration is influenced by the extracellular matrix environment[

], we

tested the effects of different ECM coatings (fibronectin, laminin, collagen type 1) to

promote NC fate. All three ECM molecules supported expression of NC genes (

NGFR,

PAX3, SOX10, and NES

) to a similar extent (Fig.S2D). However, both fibronectin and

collagen type I showed similar levels of SOX+NES+ cells (p=0.68, n=3 donors; Fig.S2E),

which were significantly higher than that observed on laminin-coated or non-coated surfaces

(fibronectin: 2.9±0.42 fold, collagen type I: 2.8±0.13 fold, p=0.004, n=3 donors; Fig.S2E).

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Cultured KC assumed typical cuboidal morphology in low calcium (0.06mM) serum free

medium (Fig.1E). Upon induction with FGF2 and IGF1 in high calcium (1.8mM) medium,

KC formed compact cell clusters (n=30 donors; Fig.1E). As early as day 3, small spindle

shaped cells started to delaminate from KC clusters, migrated outwards and started to

proliferate, while newer cells continued to delaminate from the KC clusters. Delaminated

cells detached from the surface by trypsin/EDTA treatment much faster (~1 min) than KC

clusters. Thus using short-term (~1 min) trypsin/EDTA treatment, we were able to separate

and collect delaminating cells from KC clusters, which remained intact. When analyzed for

the presence of NC markers, the cells that delaminated from KC clusters displayed

molecular characteristics of NC cells and thus termed KC derived NC cells (KC-NC). KC-

NC were highly proliferative as evidenced by a short population doubling time of ~19.6 hr

(n=2 donors; Fig.S2F) and high clonogenic ability (n=3 donors; Fig.S2G). KC-NC could be

maintained for 20 days without loss of proliferation potential. KC-NC downregulated

KRT14 and upregulated NES protein expression as determined by immunofluorescence (n=6

donors; Fig.1F) and flow cytometry (KC, 99.4±0.26% KRT14+, 0% NES+; KC-NC, 0%

KRT14+, 99.6±0.2% NES+;n=3 donors; Fig.1G). Notably, KC-NC were uniformly positive

for SOX10 (98.3±0.4%), while 75.4±1.18% were positive for NGFR (p75NTR) as

determined by immunofluorescence (n=3 donors; Fig.1H) and flow cytometry (n=4 donors,

Fig.1I). In addition, KC-NC expressed other key NC genes such as PAX3 (93.5±3.46%),

FOXD3 (90.1±3.58%) and KIT (96.8±1.04%) (n=3-6 donors; Fig.1H,J).

To determine relative efficiency of KC reprogramming towards NC fate, we plated cells at

clonal density i.e. one cell per well of a 96 well plate containing 3T3-J2 feeder cells and

grew a total 174 single cell clones from 3 different donors (Fig.1N). The 3T3-J2 feeder cells

were removed by Versene treatment leaving behind KC clones that were induced into NC

phenotype using NCIM (Fig.1K, L, M). Prior to NC induction cells from each clone were

stained for KRT14 and NES to ascertain KC identity. After induction, the NC forming

efficiency ranged from 6.06% to 7.9% among different KC donors as evidenced by loss of

KRT14 and acquisition of NES and SOX10 by immunostaining (Fig.1 L, M, S2H). These

results confirm the clonal ability of KC to convert to NC phenotype.

Detailed Molecular Profile of KC-NC

We followed the kinetics of gene expression as KC turned into KC-NC using qRT-PCR. NC

specific genes were expressed for a window of time between day 3 and 12 post-induction,

with some NC specific genes (

SOX10, FOXD3, ID2, B3GAT1 (HNK1)

) exhibiting peak

expression by day 6 and reduced expression thereafter, and others (

PAX3, NES, KIT, NGFR,

NR2F1

) exhibiting sustained expression up to 12-18 days (n=3 donors; Fig.2A,B). We also

examined the protein levels of SOX10, PAX3, FOXD3, NES and NGFR by immunostaining

and quantified relative changes in protein expression using ImageJ software. Similar to RNA

expression, KC-NC expressed NC specific proteins between day 3 and 12 (n=3 donors;

Fig.S4A,B). Concomitantly, KC specific genes

TP63, KRT14, KRT5

and

KRT8

were

downregulated, suggesting loss of epidermal phenotype in KC-NC (n=5 donors; Fig.2C).

Taken together, the data suggest that the NC phenotype of KC-NC is transient, similar to

what was previously reported for embryonic NC cells[

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NC cells delaminate from the neural tube via an epithelial to mesenchymal transition (EMT)

to acquire migratory phenotype[

]. Since KC-NC appear to undergo a similar EMT

program, we examined changes in

ZEB1

and

ZEB2 (SIP1)

[

], which mediate an E-

Cadherin (CDH1) to N-Cadherin (CDH2) switch during EMT. Both were upregulated by

day 3 of KC-NC reprogramming (n=3 donors; Fig.2D). Subsequently, CDH1 was

downregulated while CDH2 was upregulated in KC-NC both at the mRNA (n=3 donors; Fig.

2D) and protein level (n=5 donors; Fig.2E, S4C). Interestingly, we observed

-catenin,

normally at adherens junctions of epithelial cells, translocated into the nucleus of KC-NC

after undergoing EMT possibly reflecting WNT activation (n=3 donors; Fig.S4D,E). In

addition, other key mediators of EMT such as

SNAI1, TWIST1

and

FOXC2

were

significantly upregulated during KC-NC reprogramming (n=3 donors; Fig.2D). During

EMT, epithelial cells shut off the expression of keratins (class I, II intermediate filaments)

and induce expression of VIM (class III intermediate filament protein), which reportedly

mediates cell shape and motility changes[

]. Indeed, we observed that KC-NC abolished

KRT14 and expressed VIM (n=3 donors; Fig.S4F). Taken together, these results suggest that

the KC to NC conversion invokes a coordinated gene regulatory machinery to induce EMT.

The global transcription profile of KC-NC is similar to hESC derived NC

Next we performed transcriptome analysis of KC-NC and KC from three donors. Between

the two cell types, there were 3,894 differentially expressed genes; some of the

quintessential KC-NC genes were verified by qRT-PCR (Fig.S4G). The data show that

several NP border genes were expressed in KC but decreased as KC acquired NC fate

(Fig.S4H). Concomitantly, genes that were expressed in the basal epidermal layer or during

epidermal stratification were downregulated (Fig.2F). Instead, KC acquired a NC signature

as suggested by upregulation of several NC (Fig.2G), EMT (Fig.2H) and ECM (Fig.S4I)

genes. RTKs and WNTs were upregulated, while NOTCH and TGF

pathways were

downregulated (as indicated by increased expression of TGF

pathway inhibitors) (Fig.S4J)

[

]. In addition, histone demethylase (

KDM6A, KDM5D

), histone methyltransferase

(

EZH1

), de novo DNA methyltransferases (

DNMT3A/B

) and NAD-dependent deacetylase

SIRT1

were upregulated during KC-NC reprogramming (Fig.S4K), consistent with

epigenetic modifications occurring during NC induction[

]. Change in expression of a

subset of those genes (

EZH1, EZH2, DNMT3A, DNMT3B

) was also confirmed with qRT-

PCR (n=3 donors; Fig.S4L).

Promoter demethylation of the critical neural crest gene,

SOX10

, is essential for acquisition

of NC fate[

]. Therefore, we examined SOX10 promoter methylation in both KC and

the corresponding KC-NC populations using the Epitect Methyl II PCR Array (QIAGEN) by

examining 871 bp long region of Chr22 (38379093-38379964 (hg19), containing 82 CpG)

for CpG island methylation, whose status inversely correlates with SOX10 expression[

SOX10

promoter was heavily methylated in KC but severely hypomethyled in KC-NC

(methylation KC: 97±1.2% of input genomic DNA; methylation KC-NC: 0.54±0.3% of

input genomic DNA, p=5.45x10

−15

, n=5 donors), consistent with high expression of

SOX10

in KC-NC (Fig.2I).

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We next compared KC and KC-NC transcriptomes using average linkage hierarchical

clustering on all pairwise distances between their global transcriptome-wide RNA-seq

profiles (Fig.2J) and 3D multidimensional scaling analysis (Fig.2K). KC and KC-NC

transcriptome were significantly different as shown by distinct clustering (Fig.2J,K).

Similarities with published neural crest signatures were analyzed using ROCR and

verification libraries in R Bioconductor package. The results show that KC-NC are very

similar to the human embryonic stem cell derived NC cells (Mica et al. 2013[

], AUC:

0.597; p=4.304x10

−5

and Lee et al.[

], AUC: 0.644; p= 5.454 x10

−5

; Fig.2L), as opposed to

parental KC (AUC: 0.0003461405; p=1; Fig.2L).

KC-NC give rise to functional neural crest derivatives

in vitro

Peripheral neurons—

KC-NC differentiated into neurons (n=5 donors) with typical

neuronal morphology (Fig.3Ai), expressing

TUBB3

gene

(a.k.a. TUJ1)

(Fig.3Aii), as well as

proteins TUBB3, PRPH (Peripherin), NeuN (neuronal nuclei) and MAP2, indicative of their

maturity and peripheral identity (Fig.3Aiii,iv,v). Using whole-cell current-clamp method

(Fig.3Bi), the resting membrane potential (RMP) of KC-NC derived neurons was −47±5mV

(Mean±SEM; n=25 cells) and membrane capacitance was 48±8 pF (Mean±SEM;

n=25cells). On current-clamp recordings, they displayed evoked action potentials upon

injecting 50pA step currents with duration of 6.4±0.9ms (Mean±SEM; n=15 cells; Fig.3Bii).

KC-NC neurons expressed sustained outward current ranging from few hundred pA to ~4

nA (n=10 cells; Fig.3Biii,iv), which displayed voltage dependence and kinetics typical of

tetraethylammonium (TEA) sensitive delayed rectifier potassium channels (Fig.3Biv,v). We

also detected KC-NC derived neurons displaying inward currents (ranging from few

hundreds of pA to ~2 nA (n=4cells)) with kinetics typical of TTX sensitive voltage activated

sodium channels (Fig.3Biii,iv,vi). Taken together these results suggest functional maturity of

KC-NC derived neurons.

Melanocytes—

KC-NC efficiently differentiated into melanocytes (n=3 donors; Fig.3C).

After 3 weeks of differentiation, ~20% cells expressed MITF a key transcription factor for

melanocyte development and ~33% of MITF+ cells also expressed the marker of mature

melanocytes, PMEL (a.k.a. HMB45; Fig.3Ci), and synthesized melanin after 5 weeks (Fig.

3Cii,iii). They abundantly expressed melanocyte genes (

TYR, PMEL, DCT, MITF, GPNMB,

and S100B

) as determined by qRT-PCR (Fig.3Civ) and displayed functional tyrosinase

activity as evidenced by the L-DOPA assay[

] (Fig.3Cv).

Schwann cells—

KC-NC differentiated into Schwann cells as determined by qRT-PCR

analysis of key Schwann cell genes and immunostaining (n=3 donors; Fig.3D). They

upregulated

MPZ, EGR2 (a.k.a. KROX20), PMP22, S100B

and

NFATC4

(Fig.3Di) genes

and were MPZ+ (98±1.5%), S100B+ (94±1.3%), GFAP+ (92.6±1.6%) and dual positive for

CNP (CNPase)+/ PLP1 (97.5±1.8%) (Fig.3Dii,iii,iv,v).

Mesenchymal Stem Cells—

Upon differentiation KC-NC formed mesenchymal lineage

as evidenced by differentiation towards osteogenic, chondrogenic, adipogenic and smooth

muscle lineages and expression of mesenchymal markers CD73, CD90, CD44, CD105,

CD49b (n=3 donors; Fig.S5A). Upon osteogenic differentiation (n=3 donors), cells

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expressed

RUNX2

and

ALP

(alkaline phosphatase) and deposited calcium phosphate as

shown by Von Kossa staining (Fig.3E). Chondrogenic differentiation (n=3 donors) was

assessed by expression of

COL2A1

and mature marker

ACAN

(Aggrecan) as well as by

Alcian Blue staining for glycosaminoglycans (Fig.3F). Upon adipocyte differentiation (n=3

donors), they expressed

PPAR-

and

LPL

and deposited oil droplets, as shown by Red-O

staining (Fig.3G).

KC-NC derived smooth muscle cells (KC-NC-SMC) (n=3 donors) showed transcriptional

upregulation of SMC specific genes (

ACTA2, TAGLN, SMTN, CALD1, MYH11

) and

displayed fibrillar organization of contractile proteins (ACTA2, CNN1, MYH11) (Fig.3H).

KC-NC-SMC compacted fibrin hydrogels as a function of time to a similar extent as human

aortic smooth muscle cells (ASMC) (p=0.095, n=3 donors; Fig.3I), indicating development

of ability to generate force [

]. In addition, KC-NC-SMC remodeled fibrin hydrogels

possibly by synthesizing ECM (collagen and elastin) as evidenced by increase in ultimate

tensile stress (Fig.S5B). Most notably, KC-NC-SMC displayed receptor and non-receptor

mediated isometric contractions upon treatment with vasoconstrictors[

] (U46619,

Endothelin-1, KCl; n=3 donors; Fig.3J). These results suggest functional SMC

differentiation of KC-NC.

KC-NC are clonally multipotent

To ascertain the multipotency of KC-NC, we plated KC-NC at clonal density and picked 6

clones per donor from 4 different KC donors (total 24 KC-NC clones). KC from each donor

gave rise to KC-NC that differentiated into all four lineages (Table 1). Each clone was

induced to differentiate into smooth muscle (SMC), melanocytes (Mel), Schwann cells (SC)

and neurons (N) as evidenced by immunostaining for lineage specific markers.

We found that 21% of the clones (5 out of 24 clones) differentiated into all four lineages;

12% (3 out of 24 clones) were tripotent; 50% (12 out of 24 clones) were bipotent; and 17%

(4 out of 24 clones) were unipotent (Fig. 4A). Immunostaining for MPZ (P0), PLP1 and

S100B proteins showed that 62.5 ± 7.2% clones acquired Schwann cell fate (Fig. 4B,D);

while 33.3 ± 11.7% clones gave rise to melanocytes as evidenced by PMEL (SILV)

immunostaining and L-DOPA reaction positivity (Fig. 4B,E). The majority of clones (91.6

± 8.3%) differentiated into SMC as determined by ACTA2 and CNN1 positive cells (Fig.

4B,F). Finally, immunostaining for neuronal markers TUBB3, PRPH and MAP2 showed

that 50 ± 16.6% of clones differentiated into neuronal phenotype (Fig. 4B, G). Interestingly,

KC-NC derived neurons in each TUBB3+ clone were uniformly positive for 5-HT

(serotonin), a marker of enteric autonomic neurons and 32 ± 2.5% were found to be positive

for BRN3A (POU4F1), indicative of their sensory identity (Fig.4G). These results suggest

that KC-NC give rise to both enteric autonomic and sensory neurons. The differentiation

potential of KC-NC derived from each donor was summarized in Fig. 4C. Despite variations

in the multi-lineage potential at the clonal level, KC-NC derived from all donors were

capable of differentiation towards all the stereotypical lineages of NC cells.

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KC derived NC migrate and differentiate into NC lineages

in ovo

We transplanted KC or KC-NC labeled with EGFP into the head mesenchyme of 10-13

somite host chick embryos (Fig.5B) that were analyzed either 2 days (n=4 embryos) or 3

days (n=4 embryos) after the transplantation. The results show that normal KC did not

contribute to neural crest derivatives, but were found in the mesenchyme (a total of 7 cells

detected in 3 embryos). In contrast, a total of 151 transplanted human KC-NC EGFP+ cells

(from total 400 transplanted KC-NC) were found in sections from 8 embryos in locations

typical for neural crest. They contributed to the full repertoire of neural crest derivatives,

from neural and glial cells to mesenchymal and pigment cells (Fig.5A). They gave rise to

TUBB3 (TUJ1)+ neurons (Fig.5C), BLBP+ glial cells (Fig.5D) in the trigeminal ganglia as

well as putative Schwann cells around axon bundles (Fig.5E). We also detected transplanted

cells that were localized in blood vessel walls and expressed

-SMA (ACTA2), indicating

differentiation into smooth muscle cells (Fig.5F). KC-NC gave rise to presumptive

melanoblasts below the ectoderm (Fig.5G). Additionally, transplanted cells were detected in

the branchial arches, the gut wall, the heart or the mesenchyme (Fig.5A). These results

confirm that KC-NC behave similar to embryonic NC

in ovo

and can contribute to multiple

NC derivatives.

DISCUSSION

We report for the first time that KRT14+ postnatal human epidermal KC can be

reprogrammed to a NC fate upon FGF2 stimulation, further potentiated by IGF1. KC derived

NC cells displayed a global transcription profile similar to human embryonic stem cell

(hESC) derived NC cells[

] and differentiated into functional NC derivatives including

peripheral neurons, melanocytes, Schwann cells and mesenchymal stem cell derivatives

(osteocytes, chondrocytes, adipocytes and smooth muscle cells). Upon transplantation into

chicken embryos, KC-NC migrated along stereotypical pathways and gave rise to multiple

NC derivatives.

Previous work showed that WNT1 expressing cells in the bulge of the murine hair follicle

represent a NC cell population[

]. Notably, WNT1 expression was limited to the bulge and

dermal papilla, while inter-follicular epidermis was devoid of WNT1 expressing cells,

suggesting absence of NC cells in the inter-follicular epidermis[

]. Pioneering work by the

Sieber-Blum laboratory suggested the presence of melanocyte precursors in the murine

bulge region as a potential source of melanocytes responsible for hair pigmentation. Human

inter-follicular epidermis contains unipotent melanocyte precursor/melanocyte stem cells

that could potentially contaminate our KC-NC population. However, we have accumulated

evidence that strongly suggests that this is highly unlikely and that KC-NC originate from

KC. Specifically, we isolated inter-follicular KC from glabrous (lacking hair follicles)

foreskin[

], ruling out contamination from hair follicle bulge cells. We further ensured that

our starting KC population does not contain melanocytes (Fig. S1). Foreskin derived KC

expressed KRT14, TP63 and ITGB1 but lacked PMEL and MITF. Importantly, each KC

clone was examined by KRT14 immunostaining prior to NC induction to confirm the purity

of KC and lack of melanocytes. Upon induction, clonally derived KRT14+ KC gave rise to

NC cells, confirming their reprogramming potential. Finally, in our KC-NC clonal

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multipotency experiments, we employed 24 single cell clones from 4 donors and did not find

a single KC-NC clone that gave rise only to melanocytes and no other lineage, further

suggesting the lack of unipotent melanocyte precursor in our starting KC populations.

We speculate that expression of various neural plate border genes (

SNAI2, MYC, TFAP2A,

SOX9, DLX3/5, MSX1/2, IRX2

etc.) may predispose KRT14+ KC with NC potential.

Indeed, the higher propensity of KC to reprogram to the pluripotent state - 100 times more

efficiently than fibroblasts – was attributed to native expression of pluripotency factors MYC

and KLF4[

]. On the other hand, SOX10 overexpression was required to reprogram human

fibroblast into NC cells[

] and neural progenitors into oligodendrocyte progenitors[

suggesting that the molecular nature of the starting cell population may be critical to the

reprogramming process.

NC induction during embryonic development involves activation of a gene regulatory

network (GRN)[

],[

]. Similar to the transcriptional dynamics during NC formation in

vivo[

] , expression of neural plate border genes

(TFAP2A, MYC, SNAI2, MSX1/2,

DLX3/5, SOX9)

, in KC was followed by sequential upregulation of EMT

(ZEB1, ZEB2,

SNAI1, FOXC2, TWIST)

and NC specification

(SOX10, FOXD3, PAX3, TFAP2A, NR2F1,

ID2) gene module

during KC to NC reprogramming ,. Consistent with upregulation of NC

specific genes the

SOX10

promoter region[

] became hypomethylated. The drastic change

in methylation status was accompanied by transcriptional changes in several epigenetic

modifiers (

DNMT3A/B, EZH1/2, SIRT1

The ability of KC-NC to migrate along the routes of endogenous NC cells within chicken

embryos and differentiate into multiple NC derivatives provides very strong support of their

NC character [

]. Similar to endogenous and hESC derived NC, KC-NC differentiated into

functional peripheral neurons, Schwann cells, melanocytes and mesenchymal stem cell

derivatives (osteocytes, chondrocytes, adipocytes and smooth muscle cells)

in vitro

, further

supporting the NC phenotype of these cells.

Although dermal fibroblasts can be reprogrammed into specific cell types (neurons, glial

cells, cardiomyocytes and hepatocytes etc.)[

], the virally-mediated genetic modification of

these reprogrammed cells restricts their potential clinical applicability. Moreover, poor

proliferation of terminally reprogrammed cells[

] further limits their clinical utility as cell

based regenerative therapies require large number (~10

) of cells.

Given the accessibility, higher proliferative capacity and ease of reprogramming epidermal

KC without the need for genetic manipulation, KC-NC represent a potentially useful source

of functional therapeutic cells for regenerative medicine and tissue engineering applications

as well as a model for analysis of human neurocristopathies[

], similar to human iPSC.

Taken together, our study presents human epidermal KC as a novel inducible source of

functional NC cells without genetic modification. This readily accessible source of

abundant, highly proliferative, autologous NC cells has significant implications for stem cell

biology, drug discovery and regenerative medicine.

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Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

This work was supported by a grant (IMPACT Award) from the University at Buffalo to S.T.A. K.A.C. was

supported by grant F31 NS 084668. G.P. was supported by grants F31 NS 084668 and AHA 12EIA9100012.

M.E.B. was supported by R01DE024157. Authors thank Deepika Verma for her help in creating schematic diagram.

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