Vol.2, No.4, 141-154 (2012) Stem Cell Discovery
http://dx.doi.org/10.4236/scd.2012.24019
Differentiation of human epidermis-derived
mesenchymal stem cell-like pluripotent cells into
neural-like cells in culture and after transplantation
Min Zhang, Bing Huang*, Kaijing Li, Zhenghua Chen, Jian Ge, Weihua Li, Jianfa Huang,
Ting Luo, Shaochun Lin, Jie Yu, Wencong Wang, Liping Lin
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China;
*Corresponding Author:
Received 11 August 2012; revised 12 September 2012; accepted 9 October 2012
ABSTRACT
Skin is the largest organ of the human body and
a possible source of stem cells for research and
cell-based therapy. We have isolated a popula-
tion of mesenchymal stem cell-like pluripotent
cells from human epidermis, termed human (h)
EMSCPCs. This preliminary study tested if these
hEMSCPCs can be induced to differentiate into
neural-like cells. Human EMSCPCs were first
cultured for four to seven days in a serum-free
neural stem cell (NSC) medium for pre-induction.
During pre-induction, hEMSCPCs coalesced into
dense spheres that resembled neural rosettes.
In the presence of a conditioned differentiation
medium, pre-induced cells took on the morpho-
logical characteristics of neural cells, including
slender projections with inflated or claw-like
ends that contacted the soma or projections of
other cells as revealed by confocal microscopy.
Moreover, these differentiating cells expressed
the neural-specific markers β-III tubulin, MAP2,
GFAP, and synapsin I as evidenced by immu-
nocytochemistry. Both pre-induced hEMSCPCs
and uninduced hEMSCPCs were labeled with
CM-DiI and transplanted into the vitreous cavi-
ties of nude mice. Transplanted cells were ex-
amined four weeks later in frozen eyeball sec-
tions by immunofluorescence staining, which
demonstrated superior retinal migration and
neural differentiation of pre-induced cells. Our
study is the first to demonstrate tha t hEMSCPC s
possess the capacity to differentiate into neu-
ral-like cells, suggesting potential uses for the
treatment of retinal di seases such as age-r elated
macular degeneration.
Keywords: Human Epidermis; Pluripotent Cells;
Differentiation; Neural Cells; Cell Therapy
1. INTRODUCTION
Neurological degenerative diseases like Alzheimer’s
disease, Parkinson’s disease, and age-related macular
degeneration are a group of chronic, diverse and pro-
gressive disorders. Studies have shown heredity, oxida-
tive stress, neurotrophic factor deficiency, dysbolism and
other unknown factors could cause a main pathological
change of special neurons degeneration and loss, fol-
lowed by demyelination of nerve fibers [1]. These patho-
physiology leads to a decreased activity in the pathway
of neural conduction, and results in disturbance of me-
mory, learning, moving and other activities, which causes
severe public health burden, particularly in an aging po-
pulation. Current treatments for these diseases include
neuroprotective agents, surgery, physical stimulation, gene
therapy, and cell replacement [2-4]. However, pharma-
cologic, surgical, and physical therapies cannot cure
these diseases. Recent reports have shown that gene and
cell-replacement therapies are promising alternatives for
treating or even curing neurological disorders [5-7]. How-
ever, the complexity of the human genome and proteome
limits the therapeutic effects of single gene therapy. In-
deed, the results of clinical trials testing single gene
therapies for Parkinson’s disease were less than ideal
[8-9].
Theoretically, cell-replacement therapy can cure neu-
rodegenerative diseases by replacing lost cells and re-
constructing tissues, leading to functional recovery. Mul-
tipotent stem cells are widely used for research on cell-
replacement therapy and can be derived from both the
embryo (embryonic stem cells, ESCs) and adult tissue
(adult stem cells, ASCs). The strong plasticity of ASCs
enables directional differentiation into multiple cell types.
Numerous studies have revealed that ESCs, neural stem
cells (NSCs), bone marrow stem cells (BMSCs), and

M. Zhang et al. / Stem Cell Discovery 2 (2012) 141-154
142
precursor cells derived from peripheral blood, umbilical
cord blood, and fat tissue can differentiate into neural
cells [10-17]. However, there are ethical issues sur-
rounding the harvesting of ESCs; moreover, these cells
are potentially oncogenic [18-19]. Neural stem cells exist
in several adult human tissues but are difficult to isolate
[20]. The process of isolating BMSCs is invasive and
painful, and the quantities obtained are generally not suf-
ficient for therapeutic applications [21]. Moreover, more
accessible peripheral blood and umbilical cord blood
contain relatively low numbers of precursor cells, and it
is still disputed whether these precursor cells can inte-
grate with the host tissue and differentiate into the ap-
propriate cell types after transplantation [22-24]. Al-
though fat tissue is easily extracted, techniques for the
isolation and purification of precursor cells from adipose
tissue are still not fully developed [25].
Skin is the largest human organ and its cells are easily
harvested. Several groups have isolated pluripotent cells
from mammalian skin that can differentiate into neural
cells [26-32]. After transplantation into animal models,
these skin-derived pluripotent cells were able to promote
nerve regeneration and functional improvement after
injury [33-36]. It is known that epidermal stem cell-like
pluripotent cells are present in the epidermal-basal layer
and function in the repair and regeneration of the epi-
dermis [37-38]. We have isolated a population of me-
senchymal stem cell-like pluripotent cells from mixed
cultures of human epidermal cells that we refer to as
human epidermis-derived mesenchymal stem cell-like
pluripotent cells (hEMSCPCs) (national patent number:
201010282388.0) [39]. In this exploratory study, we exa-
mined whether hEMSCPCs have the capacity to dif-
ferentiate into neural-like cells. Our data revealed that
hEMSCPCs can be induced to differentiate into cells
with neural cell characteristics in vitro and express some
neural cell-specific markers in vivo when transplanted
into the mouse eye, suggesting the hEMSCPCs may be
used for autologous cell-based therapies to treat neuro-
logical disorders.
2. MATERIALS AND METHODS
2.1. Isolation of hEMSCPCs and Cultured in
Growth Medium
The hEMSCPCs were isolated from foreskin tissue
obtained from circumcision surgery. Tissue donors were
healthy as defined by normal blood and urine test results,
normal liver and lung function, no history of genetic
disease, and the absence of current infectious disease.
Written informed consent was provided by the partici-
pants. The study was approved by the Medical Ethics
Committee of Zhongshan Ophthalmic Center, Sun Yat-
sen University (No. 2008-30).
Briefly, foreskin tissue was rinsed in phosphate buff-
ered saline (PBS) containing gentamycin (1000 U/ml) for
subsequent treatment, the tissue was cut into pieces of 3
mm × 3 mm in size using scalpes and transferred into a
sterilized 15 ml centrifugation tube. Then DispaseⅡ (2
U/ml; GIBCO, USA) was added into the tube, incu-
bated at 6˚C - 8˚C for 15 hours and then 37˚C for 1 hour
to remove the dermis. The epidermis were transferred
into a new sterilized 15 ml centrifugation tube, washed
with PBS for 5 times and crushed. Then suspended with
PBS containing 0.25% trypsin and gently pippetted, in-
cubated at 37˚C for 30 min. Then washed with PBS
twice more and centrifugated at 1200 rpm for 5 min,
discarded the supernatant. Cell precipitation was sus-
pended in growth medium consisting of 80% DMEM
(GIBCO, USA), 18% fetal bovine serum (FBS) (Si
Jiqing Ltd., China), 10 ng/ml basic fibroblast growth
factor (bFGF) (PERPO-TECH, USA), 2 ng/ml stem cell
factor (SCF) (PERPO-TECH, USA), and 1% MEM non-
essential amino acids (NEAA) (100 × solution, GIBCO,
USA), and plated in T-25 cell culture flask, incubated at
37˚C in a 5% CO2 atmosphere. The flask remained un-
moved within 48 hours, then the medium was replaced
according to the rate of cell growth, and the un-adherent
cells were removed. Ten days later, small hEMSCPCs
appeared; three weeks later, they were deplated using
0.25% trypsin-0.02% ethylene diamine tetraacetic acid
(EDTA) and passaged at 1:3. They were continuously
cultured and passaged over 30 times in vitro [39]. The
hEMSCPCs from passages 17 to 19 derived from same
biopsy (fore-skin of a 21-year-old male) were used for
this study.
Cryopreserved hEMSCPCs were resuscitated from
liquid nitrogen and suspended in growth medium (men-
tioned above). Cell suspensions (8 ml of 1.0 × 104 cells/
ml) were plated in T-25 cell culture flasks and incu-
bated at 37˚C in a 5% CO2 atmosphere. The medium was
replaced according to the rate of cell growth. When cells
reached confluence, the hEMSCPCs were deplated using
0.25% trypsin-0.02% EDTA and passaged at 1:3. Cul-
tures were observed and photographed using an in-
verted microscope (Leica DMIRB, Germany).
2.2. Pre-Induction Culture of hEMSCPCs in
NSC Medium
For pre-induction, hEMSCPCs were deplated and
suspended in NSC medium consisting of 96% DMEM/
F12 (GIBCO, USA), 2% B27 (GIBCO, USA), 20 ng/ml
bFGF (PERPOTECH, USA), 20 ng/ml epidermal growth
factor (EGF) (PERPOTECH, USA), 2 ng/ml SCF (PER-
POTECH, USA), and 1% MEM NEAA (100 × solution,
GIBCO, USA). Cell suspensions (8 ml of 1.0 × 104
cells/ml) were replated in T-25 cell culture flasks and
incubated at 37˚C under 5% CO2. The medium was re-

M. Zhang et al. / Stem Cell Discovery 2 (2012) 141-154 143
placed everyday. Cultures were observed and photo-
graphed as described above.
2.3. Differentiation of hEMSCPCs in
Conditioned Differentiation Medium
After 6 days, the NSC medium was replaced with a
conditioned differentiation medium consisting of 88%
DMEM/F12 (GIBCO, USA), 10% FBS (Si Jiqing Ltd.,
China), 20 ng/ml bFGF (PERPOTECH, USA), 20 ng/ml
EGF (PERPOTECH, USA), and 1% MEM NEAA (100
× solution, GIBCO, USA). Cells were incubated for 1 or
3 weeks depending on the experiment. In addition, hEM-
SCPCs cultured in growth medium but not pre-induced
in NSC medium were cultured in differentiation medium
as a control. Cultures were observed and photographed
under an inverted microscope (Leica DMIRB, Germany).
The hEMSCPCs pre-induced in the NSC medium grew
slender projections during differentiation, so some cul-
tures were plated at lower density (1.0 × 103 cells/ml) to
aid in morphological observation.
2.4. Subculturing for Immunofluorescence
and CM-DiI Staining
To detect the expression of cell-specific markers in
hEMSCPCs during differentiation, cells were seeded
onto cover slips and immunostained (below). Other cul-
tures were labeled with CM-DiI to observe cell-cell con-
tacts. Seeded cover slips were divided into five groups of
10 slides each. The hEMSCPCs cultured in the growth
medium but not pre-induced by NSC medium constituted
group A (GM, control). The hEMSCPCs cultured in
growth medium and then pre-induced in the NSC me-
dium for six days constituted group B (GM + NSC). Hu-
man EMSCPCs cultured in growth medium, pre-induced
in NSC medium for six days, and then cultured in the
conditioned differentiation medium for three weeks were
group C (GM + NSC + CM 3 weeks). Human EMSCP-
Cs cultured in the growth medium but not pre-induced in
the NSC medium for six days before culture in the con-
ditioned differentiation medium for three weeks were
group D (GM + CM 3 weeks) and served as the control
for group C. Group E consisted of hEMSCPCs cultured
in growth medium, pre-induced in the NSC medium for
six days, and then cultured in the conditioned differentia-
tion medium for one week (GM + NSC + CM 1 week).
At the beginning, cell densities of group A and group B
were adjusted to 1.0 × 104 cells/ml, while those of groups
C, D, and E were adjusted to 1.0 × 103 cells/ml for im-
proved morphological observation of differentiating cells.
Human EMSCPCs suspensions were plated onto sterile
22 × 22 mm2 cover slips in 35 mm culture dishes. Each
culture dish contained one hEMSCPCs cover slip. All
cultures were incubated at 37˚C under 5% CO2. After the
treatments described above, cover slips were collected
for staining. Groups A, B, C, and D cells were stained by
immunofluorescence and group E cultures were labeled
with CM-DiI.
2.5. CM-DiI Labeling of the Differentiating
hEMSCPCs
Cells were labeled with CM-DiI rather than processed
for electron microscopy (EM) because EM fixation and
processing/staining tend to cause contraction of pro-
cesses [40,41]. Furthermore, EM is laborious and expen-
sive. In contrast, CM-DiI is a lipid-soluble biomembrane
stain that allows for clear visualization of cell morpho-
logy and cell-cell contacts [42].
Ten hEMSCPCs-seeded cover slips of group E (de-
fined above) were collected, washed twice in PBS, and
stained with 5 μl/ml CM-DiI (Molecular Probes, USA) in
200 μl PBS for 3 min at 37˚C. Stained cover slips were
washed twice quickly in PBS and then fixed in 4% para-
formaldehyde for 40 min. The nuclei were counterstained
by Hoechst (Sigma, USA) for 5 min at room temperature
(RT). Slides were then treated by an anti-fade solution
(Applygen, China) and imaged under a laser confocal
scanning microscope (Zeiss, Germany). Ten different vi-
sual fields were observed in each cover slip.
2.6. Immunofluorescence Staining of
hEMSCPCs Cultured in Vitro
Immunofluorescence staining was used to detect the
expression of cell-specific antigens in hEMSCPCs cul-
tured in vitro. The hEMSCPCs-seeded cover slips of
groups A, B, C, and D were collected and fixed in 4%
paraformaldehyde for 15 min, permeabilized in 0.3%
Triton-X for 15 min, and then incubated at 37˚C for 40
min in the following primary antibodies: human nestin,
MAP2, synapsin I (Abcam, USA), vimentin (ZSGB-BIO,
China), β-III tubulin (Millipore, USA), and GFAP (Ep-
tomics, USA). Slides were then incubated in secondary
antibodies, either Cy3-conjugated goat anti-mouse (Mil-
lipore, USA) or FITC-conjugated goat anti-rabbit (South-
ern Biotech, USA) for 30 min at RT. The nuclei were
stained by Hoechst (Sigma, USA) for 5 min at RT, and
then all slides were treated with an anti-fade solution
(Applygen, China) and imaged under a laser confocal
scanning microscope (Zeiss, Germany). Ten different vi-
sual fields were observed in each cover slip.
2.7. Flow Cytometry
Flow cytometry was used to quantify the expression of
cell-specific markers in GM + NSC 4 days and GM
hEMSCPCs groups. Both direct labeling and indirect
labeling were used. Antibodies used for direct labeling
were specific for human CD73 and its PE-iso-type con-

M. Zhang et al. / Stem Cell Discovery 2 (2012) 141-154
144
trol (BD, USA). Primary antibodies used for indirect
labeling were specific for human nestin (Abcam, USA),
vimentin (ZSGB-BIO, China), MAP2, and GFAP (Ab-
cam, USA). The secondary antibodies were R-PE-con-
jugated goat anti-mouse and FITC-conjugated goat
anti-rabbit (Southern Biotech, USA). Fix and Perm Cell
Permeabilization reagents (Invitrogen, USA) were used
for labeling intracellular antigens. Cell suspensions were
stained and counted by flow cytometry according to the
manufacturer’s directions. Cell suspensions treated with
secondary antibodies but not primary antibodies served
as iso-type controls for indirect labeling. Cell suspend-
sions were tested immediately by flow cytometry (BD
FACSAriaTM, USA) using FCS Express V3 software for
data analysis. The positive value of iso-type controls was
maintained at 0% to 1%.
Prior to cell transplantation in vivo (below), the ex-
pression levels of immunogenic markers (HLA-I and
HLA-DR) in GM + NSC 4 days and GM hEMSCPCs
were first detected by direct labeling and flow cytometry.
Cell suspensions were treated with antibodies specific for
human HLA-I (Invitrogen, USA), HLA-DR, and their
FITC-iso-type control (BD, USA). Protocols were in
accordance with the manufacturer’s directions.
2.8. Transplantation of hEMSCPCs into the
Vitreous Cavities of Nude Mice
The retina, an extension of the central nervous system
containing a variety of highly differentiated cell types,
was chosen to provide the internal microenvironment for
hEMSCPCs differentiation. Human EMSCPCs were trans-
planted into the vitreous cavities of nude mice, and mi-
gration and differentiation were observed after four
weeks.
The GM + NSC 4 days and GM cultures were termed
groups A and B in the transplantation study. Before
transplantation, both groups were labeled by CM-DiI
(Molecular Probes, USA) according to the manufac-
turer’s instructions. In addition, one 500 μl sample from
each cell suspension labeled by CM-DiI was analyzed by
flow cytometry to test CM-DiI labeling efficiency. Cell
suspensions (1 × 107 cells/ml) with high labeling effi-
ciency were immediately injected into the eyes of nude
mice.
Sixteen 6-week-old BLAB/c nude mice of both gen-
ders were provided by the Laboratory Animal Center,
Sun Yat-sen University (Quality certificate number:
0061839). The mice were cared for in accordance with
the Regulations on Administration of Experimental Ani-
mals in Guangdong Province, China. They were housed
in the specific pathogen-free (SPF) area of the Ophthal-
mology Animal Experimental Center, ZhongShan Oph-
thalmic Center, Sun Yat-sen University, with a 12 h light-
dark cycle (23˚C - 25˚C, humidity 55%). These trans-
plantation experiments were approved by the Laboratory
Animal Administration and Ethics Committee of Zhong-
shan Ophthalmic Center, Sun Yat-sen University (No.
2010-024). The 16 nude mice were randomly divided
into three groups (group A, B, and C), each with a 1:1
sex ratio. The right eyes of all group A (n = 6) mice were
injected with hEMSCPCs that had been pre-induced in
the NSC medium for four days, while the right eyes of
group B were injected with hEMSCPCs cultured in the
growth medium but not pre-induced by NSC medium.
The remaining four mice (group C) served as normal
controls.
Before transplantation, experimental mice were anes-
thetized by intraperitoneal injection of 4.3% chloral hy-
drate (0.01 ml per 1 g body weight) obtained from the
Ophthalmologic Hospital, Sun Yat-sen University. To-
bramycin eye drops were used to disinfect the experi-
mental eye, followed by dicaine hydrochloride eye drops
(both drugs obtained from the Ophthalmologic Hospital,
Sun Yat-sen University) for superficial anesthesia. A
1-ml-injector needle was used to pierce the central cor-
nea and drain part of the aqueous humor to lower the
intraocular pressure. Under an operating microscopy
(Topcon, Japan), a second 1-ml injector containing 10 μl
of either cell suspension (pre-induced with NSC medium
or untreated) was placed 1 mm outside the corneoscleral
junction on the temporal side. The needle penetrated at a
15˚C acute angle relative to the eyeball coronal plane.
When the needle reached the vitreous cavity, cell sus-
pendsions were injected. The needle was immediately
withdrawn at the first signs of eyeball puffing. The ex-
perimental eye was then washed with tobramycin eye
drops, followed by tobramycin eye ointment. All proce-
dures were performed in the SPF area operating room.
Appropriate body temperature was maintained during the
operation and intraoperative animal care conformed to
institutional guidelines. After mice regained conscious-
ness, they were sent back to the feeding room. To prevent
infection, tobramycin eye drops were applied three times
daily for three days after the operation.
2.9. Frozen Eyeball Sectioning and
Immunofluorescence Staining
Four weeks after cell transplantation, all the mice were
anesthetized by intraperitoneal injection of 4.3% chloral
hydrate and sacrificed by cervical dislocation. All right
eyes were enucleated, frozen in OCT embedding com-
pound (Sakura, USA), and stored at –20˚C before cry-
ostat sectioning at 6 μm on a freezing microtome (Leica,
Germany). Sections were then processed for immunofl-
uorescence labeling as described (see Immunofluores-
cence staining of hEMSCPCs cultured in vitro). Primary
antibodies were specific for human nestin, MAP2,
rhodopsin (Abcam, USA), β-III tubulin (Sigma, USA),

M. Zhang et al. / Stem Cell Discovery 2 (2012) 141-154 145
and GFAP (Epitomics, USA). An FITC-conjugated goat
anti-rabbit antibody (Southern Biotech, USA) was used
for fluorescence tagging. The migration and differentia-
tion of the transplanted hEMSCPCs in nude mice were
examined by laser confocal scanning microscopy (Zeiss,
Germany). Ten different visual fields were observed in
each section.
2.10. Statistical Analysis
Analysis of variance (ANOVA) with repeated meas-
ures was used to compare treatment means. A value of P
< 0.05 was considered statistically different. The SPSS
v13.0 software package was used for all statistical ana-
lyses.
3. RESULTS
3.1. Morphology of hEMSCPCs Cultured in
Growth Medium
When cultured in growth medium, hEMSCPCs ap-
peared as small spindle-shaped cells that adhered to the
bottoms of culture flasks in a monolayer arranged in a
vortex pattern (Figures 1(A) and (B)). They proliferated
rapidly in growth medium; when seeded at 1.0 × 104
cells/ml × 8 ml in T-25 plastic culture flasks, they rea-
ched confluence in two days [39].
3.2. Changes in hEMSCPCs Morphology
during Pre-Induction in NSC Medium
To direct hEMSCPCs toward the neuronal lineage, we
provided a conducive environment using a neural stem
cell (NSC) medium. One day after culture in NSC me-
dium, the hEMSCPCs somata aggregated to form small
dense light-reflective spheres that were fully or partly
adherent to the bottoms of the flasks. A few small spin-
dle-shaped cells began to stretch out from the edges of
the spheres (Figures 1(C) and (D)). After three days in
NSC medium, many spindle-shaped cells stretched out
from the edges of the spheres and formed strongly light
reflective rosette-shaped clusters at the edges (Figures
1(E) and (F)). As days went on, the spindle-shaped cells
stretched out further and the rosettes enlarged (Figures
1(G) and (H)).
3.3. Changes in hEMSCPCs Morphology
during Culture in Conditioned
Differentiation Medium as Revealed by
Light Microscopy
At day 6, NSC medium was replaced with a condi-
tioned differentiation medium. Within one day, most
cells began to grow one or more slender projections that
resembled neurites (Figures 2(B)- (D)). Three days later,
as these projections became longer and continued to
extend, contacts were formed between cells (Figures
2(E)-(G)). After one week in conditioned medium, the
projections continued to extend and branches emerged at
the ends (Figure 2(H)). In contrast, hEMSCPCs cultured
in the growth medium but not in NSC medium showed
no obvious changes in morphology during incubation in
the conditioned differentiation medium (Figure 2(A)).
3.4. Changes in hEMSCPCs Morphology
during Culture in Conditioned
Differentiation Medium as Revealed
by CM-DiI Labeling
To examine cell morphology and cell-cell contacts in
detail, cultures were stained with the membrane dye
CM-DiI and viewed under laser confocal scanning mi-
croscopy. The profiles of differentiated hEMSCPCs were
clearly distinguished by CM-DiI, including cell bodies
and slender projections that exhibited inflated ends or
even claw-like ends (Figures 3(A)-(C)). Many of these
CM-DiI-labeled differentiated cells contacted each other,
either through projection-soma or projection-projection
contacts (Figures 3(A)-(C)).
3.5. Immunofluorescence Staining of
Cultured hEMSCPCs
Immunofluorescence staining was used to detect the
expression of cell-specific markers during culture in the
three culture media (growth, NSC, and conditioned dif-
ferentiation media). When cultured only in the growth
medium, hEMSCPCs were positive for the NSC marker
nestin as well as the neural precursor cell and mesen-
chymal cell marker vimentin (Figures 3(D) and (E)), but
negative for neural cell markers β-III tubulin, micro-
tubule-associated protein-2 (MAP2), glial fibrillary
acidic protein (GFAP), and the synaptic marker synapsin
I (data not shown). After pre-induction in the NSC me-
dium, the expression of these markers was not signifi-
cantly changed, though cells remained positive for nestin
and vimentin (Figure 3(F)). Consistent with the marked
morphological transformation (Figures 2 and 3(A)-(C)),
cells cultured in the conditioned differentiation medium
following pre-induction in NSC medium expressed neu-
ral cell markers β-III tubulin, MAP2, GFAP, and synap-
sin I (Figures 3(G)-3(J)). In contrast, hEMSCPCs cul-
tured in the growth medium but not pre-induced in NSC
medium did not express these neural cell markers during
culture in conditioned differentiation medium (data not
shown).
3.6. Flow Cytometry Analysis
Flow cytometry was used to quantify the different
protein expression phenotypes. During culture in growth
medium, most hEMSCPCs stably expressed the mesen-

M. Zhang et al. / Stem Cell Discovery 2 (2012) 141-154
146
Figure 1. Morphological changes of hEMSCPCs during culture in growth medium and NSC
medium. (A) and (B) The morphology of hEMSCPCs cultured in growth medium. Plated
cells formed monolayers in a vortex pattern within two days ((A), magnification ×50; (B),
magnification ×200). (C)-(H) Change in morphology during pre-induction in NSC medium.
One day after changing to the NSC medium, hEMSCPCs formed small dense spheres and a
few small spindle-shaped cells began to stretch out from the edges ((C), magnification ×50;
(D), magnification ×200). After three days in NSC medium, many spindle-shaped cells
stretched out from the spheres ((E), magnification ×50; (F), magnification ×200). After six
days, many cells were observed migrating outward ((G), magnification ×50; (H), magnifica-
tion ×200).

M. Zhang et al. / Stem Cell Discovery 2 (2012) 141-154 147
Figure 2. Morphology of hEMSCPCs during culture in conditioned differentiation medium. (A) Human
EMSCPCs not pre-induced in NSC medium showed no significant changes in morphology when cultured in
the conditioned differentiation medium (magnification ×200). (B)-(H) After one day in the conditioned differ-
entiation medium, hEMSCPCs that were pre-induced began to grow one (B), two (C) or more (D) slender pro-
jections (magnification ×200); (E)-(G) After three days in conditioned differentiation medium, these projec-
tions were longer and continued to extend toward neighboring cells. Contacts (red arrows) were formed be-
tween cells (magnification ×200; (e), (f), (g1), and (g2) are the magnified boxed areas in panels (E), (F), and
(G). (H) After one week, the projections continued to extend and branches (red arrows) formed at the ends
(magnification ×100, h is the magnified region in (H)).

M. Zhang et al. / Stem Cell Discovery 2 (2012) 141-154
148
Figure 3. CM-DiI labeling of differentiating hEMSCPCs and immunofluorescence staining of hEMSCPCs cultured
in vitro. (A)-(C) The profile of differentiated hEMSCPCs labeled by red CM-DiI, showing cell bodies and slender
projections with inflated or claw-like ends (white arrows in (C)). These CM-DiI-labeled differentiated cells contacted
each other and CM-DiI-labeled cell membranes could be seen between these contacts (white arrows) ((a), (b), and (c)
are high magnification zones from panels (A), (B), and (C)). (D)-(E) When cultured in growth medium, many
hEMSCPCs were positive for nestin ((D), green) and vimentin ((E), red). (F) After pre-induction in NSC medium,
cells were also positive for nestin (green) and vimentin (red). (G)-(J) Cells pre-induced for six days could express the
neural cell markers β-III tubulin ((G), red), MAP2 ((H), green), GFAP ((I), green), and the synaptic marker synapsin
I ((J), green) during culture in differentiation medium. Nuclei were counterstained with blue Hoechst. All scale bars
are 20 μM.
chymal stem cell marker CD73 (94.7%) and the NSC
marker nestin (81.4%). A smaller fraction expressed the
neural precursor cell and mesenchymal cell marker
vimentin (30.8%), while none expressed the neural cell
markers MAP2 or GFAP (Figure 4(A)). Daily assess-
ment of marker expression during seven days of pre-
induction in NSC medium showed that CD73 expression
was maintained, while MAP2 and GFAP were still not
expressed (Figure 4(A)). No statistically significant dif-
ference in the expression of CD73, MAP2, and GFAP

M. Zhang et al. / Stem Cell Discovery 2 (2012) 141-154 149
was found between days in NSC medium (n = 3, P >
0.05) (Figure 4(A)). During the first three days of
pre-induction, the expression levels of nestin (80.5% ±
0.98% of all cells) and vimentin (34.5% ± 1.08%) were
stable (n = 3, P > 0.05). From day 3 to day 7, however,
the expression of nestin and vimentin fluctuated. Nestin
expression was 74.5% ± 0.91% on day 4, 71.2% ± 1.09%
on day 5, and 79.6% ± 0.86% on day 6, before decreas-
ing again on day 7 (60.9% ± 1.39%). Vimentin expres-
sion first increased from day 3 to 4 (44.5% ± 2.05%),
decreased from day 4 to 5 (20.5% ± 1.18%), increased
from day 5 to 6 (26.6% ± 1.68%), then decreased again
from day 6 to 7 (18.5% ± 0.68%). Changes in expression
of both proteins were statistically significant between
days (n = 3, P < 0.001) (Figure 4(A)).
Prior to cell transplantation in vivo, the expression of
the immunogenic markers HLA-I and HLA-DR were
also detected in hEMSCPCs by flow cytometry. When
cultured in the growth medium, the hEMSCPCs mode-
rately expressed HLA-I (35.3%) but not HLA-DR (Fig-
ure 4(B)). After pre-induction for four days, they con-
tinued to moderately express HLA-I (38.4%) but not
HLA-DR, indicating that hEMSCPCs retained the same
immunogenic status in NSC medium (Figure 4(C)). In
addition, hEMSCPCs were also stained with CM-DiI
prior to cell transplantation in vivo. Staining efficiency
was assessed by flow cytometry and revealed a labeling
rate of 99.7% (Figure 4(D)).
3.7. Migration and Differentiation of
Transplanted hEMSCPCs in the
Retinas of Nude Mice
To examine neural differentiation in vivo, CM-DiI-
stained hEMSCPCs were implanted into the vitreous
cavities of nude mice. Two groups of cells were trans-
planted, hEMSCPCs pre-induced in the NSC medium for
four days (group A) and hEMSCPCs cultured in the
(A) (B)
(C) (D)
Figure 4. Flow cytometry analysis of cell-specific marker expression. (A) Cell-specific markers (CD73-blue,
nestin-cyan, vimentin-gray, MAP2-purple, GFAP-yellow) expressed by hEMSCPCs. During seven days in NSC
medium, most hEMSCPCs stably expressed CD73 and nestin, few expressed vimentin, while none expressed
MAP2 or GFAP. (B) In growth medium, hEMSCPCs moderately expressed HLA-I (35.3% of cells) but not
HLA-DR. (C) After four days in NSC medium, hEMSCPCs still moderately expressed HLA-I (38.4%) but not
HLA-DR. (D) The efficiency of CM-DiI labeling prior to transplantation was up to 99.7%.

M. Zhang et al. / Stem Cell Discovery 2 (2012) 141-154
150
growth medium but not pre-induced in the NSC medium
(group B). Cells of the two groups were separately trans-
planted into the vitreous cavities of nude mice. Four
weeks later, migration and differentiation in the retina
were assessed by immunostaining in frozen sections of
the eyes. The retinas of normal uninjected nude mice
were clear and regular, with no fluorescent (red- or
green-labeled) cells (Figure 5(A)). In mice injected with
group A cells, five of six experimental eyes showed mi-
gration of transplanted cells after four weeks (Figures
5(B)-(F)). In addition, pre-induced CM-DiI-stained cells
expressed the NSC marker nestin and the neural cell
markers β-III tubulin and GFAP (Figures 5(I)-(K)), but
not MAP2 (data not shown). In mice injected with group
B cells, however, only one of six experimental eyes
showed migration of transplanted cells (Figures 5(G)
and (H)) as well as expression of β-III tubulin (Figure
5(L)). No nestin-, MAP2-, or GFAP-positive cells were
found (data not shown). Cells of group A exhibited supe-
rior migration and differentiation compared to cells of
group B. In both experimental groups, no transplanted
cells expressed the photoreceptor cell marker rhodopsin
(data not shown). The transplanted cells that migrated
into the retinas presented as either single cells or as ag-
glomerates (Figures 5(B)-(H)). Most cells concentrated
in the subretinal cavities, the retinal pigment epithelium
(RPE) layer, and other nearby areas (Figures 5(D)-(F),
and 5(H)). Thus, injected cells migrated across all retinal
layers to reach the RPE.
4. DISCUSSION
The shear size and reparative capacity of human skin
makes it an ideal source of pluripotent cells for research
and possible autologous cell-based therapies. As a first
step toward utilizing these pluripotent cells for neural
regeneration therapy, we developed a two step-culture
method that gradually induced the appearance of a neu-
ral-like morphology and the expression of several neu-
ral-specific cell markers. When introduced into the retina,
cells from the first culture step (pre-induction) migrated
cross multiple cell layers and expressed neural-specific
cell markers. Although they were not functionally inte-
grated into the healthy retina (at least after four weeks), it
is possible that these pre-neural-like cells may be in-
duced to replenish lost cells in the degenerating or dam-
age retina, such as photoreceptor cells. The capacity of
these hEMSCPCs to express neuron-like and glia-like
phenotypes in vitro and in vivo suggests that these cells
are a potential source for neural stem cells to repair
damaged neural tissue.
A number of research groups have isolated precursor
cells from skin and shown that these cells can differenti-
ate into multiple cell types under appropriate conditions.
Miller et al. [27,31,43-49] isolated precursors from neo-
natal mammalian skin that could differentiate into other
cell types, and even mediate regeneration after injury in
animal models. They retained a normal karyotype and
capacity to differentiate even after regular passage for
one year in vitro, but pluripotency was markedly lower in
skin-derived precursors from adult mammals. Further-
more, the biosafety of adult-derived cells is unknown.
Katsuoka et al. [33-35,50-52] isolated a population of
stem cells from mammalian dermal hair-follicles that
could also differentiate into other cell types and pro-
mote regeneration after injury, but they were difficult to
isolate in sufficient quantities for clinical applications.
In contrast, the hEMSCPCs that we isolated from adult
human epidermis were easily obtained in large quantities,
could be continuously passaged over fifty times without
changes to the normal karyotype, and demonstrate good
biosafety in vitro [39].
The growth patterns of hEMSCPCs changed markedly
when cultured in NSC medium; individual cells coa-
lesced into dense, highly light-reflective spheres. Spin-
dle-shaped cells educed from these spheres began to
spread out so that the aggregates resembled neural ro-
settes. Once these cells were cultured in a conditioned
differentiation medium, many differentiated into cells
with a neural phenotype. Some cells grew slender pro-
jections with inflated or claw-like ends that contacted the
soma or projections of other cells as revealed by the cell
tracker CM-DiI, a lipid soluble biomembrane stain that
stably labels growing cells for clear imaging of fine
morphological features [40-42]. Many also expressed
neuronal or glial markers, including β-III tubulin, MAP2,
GFAP, and synapsin I. Flow cytometry and immunofluo-
rescence staining showed that hEMSCPCs expressed the
NSC marker nestin, the neural precursor and mesenchy-
mal cell marker vimentin, but not the neural cell markers
β-III tubulin, MAP2, GFAP, or synapsin I during sequen-
tial culture in the growth medium and NSC medium.
However, expression of neural cell markers was de-
pendent on pre-induction in NSC medium, as hEMSCP-
Cs cultured only in differentiation medium did not ex-
press neural markers (β-III tubulin, MAP2, GFAP, or
synapsin I). Moreover, uninduced cells showed inferior
migration and differentiation compared to pre-induced
cells after transplantation into the vitreous cavities of
nude mice. Thus, pre-induction did not markedly alter
neural marker expression in hEMSCPCs but was neces-
sary to allow differentiation in a special conditioned me-
dium and in the mouse eye (part of the central nervous
system).
Flow cytometry was used to quantify the expression of
cell-specific markers. The cell surface glucoprotein
CD73 is a marker for mesenchymal stem cells [53].
Nestin is a class VI intermediate filament protein once
thought to be a specific marker of NSCs, but recent re-

M. Zhang et al. / Stem Cell Discovery 2 (2012) 141-154 151
Figure 5. Migration and differentiation of transplanted hEMSCPCs in the retinas of
nude mice. (A) The retinas of normal nude mice were clear and regular, and no red- or
green-labeled cells were observed. (B)-(L) Four weeks after transplantation into the
vitreous cavities of nude mice, many pre-induced hEMSCPCs (white arrows) had mi-
grated into the retinal ganglion cell layer (B), (C), the sub-retinal space (D), (E), and
the retinal pigment epithelium layer (E), (F). In addition, pre-induced cells expressed
β-III tubulin (I), GFAP (J), and nestin (K). Transplanted cells that had not been
pre-induced by NSC medium in vitro (white arrows) could migrate into the retinal
ganglion cell layer (G) and the sub-retinal space (H), and express β-III tubulin (L). “V”
represents the vitreous cavities or nearby areas. Nuclei were counterstained with blue
Hoechst. All scale bars represent 20 μM.

M. Zhang et al. / Stem Cell Discovery 2 (2012) 141-154
152
ports indicated that it is also expressed by multipotent
precursor cells and that expression correlates with the
potential for proliferation, migration, and differentiation
[54,55]. Vimentin is a class III intermediate filament
protein and a marker for neural precursor cells and mes-
enchymal-derived cells [56-59]. MAP2 is a mature neu-
ronal marker, while the intermediate filament protein
GFAP is expressed almost exclusively by astrocytes.
Flow cytometry showed that the expression levels of
these markers by hEMSCPCs were not significantly
changed during culture in growth medium or brief (2 - 3
day) culture in NSC medium. However, from the 3rd to
7th day in NSC medium, expression of nestin and vi-
mentin began to fluctuate, possibly indicating the be-
ginning of a phenotypic transition. However, hEMSCPCs
pre-induced in NSC medium grew slender projections
that could be easily sheared off the soma during harvest-
ing for flow cytometry, so this fluctuation in expression
may reflect variable loss of markers localized to pro-
cesses. A more accurate determination of marker expres-
sion patterns during NSC culture will require the use of
alternative methods like real time PCR or Western blot-
ting.
Approximately the same percentage of pre-induced
and uninduced hEMSCPCs expressed HLA-I, indicating
that these cells have a stable immunogenicity. Nude mice
were chosen for hEMSCPC transplantation experiments
because they are immunologically defective and so
would not generate an immune response to transplanted
cells. Four weeks after injection of hEMSCPCs, both in-
duced and uninduced CM-DiI-labeled cells had migrated
primarily to the subretinal space, retinal pigment epithet-
lium layer, and other nearby areas. The reasons for this
selective migration are still obscure. Both hEMSCPCs
and retinal pigment epithelium originate from the ec-
toderm, and this homology may have allowed for better
survival of transplanted hEMSCPCs. The subretinal ca-
vity may also accumulate transplanted cells even in im-
munologically active mice because it is an immunopri-
vileged zone. While both induced and uninduced cells
migrated, pre-induced cells showed superior migration
and neural differentiation.
There were differences in neural differentiation in vi-
tro versus in vivo. In contrast to observations in vitro, the
mature neuron marker MAP-2 was not expressed by ei-
ther group of transplanted hEMSCPCs, although this
might reflect the short transplantation time (four weeks).
In addition to common markers like MAP-2, transplanted
cells did not express retina-specific markers. For exam-
ple, rhodopsin, a marker of retinal photoreceptors, was
not expressed by transplanted hEMSCPCs that migrated
into the retina, possibly because the healthy retina re-
leases no factors that would induce differenttiation. A
longer transplantation time or induction of growth factor
expression may be required for full expression of neural
markers, but these questions require further study.
The epidermis is easily harvested, so hEMSCPCs may
be a productive source of cells for autologous cell-based
therapy against neurological diseases. Our results show
that hEMSCPCs possess the capacity to differentiate into
neural-like cells if pre-induced in NSC medium. Whether
hEMSCPCs can replenish endogenous neural cells after
induction and differentiation requires further study. To
reach this goal, further studies are required to understand
the neural lineage capacity of these cells, including tests
of differentiation efficiency and function in vitro, and to
assess the biological safety of these cells in vivo. Ex-
periments testing the restorative efficacy of these cells in
animal models of neurodegenerative diseases are clearly
warranted.
5. ACKNOWLEDGEMENTS
We are grateful to members of the State Key Laboratory of Oph-
thalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, for
discussion and advice. This work was supported by the Science and
Technology Projects of Guangdong Province, China (2009B060600002,
2010B060500006).
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