Large-scale expansion and characterization of human adult neural crest-derived multipotent stem cells from hair follicle for regenerative medicine applications
Aim: The purpose of this work was to obtain, multiply and characterize the adult neural crest-derived multipotent stem cells from human hair follicle for their further clinical use. Materials and Methods: Adult neural crest-derived multipotent stem cells were obtained from human hair follicle by exp...
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| Published in: | Experimental Oncology |
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| Date: | 2017 |
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
2017
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| Cite this: | Large-scale expansion and characterization of human adult neural crest-derived multipotent stem cells from hair follicle for regenerative medicine applications / R.G. Vasyliev, A.E. Rodnichenko, O.S. Gubar, A.V. Zlatska, I.M. Gordiienko, S.N. Novikova, D.O. Zubov // Experimental Oncology. — 2017 — Т. 39, № 3. — С. 171–180. — Бібліогр.: 29 назв. — англ. |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine| _version_ | 1860017349582651392 |
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| author | Vasyliev, R.G. Rodnichenko, A.E. Gubar, O.S. Zlatska, A.V. Gordiienko, I.M. Novikova, S.N. Zubov, D.O. |
| author_facet | Vasyliev, R.G. Rodnichenko, A.E. Gubar, O.S. Zlatska, A.V. Gordiienko, I.M. Novikova, S.N. Zubov, D.O. |
| citation_txt | Large-scale expansion and characterization of human adult neural crest-derived multipotent stem cells from hair follicle for regenerative medicine applications / R.G. Vasyliev, A.E. Rodnichenko, O.S. Gubar, A.V. Zlatska, I.M. Gordiienko, S.N. Novikova, D.O. Zubov // Experimental Oncology. — 2017 — Т. 39, № 3. — С. 171–180. — Бібліогр.: 29 назв. — англ. |
| collection | DSpace DC |
| container_title | Experimental Oncology |
| description | Aim: The purpose of this work was to obtain, multiply and characterize the adult neural crest-derived multipotent stem cells from human hair follicle for their further clinical use. Materials and Methods: Adult neural crest-derived multipotent stem cells were obtained from human hair follicle by explant method and were expanded at large-scale up to a clinically significant number. The resulted cell cultures were examined by flow cytometry and immunocytochemical analysis. Their clonogenic potential, ability to self-renewal and directed multilineage differentiation were also investigated. Results: Cell cultures were obtained from explants of adult human hair follicles. Resulted cells according to morphological, phenotypic and functional criteria satisfied the definition of neural crest-derived multipotent stem cells. They had the phenotype Sox2⁺Sox10⁺Nestin⁺CD73⁺CD90⁺CD105⁺CD140a⁺CD 140b⁺CD146⁺CD166⁺CD271⁺CD349⁺ CD34⁻CD45⁻CD56⁻HLA⁻DR⁻, showed high clonogenic potential, ability to self-renewal and directed differentiation into the main derivatives of the neural crest: neurons, Schwann cells, adipocytes and osteoblasts. Conclusion: The possibility of a large-scale expansion of adult neural crest-derived multipotent stem cells up to 40–200·106 cells from minimal number of hair follicles with retention of their phenotype and functional properties are the significant step towards their translation into the clinical practice.
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| first_indexed | 2025-12-07T16:45:33Z |
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Experimental Oncology 39, 171–180, 2017 (September) 171
LARGE-SCALE EXPANSION AND CHARACTERIZATION OF HUMAN
ADULT NEURAL CREST-DERIVED MULTIPOTENT STEM
CELLS FROM HAIR FOLLICLE FOR REGENERATIVE MEDICINE
APPLICATIONS
R.G. Vasyliev1, 2, *, A.E. Rodnichenko1, 2, O.S. Gubar2, 3, A.V. Zlatska1, 2, I.M. Gordiienko2, 4,
S.N. Novikova1, D.O. Zubov1, 2
1State Institute of Genetic and Regenerative Medicine, National Academy of Medical Sciences of Ukraine,
Kyiv 04114, Ukraine
2Biotechnology Laboratory ilaya.regeneration, Medical Company ilaya®, Kyiv 03115, Ukraine
3Institute of Molecular Biology and Genetics, NAS of Ukraine, Kyiv 03680, Ukraine
4R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NAS of Ukraine,
Kyiv 03022, Ukraine
Aim: The purpose of this work was to obtain, multiply and characterize the adult neural crest-derived multipotent stem cells from
human hair follicle for their further clinical use. Materials and Methods: Adult neural crest-derived multipotent stem cells were
obtained from human hair follicle by explant method and were expanded at large-scale up to a clinically significant number. The
resulted cell cultures were examined by flow cytometry and immunocytochemical analysis. Their clonogenic potential, ability
to self-renewal and directed multilineage differentiation were also investigated. Results: Cell cultures were obtained from explants
of adult human hair follicles. Resulted cells according to morphological, phenotypic and functional criteria satisfied the definition
of neural crest-derived multipotent stem cells. They had the phenotype Sox2+Sox10+Nestin+CD73+CD90+CD105+CD140a+CD
140b+CD146+CD166+CD271+CD349+ CD34-CD45-CD56-HLA-DR-, showed high clonogenic potential, ability to self-renewal
and directed differentiation into the main derivatives of the neural crest: neurons, Schwann cells, adipocytes and osteoblasts.
Conclusion: The possibility of a large-scale expansion of adult neural crest-derived multipotent stem cells up to 40–200·106 cells
from minimal number of hair follicles with retention of their phenotype and functional properties are the significant step towards
their translation into the clinical practice.
Key Words: regenerative medicine, neural crest, hair follicle, neural crest-derived multipotent stem cells, directed differentiation,
large-scale expansion.
Regenerative medicine is a rapidly developing field
that promises to significantly improve the quality of the
clinical treatment and to develop the cure for incur-
able diseases and pathological conditions. The main
tools of regenerative medicine are tissue engineering,
cell therapy and gene therapy. Nowadays there are
several tissue-engineering, cellular and gene medi-
cal products in the world, successfully passed clinical
trials and emerged on the market [1]. Most methods
of regenerative medicine are based on in vitro cultur-
ing of human stem, progenitor or differentiated cells
to increase their number up to required for clinical use,
as well for the genetic correction, modifications of their
biological properties or to create 3D living equivalents
of different tissues and organs. Despite the significant
progress in the field of pluripotent stem cells (embry-
onic stem cells and induced pluripotent stem cells),
a number of ethical and biosafety issues remain un-
resolved. Thus, the identification and characterization
of new types of adult stem cells is an actual challenge.
In addition, the medical use of in vitro cultured human
cells requires the development of criteria for the safety
and efficacy of cell-based medicinal products in terms
of identity, purity and potency. For example, such
criteria were developed for multipotent mesenchymal
stem/stromal cells (MSCs) from bone marrow and
adipose tissue [2, 3].
Adult neural crest-derived multipotent stem cells
(NC-MSCs) for a number of reasons are a promising
cell type for regenerative medicine applications [4].
During the embryonic development, neural crest cells
generate a wide variety of cell types and tissues, such
as: bone, cartilage and connective tissue of the head
and neck; melanocytes; endocrine cells (thyroid gland
C-cells, adrenal medulla); neurons and glia of the pe-
ripheral nervous system and others. Adult NC-MSCs
are identified and isolated from many tissues and
organs of the adult mammalian organism, including
Submitted: August 08, 2017.
*Correspondence: E-mail: rvasyliev.ilaya@gmail.com
Tel.: +380992750434
Abbreviations used: BDNF — brain-derived neurotrophic factor;
bFGF — basic fibroblast growth factor; BSA — bovine serum albu-
min; CFU — colony forming units; EGF — epidermal growth factor;
ELISA — enzyme-linked immunosorbent assay; FBS — fetal bovine
serum; GDNF — glial cell line-derived neurotrophic factor; GMP/
GTP — good manufacturing practice/good tissue practice; HF — hair
follicle; IGF — insulin-like growth factor; ITS — insulin-transferrin-
selenite supplement; LNGFR — low-affinity nerve growth factor re-
ceptor; MSCs — multipotent mesenchymal stem/stromal cells; NC-
MSCs — neural crest-derived multipotent stem cells; NGF — nerve
growth factor; P — passage; PCR — polymerase chain reaction;
PDGF — platelet-derived growth factor; PDN — population doubling
number; PDT — population doubling time; PE — plating efficiency;
SCI — spinal cord injury; SKPs — skin-derived precursors cells.
Exp Oncol 2017
39, 3, 171–180
ORIGINAL CONTRIBUTIONS
172 Experimental Oncology 39, 171–180, 2017 (September)
humans [5]. It is shown that adult NC-MSCs retain
the ability to directed multilineage differentiation into
the main derivatives of the neural crest, similar to their
embryonic counterparts [5]. In a number of studies
on animal models the positive effect of adult NC-MSCs
has been shown to restore the critical size of calvarial
bone defects [6], stimulation of peripheral nerve re-
generation [7, 8], spinal nerve dorsal root avulsion [9]
and spinal cord injury treatment [10].
One of the most attractive from the possible
sources of adult NC-MSCs is the hair follicle (HF) due
to the minimal invasiveness of the biopsy. For the first
time, adult NC-MSCs were identified and isolated
from mouse whisker follicles [11]. The method was
based on the use of the explant technique and a mi-
gratory behavior of NC-MSCs. Later, adult NC-MSCs
were obtained from skin HFs of mice and another
species, including human [12–14]. For the production
of human adult NC-MSCs, several techniques have
been proposed: the culturing of HF explants plucked
[15] or dissected from a skin biopsy [16], selection
of cells from dissociated HFs based on surface
markers by fluorescence activated cell sorting or im-
munomagnetic separation [17], culturing dissociated
cells under sphere-forming conditions, etc [18]. Each
of these techniques has its advantages and disadvan-
tages. However, even when using similar techniques
for the production of human adult NC-MSCs, the re-
sulting populations differ in the expression of a num-
ber of key neural crest markers such as Sox10 and
p75 (CD271, LNGFR) [15, 16]. Also, minimal data are
available on the expression of surface markers by cul-
tured human adult NC-MSCs and on the possibility
of their large-scale expansion to obtain the significant
cell number required for clinical applications.
Thus, the goal of our work was to obtain human
adult NC-MSCs cultures from the minimal skin biopsy
of patients, to master their large-scale expansion, and
to determine their phenotype and functional properties.
MATERIALS AND METHODS
All procedures for obtaining human skin biopsies,
cell isolation and culturing were performed with writ-
ten informed consent of patients and in accordance
with the laws of Ukraine. The study protocol and the
local clinical protocol were approved by the Bio-
ethics Committee of the Medical company ilaya®. Cell
culturing was carried out in the GMP/GTP-compliant
biotechnological laboratory ilaya.regeneration (Li-
cense to operate the Banks of human cord blood,
other tissues and cells, issued by the Ministry of Health
of Ukraine AE No. 186342 from 11.07.2013).
The donor skin specimens were obtained from
3 patients with spinal cord contusion injuries and
2 patients with critical sized calvarial bone defects.
Donors and cells screening. All patients/
donors (peripheral blood — ELISA, PCR) and cell
cultures (PCR) were screened for absence of human
immunodeficiency virus type 1/2, hepatitis B virus,
hepatitis C virus, herpes simplex virus type 1/2,
cytomegalovirus, Epstein — Barr virus, Treponema
pallidum and Mycoplasma ssp. The cell cultures were
also tested for common mycoplasma contamination
by PCR with Mycoplasma Test Kit I (AppliChem, Ger-
many). The normal karyotype of cultured cells was
confirmed by GTG-banding method.
The isolation and culturing of NC-MSCs from
human HF. The NC-MSCs cultures were obtained
from HF explants according to M. Sieber-Blum
method [11] in our modification. One skin specimen
per donor was obtained under local anesthesia with
use of a dermal punch (diameter 5 mm). The biopsies
were placed in DMEM:F12 transport medium (Sigma,
USA) supplemented with 15 mM HEPES and antibi-
otic/antimycotic solution. Skin specimen was minced
and incubated overnight at 4 °C in Dispase II solution
(0.4 U/ml) (Sigma-Aldrich, USA). The next day, under
the control of Stemi 2000 stereomicroscope (Carl
Zeiss, Germany), a layer of epidermis was removed
using tweezers, and then HFs were isolated. HFs
were explanted over a thin layer of collagen gel in two
35 mm Petri dishes and incubated for 40 min. After
attachment, the explants were covered with a growth
medium and cultured in a multi-gas incubator Binder
CB 210 (Binder, Germany) at 37 °C with 5% CO2 and
5% O2 and a saturating humidity. For expansion
of adult NC-MSCs, the following growth medium
was used: αMEM w/o nucleosides (Sigma-Aldrich,
USA), 5% FBS (Sigma-Aldrich, USA), 5 ng/ml bFGF
(Gibco, UK), 10 ng/ml EGF (Gibco, UK), 1% ITS
supplement (Gibco, UK), 2 mM stable L-glutamine
(BioWest, France) and 1% antibiotic/antimycotic
solution (BioWest, France). The primary culture was
passaged on Day 14–20 into the collagen-coated
T25 flasks (SPL, Korea). Starting at P2, the cells were
seeded into the collagen-coated 875 cm2 surface
area multi-flasks (Corning, USA) with a seeding den-
sity of 1000 cells/cm2. The cultures were subcultured
using 0.01% trypsin solution in 0.53 mM Na2EDTA
solution (Sigma-Aldrich, USA). The cell population
doubling number (PDN) and cell population doubling
time (PDT) were calculated according to the following
standard formulas [19]:
PDT = T / 3.31 lg (Xk / X0);
PDN = 3.31 lg (Xk / X0),
where Xk — number of obtained cells; X0 — number
of plated cells; T — cell culture time.
Colony forming units (CFU) assay and self-re-
newal ability. To assess the clonogenic potential, the
NC-MSCs at the last passage (P) were seeded in num-
ber of 100-300 cells in collagen-coated 100 mm Petri
dishes (SPL, Korea) in growth medium supplemented
with 20% FBS and cultured for 14 days. The cells were
either fixed and stained for CFU assay or subcloned
for self-renewal analysis. The effectiveness of colony
formation (plating efficiency) was calculated using the
standard formula [19]:
PE, % = (no. colonies counted / no. cells
inoculated) × 100,
where PE — plating effciency.
Experimental Oncology 39, 171–180, 2017 (September) 173
Transfer of the clonal colony (subcloning) was car-
ried out using cloning cylinders (Sigma-Aldrich, USA).
The cells were cultured for 14 days, fixed and stained
for analysis for formation of secondary CFUs.
Analysis of the expression of cell surface and
intracellular markers by flow cytometry. NC-
MSCs immunophenotype was determined at the final
passage using a flow cytometer-sorter BD FACSAria
(BD Biosciences, USA) with fluorochrome-conjugated
mouse monoclonal antibodies to CD34, CD45, CD56,
CD73, CD90, CD105, CD140a, CD140b, CD146,
CD166, CD271 and Nestin (BD Pharmingen, USA)
in accordance with the manufacturer’s instruc-
tions. For detection of Sox10, primary unconjugated
mouse monoclonal antibodies (Novus, USA) and
secondary Alexa-647 donkey anti-mouse monoclonal
antibo dies (Thermo Fisher, USA) were used. For the
detection of CD349 (Frizzled-9), primary unconju-
gated rabbit antibodies (Bioss, USA) and secondary
Alexa-647 donkey anti-rabbit monoclonal antibodies
were used. For intracellular staining cells were fixed
with Cytofix solution, permeabilized and stained
in PhosFlow buffer (all — BD Biosciences, USA) ac-
cording to the manufacturer’s instructions. The as-
say was performed using BD FACS Diva 6.1 software
(BD Biosciences, USA). Histogram generation was
performed with use of software Cyflogic v.1.2.1 (CyFlo
Ltd., USA).
Directed adipogenic differentiation. NC-MSCs
were seeded with density of 40•103 cells/cm2 in growth
medium and the following day were switched to the
following differentiation medium: DMEM-HG (4.5 g/l)
supplemented with 10% FBS, 1 µM dexamethasone,
200 µM indomethacine, 500 µM isobutylmethylxan-
thine, 5 µg/ml insulin (all — Sigma-Aldrich, USA) and
5% horse serum (BioWest, France). The medium was
changed 2 times a week. The duration of differentia-
tion was 14 days.
Directed osteogenic differentiation. NC-MSCs
were seeded at 20•103 cells/cm2 in growth medium
and the next day switched to a following differentiation
medium: DMEM low glucose (1.0 g/l) with 10% FBS,
100 nM dexamethasone, 10 mM β-glycerophosphate
and 50 µg/ml ascorbate-2-phosphate (all — Sigma-
Aldrich, USA). The medium was changed 2 times
a week during 21 days.
Directed neuronal differentiation. NC-MSCs
were seeded at a concentration of 50•103 cells
on coated by poly-L-lysine (Sigma-Aldrich, USA)
and laminin (Gibco, UK) coverslips in 4 well plates
(SPL, Korea) in growth medium. The following
day cells were switched to the following differen-
tiation medium: Neurobasal medium (Gibco, UK),
2% B27 supplement (Gibco, UK), 1% N2 supple-
ment (Gibco, UK), 5 µM ec32 synthetic retinoid
(AMSBIO, UK), 1 µM forskolin (Sigma-Aldrich, USA),
20 ng/ml NGF (PeproTech, USA), 20 ng/ml BDNF
(PeproTech, USA), 20 ng/ml GDNF (PeproTech, USA)
and 20 ng/ml IGF (Gibco, UK). The duration of diffe-
rentiation was 14 days.
Differentiation in the glial direction (Schwann
cells). NC-MSCs were seeded at a concentration
of 50•103 cells on coated by poly-L-lysine (Sigma-
Aldrich, USA) and laminin (Gibco, UK) glass coverslips
in 4 well plates (SPL, Korea) in growth medium. The
following day cells were switched to the following dif-
ferentiation medium: 1:1 of Neurobasal (Gibco, UK)
and DMEM:F12 (Gibco, UK) media, 2% B27 supple-
ment (Gibco, UK), 1% N2 supplement (Gibco,
UK), 1 µM ec32 synthetic retinoid (AMSBIO, UK),
5 µM forskolin (Sigma-Aldrich, USA), 20 ng/ml Neu-
regulin-1 (Gibco, UK), 10 ng/ml PDGF-BB (PeproTech,
USA) and 20 ng/ml IGF (Gibco, UK). The duration
of differentiation was 14 days.
Cytochemistry. For CFU staining, the cell colonies
were fixed for 20 min with cold ethanol and azure-eosin
stained by Romanowsky (Makrokhem, Ukraine) for
20 min. To confirm the osteogenic and adipogenic dif-
ferentiation, the cells were fixed for 20 min in 10% for-
malin (Makrokhem, Ukraine), washed with phosphate
buffered saline (Sigma-Aldrich, USA) and stained for
20 min with 2% solution of Alizarin Red S (pH 4.1; for
detecting calcified extracellular matrix) and 0.5%
solution of Oil Red O (for staining of neutral lipids)
respectively (all — Sigma-Aldrich, USA).
Immunocytochemistry. The following primary
antibodies were used for immunocytochemical stain-
ing: rabbit polyclonal against S100β (Invitrogen,
USA), p75 (CD271) and Sox2 (all — BioLegend, USA);
mouse monoclonal against Sox10 (R&D, USA), Nestin
(Santa Cruz, USA), β-III-tubulin (Sigma-Aldrich, USA).
Secondary antibodies were donkey anti-mouse and
donkey anti-rabbit Alexa-488 or Alexa-647 conju-
gated (Thermo Fisher, USA). The cells were fixed for
20 min with cold 4% paraformaldehyde, permeabilized
with intracellular staining for 15 min with 0.1% Triton
X-100, blocked for 30 min in phosphate buffered saline
with 0.1% Tween-20, 1% BSA, 5% FBS. The slides
were incubated with primary antibodies overnight
at 40 °C and with secondary antibodies for 1 hour
at room temperature.
Microscopy. Intravital microscopy and ex-
amination of cytological slides were carried out with
inverted Axio Observer A1 microscope equipped
with an AxioCam ERc 5s digital camera and ZEN
2012 software. Confocal microscopy was performed
with Zeiss LSM 510 META microscope (all — Carl Zeiss,
Germany).
Statistics. The data are presented as Mean and
Standard Deviation (M ± SD).
RESULTS AND DISCUSSION
We successfully obtained and expanded up to the
therapeutic dose the adult NC-MSCs from all 5 donors.
The characteristics of donors, number of explanted
HF and terms of growth of primary cell cultures are
presented in Table 1. On average, 15.8 ± 4.5 HFs were
obtained from one skin sample of 5 mm in diameter.
It should be noted that part of HFs (1–5 per culture,
16.4 ± 7.1% of explanted HFs) was detached, despite
174 Experimental Oncology 39, 171–180, 2017 (September)
the use of a thin layer of collagen hydrogel as a sub-
strate. Outgrowth of human NC-MSCs was observed
from more than half of the explanted HFs. Herewith,
most of the HFs revealed the mixed outgrowth of NC-
MSCs and keratinocytes (34.9 ± 11.6% HFs vs 25.3 ±
8.8% HFs with pure outgrowth of NC-MSCs). In our
case, the emigration and proliferation of NC-MSCs
from explants have started 5–10 hours, the culture
time of the primary culture was 16.2 ± 2.5 days on the
average and 104 NC-MSCs were obtained from a single
HF, which corresponds to the data published by other
groups [15, 16]. Fig. 1, a shows the HF explant with
a pure outgrowth of NC-MSCs just before subcultur-
ing. Fig. 1, b shows the NC-MSCs with characteristic
stellate morphology and migratory behavior. For fur-
ther expansion of pure cultures, the NC-MSCs were
successfully separated from the primary keratinocyte
impurity by a differential trypsinization technique while
subculturing. The use of 0.01 trypsin solution allowed
the NC-MSCs to be effectively collected, while dense
colonies of keratinocytes remained attached to the
substrate. The absence of NC-MSCs contamination
by keratinocytes at P1 was verified by immunostain-
ing for pan-cytokeratin, which gave a negative result
(data not shown).
Beginning the P1 in our culture conditions, a stable
rapid growth of NC-MSCs was established. Cell growth
parameters at P1-P3 are shown in Table 2. Thus,
when the seeding concentration was in the range
of 500–1300 cells per 1 cm2, the cultures reached
70–100% confluency within 5–7 days, and been sub-
cultured every week. At the same time, an average
6.04 ± 0.35 PDN took place within a passage, with
averaged PDT 27.90 ± 1.58 hours. These parameters
during the early passages (P1–P3) showed weak
differences both within the culture from one donor
at different passages, and between donors.
Determination parameters of safety and efficacy
of human cell-based medicinal product mean the sta-
bility of the cell karyotype after in vitro expansion and
the number of CFU. Cultures from all donors at the end
of expansion were subjected to cytogenetic analysis
and showed normal karyotype. Analysis of the CFU fre-
quency showed that after large-scale expansion of cul-
tures of human adult NC-MSCs from HF, a significant
number of cells maintained the clonogenic potential
and CFU frequency was 25.9 ± 7.7%. An important
feature of stem cells is the ability to self-renew. To as-
sess the self-renewal, five CFUs per each donor were
subcloned. In all cases the formation of seconda ry
colonies was detected. Thus, human adult NC-MSCs
from HF retain their clonogenic potential and ability
to self-renew after large-scale expansion under our
cell culture cultural conditions.
We also examined the phenotype of the obtained
cultures by flow cytometry and immunocytochemistry
for key markers of neural crest (Sox10, p75), com-
mon markers of stem cells (nestin, Sox2 and CD34),
markers of mesenchymal stem/stromal cells (CD73,
CD90 and CD105), receptors for some growth factors
Fig. 1. Adult NC-MSCs from HF: a — explant of HF with pure outgrowth of NC-MSCs just before passaging. Phase contrast, scale
bar — 200 µm; b — characteristic stellate morphology of NC-MSCs. The small rounded bright cells — NC-MSCs in mitosis. Phase
contrast, scale bar — 50 µm
Table 1. Parameters of primary cell cultures (P0)
Donor
ID Age, sex and condition Total number
of explanted HFs
HFs with NC-
MSCs out-
growth, n (%)
HFs with mixed
outgrowth, n (%)
HFs with kera-
tinocytes out-
growth, n (%)
Detached
HFs, n (%)
Culture
time, days Cell yield
1 24 years, male, SCI 14 3 (21.4) 7 (23.8) 2 (23.8) 1 (23.8) 14 29,000
2 25 years, male, SCI 9 2 (33.3) 3 (23.8) 3 (23.8) 1 (23.8) 20 16,000
3 31 years, male, SCI 17 6 (35.3) 4 (23.8) 4 (23.8) 3 (23.8) 14 52,000
4 27 years, male, bone
defect 21 3 (14.3) 6 (28.6 5) 7 (33.3) 5 (23.8) 16 31,000
5 62 years, female, bone
defect 18 4 (22.2) 5 (23.8) 5 (23.8) 4 (23.8) 17 43,000
M ± SD 15.8 ± 4.5 3.8 ± 1.3
(25.3 ± 8.8)
5.2 ± 1.3
(34.9 ± 11.6)
3.8 ± 2.4
(22.0 ± 9.2)
2.8 ± 1.8
(16.4 ± 7.1) 16.2 ± 2.5 34,200.0 ± 13,809.4
Note: SCI — spinal cord injury.
Experimental Oncology 39, 171–180, 2017 (September) 175
(CD140a/PDGFRα, CD140b/PDGFRβ and CD349/
Frizzled-9), adhesion molecules (CD56/NCAM,
CD146/MCAM and CD166/ALCAM), and markers
of hematopoietic (CD45) and antigen-presenting cells
(HLA-DR). Representative flow cytometry histograms
of cell culture from donor # 2 are shown in Fig. 2. Sum-
mary data for all donors are presented in Table 3.
All expanded cultures of NC-MSCs contain more
than 90% Sox10+ and Nestin+ cells, but the number
of cells expressing p75 (CD271) varied significantly
from 45.9 to 93.8% positive cells (73.32 ± 18.39%).
Interesting, NC-MSCs homogeneously expressed
the key mesenchymal stem/stromal markers CD73,
CD90 and CD105 [2, 3]. All these markers were
presented on more than 90% cells in average. Also
in average more than 90% of the cells were positive
for CD140b, CD166 and CD349. More heterogeneous
cultures were by expression of CD140a (67.68 ± 14.26)
and CD146 (87.92 ± 8.42). Adult NC-MSCs were nega-
tive for hematopoietic markers CD34 and CD45 (the
number of positive cells did not exceed 1%). Some-
times a small number of CD56+ (up to 7.8%) and HLA-
DR+ (up to 4.2%) cells was noted.
Taking into account the key role of Sox10,
p75 (CD271) and nestin in the determination of the
identity of NC-MSCs [4, 5], we also examined them
using the immunocytochemical analysis. Additionally,
Sox2 has also been studied because of its important
role in stemness maintenance and functional prop-
erties of NC-MSCs [20, 21]. As shown in Fig. 3 and
Fig. 2. Representative results of NC-MSCs phenotype analysis by flow cytometry
Table 2. Parameters of cell cultures at P1–P3
Donor ID
Growth sur-
face area,
cm2
Plating
density,
cell/cm2
Cell yield,
106 PDN PDT, hours Cumula-
tive PDN
Average PDT,
hours
CFU frequency
at final
passage, %
Secondary CFU
formation
(self-renewal)
Karyotype
1 P1 25 1160 1.73 5.9 28.6 11.7 28.8 20.3 ± 5.9 + Normal, 23XYP2 875 1000 49.00 5.8 29.0
2 P1 25 640 1,85 6.8 24.6 13.0 26.0 36.7 ± 5.7 + Normal, 23XYP2 875 1000 63.00 6.1 27.3
3 P1 50 1040 4,33 6.4 26.4 12.5 26.8 30.7 ± 2.1 + Normal, 23XYP2 875 1000 64.8 6.2 27.1
4 P1 25 1240 2.10 6.1 27.7
18.2 27.8 24.3 ± 3.5 + Normal, 23XYP2 875 1000 53.40 5.9 28.4
P3 2625 1000 193.50 6.2 27.2
5 P1 50 860 1.93 5.5 30.7
17.1 29.5 17.7 ± 5.5 + Normal, 23XXP2 875 1000 56.00 6.0 28.1
P3 3500 1000 178.00 5.6 29.7
M ± SD n/a n/a n/a 6.04 ± 0.35 27.90 ± 1.58 n/a 27.78 ± 1.43 25.9 ± 7.7 n/a n/a
Note: n/a — not applicable.
176 Experimental Oncology 39, 171–180, 2017 (September)
Fig. 4, most of the cells in the culture were Sox10+p75+
and Sox2+Nestin+, which confirms the flow cytometry
data. As described earlier, for human adult NC-MSCs
from HF [16, 22] the Sox10 showed a characteristic
nuclear- cytoplasmic localization, consistent with
its role as a transcription factor and active nucleo-
cytoplasmic shuttle protein [23]. Sox2 had predomi-
nantly nuclear localization, which is characteristic of its
function as a transcription factor. Nestin and p75 also
showed the characteristic staining for the intermedi-
ate filament protein and intracellular domain of growth
factor receptor respectively.
In addition to the ability to self-renew another
important functional property of stem cells is the
abili ty to multilineage differentiation. Considering that
HF from the head area were used in our study, these
NC-MSCs were derived from the cranial neural crest
during the embryonic development. Cranial neural
crest gives rise to the widest variety of tissues and cell
types from all the axial domain of neural crest [5]. So,
in addition to melanocytes, neurons and glia of the
peripheral nervous system, the cranial neural crest
gives rise to ectomesenchyme, from which a part of the
bone, cartilage and connective tissue of the head and
neck is formed. Based on this, we verified the identity
of the obtained adult NC-MSCs by ability to differenti-
ate into both mesenchymal cell types (adipocytes and
osteoblasts), and into neurons and Schwann cells.
Adult NC-MSCs were cultured for 14 days in an ad-
ipogenic induction medium and have successfully
differentiated into the adipocytes containing lipid vacu-
oles (Fig. 5, a). When cultured in osteogenic medium
during 14 days, NC-MSCs were converted to alkaline
phosphatase-positive osteoblasts (Fig. 5, b) and
after 21 days the mineralized extracellular matrix was
produced (Fig. 5, c). When NC-MSCs were cultured
in the presence of a synthetic analogue of retinoic acid
and neurotrophins (NGF, BDNF and GDNF), they were
converted into the homogeneous population of neu-
ronal β-III-tubulin positive cells with spherical or oval
soma and long thin processes (Fig. 5, d). Under the
influence of neuregulin-1, PDGF-BB and the activation
of cAMP signaling by forskolin, NC-MSCs differenti-
ated into S100β-positive cells with the characteristics
for Schwann cells bipolar morphology (Fig. 5, e).
As mentioned in the Introduction, the existence
of adult NC-MSCs in the bulge region of HF was first
shown by M. Siber-Blum using whisker follicles from
Wnt1-Cre transgenic mice for tracing developmental
origin of this cell population from neural crest [11]. In the
study NC-MSCs obtaining was based on the migratory
behavior of this cell type. NC-MSCs emigrated and
began to proliferate rapidly while culturing the explants
of the bulge region on collagen substrate in a special
neural crest cell growth medium. Mouse NC-MSCs
from bulge region of HF were Sox10+Nestin+ and had
the ability for directed differentiation into neurons,
Schwann cells, melanocytes, smooth muscle cells and
chondrocytes. In parallel, Fernandes et al. using the
same Wnt1-Cre transgenic mouse line reported the
isolation of another cell population of the neural crest
origin from derma of facial skin, which was called skin-
Table 3. Phenotype of NC-MSCs at last passage (P2–P3) by flow cytometry
Donor ID Sox10 CD271 (p75) Nestin CD73 CD90 CD105 CD140a CD140b CD146 CD166 CD349 CD34 CD45 CD56 HLA-DR
1 95.3 65.3 97.8 99.5 98.7 96.3 62.8 96.9 82.3 91.5 90.7 0,3 0,6 1,5 1,2
2 99.9 82.5 99.1 99.9 99.5 99.2 91.4 98.4 96.8 89.9 98.3 0,9 0,2 0,0 1,1
3 96.2 79.1 92.1 95.3 92.7 89.9 58.9 87.3 77.9 87.7 92.3 0,9 0,2 7,8 0,9
4 98.3 93.8 97.5 99.2 96.8 98.5 55.6 94.9 96.3 98.8 96.5 1,2 0,5 3,5 0,4
5 91.8 45.9 88.7 96.9 97.3 96.4 69.7 81.7 86.3 97.5 87.5 0,4 0,1 2,4 4,2
M ± SD 96.30 ±
3.09
73.32 ±
18.39
95.04 ±
4.44
98.16 ±
1.98
97.00 ±
2.63
96.06 ±
3.09
67.68 ±
14.26
91.84 ±
7.10
87.92 ±
8.42
93.08 ±
4.84
93.06 ±
4.37
0.74 ±
0.38
0.32 ±
0.22
3.08 ±
2.90
1.56 ±
1.51
Fig. 4. Immunocytochemical analysis of NC-MSCs for p75 (CD271)
(red) and Sox10 (green) expression. Scale bar — 20 µm
Fig. 3. Immunocytochemical analysis of NC-MSCs for
Sox2 (green) and Nestin (red) expression. Scale bar — 20 µm
Experimental Oncology 39, 171–180, 2017 (September) 177
derived precursors cells (SKPs) [24]. It turned out that
the tissue niche for SKPs is dermal papilla of HFs from
facial skin and whisker follicles [24]. The procedure for
SKPs obtaining was based on their ability to grow in the
form of spheres (resembling neurospheres) under
serum-free and non-adherent conditions. At the same
time, SKPs showed a similar differentiation potential
to NC-MSCs from the bulge region of HF. However,
they were Sox10 and p75 negative, but they expressed
other characteristic neural crest markers — Nestin,
Slug, Snail, Twist and Sox9 [24]. Later, a more in-depth
analysis of the localization of NC-MSCs in whisker fol-
licles and HF from dorsal and ventral skin of mice has
been made with use of Wnt1-Cre, Ht-PA-Cre, Sox10lacZ,
Dhh-Cre and Dct-Cre transgenic mouse lines [12].
In those paper it was confirmed that many structures
of whisker follicle derived from neural crest, harbor
cells with stem cells-like properties and this cells linked
to mesenchymal, glial and melanocyte lineages. Such
structures in whisker follicles are: bulge region, dermal
sheath, capsule, dermal papilla, nerve terminals and
regions above sebaceous glands. Analysis of mouse
HF from back skin showed that neural crest-derived
cells are localized in the bulge region and in the bulb
above the dermal papilla [12]. The dermal papilla of the
back skin itself does not originate from the neural
crest. Lineage tracing analysis showed that neural
crest-derived cells in trunk HFs are linked to glial and
melanocyte lineages. Using somewhat different from
Fernandes et al. [24] growth conditions, the authors
obtained spheres from mouse back skin and human
skin (face and thigh) samples [12]. Analysis of the cell
composition of the spheres obtained from transgenic
mice showed that they are formed by cells that have
neural crest origin. It should be noted, 100% mouse
and human skin-derived spheres contains p75+ and
Sox10+ cells. In mouse spheres 67.0 ± 10.5% of all
cells expressed p75; 76.6 ± 4.5% of all cells expressed
Sox10; and 58.6 ± 10.5% of all cells were double posi-
tive for p75 and Sox10. Then, 15.0 ± 6.2% of all sphere
cells were negative for these markers, which indicates
cell heterogeneity in the composition of spheres, which
may be associated with spontaneous cell differen-
tiation. Similar to mouse spheres, all human spheres
contained p75/Sox10 double positive cells, which
accounted for > 60% of all cells. However, the cells
were negative for other neural crest markers, such
as Sox9 and HNK-1 (CD57). Both mouse and human
neural crest-derived cells from HF showed the ability
to multilineage directed differentiation into the main
derivatives of the neural crest: peripheral neurons,
Schwann cells, melanocytes, adipocytes, chondro-
cytes and smooth muscle cells [12].
Thus, lineage tracing experiments using trans-
genic mouse lines showed that the HF contains various
Sox10 and p75 positive and negative cells of neural
crest origin with multilineage differentiation potential
and stem cell-like properties.
Fig. 5. Detection of NC-MSCs directed multilineage differentiation: a — adipogenic differentiation, Oil Red O staining of lipid vacuo-
les (red). Scale bar — 50 µm; b — osteogenic differentiation, BCIP/NBT staining for ALP activity (dark blue). Scale bar — 100 µm;
c — osteogenic differentiation, Alizarin Red S staining of mineralized extracellular matrix (red). Scale bar — 200 µm; d — neuronal
differentiation, immunocytochemical detection of β-III-tubulin (green). Scale bar — 20 µm; e — glial differentiation (Schwann cells),
immunocytochemical detection of S100β protein (green). Scale bar — 20 µm
178 Experimental Oncology 39, 171–180, 2017 (September)
The existence of NC-MSCs in the bulge region
of human HF was also confirmed by other authors and
several variants of growth media and methods for their
isolation were proposed for the isolation of NC-MSCs
from dissociated HFs or skin samples and their growth
under the floating sphere conditions [14, 17, 18].
Although it has been shown in [12] that the human
skin-derived spheres could be serially subcultured for
more than 3 months and generates more than 109 cells
during this time, the disadvantage of human NC-MSCs
culturing under sphere conditions is its high cost and
relatively low cell growth rate.
For these reasons, attempts have been made
to modify the original M. Siber-Blum protocol to obtain
NC-MSCs cultures from human HF explants. In one
of the first reports it was shown that NC-MSCs do not
provide cell emigration and growth in a monolayer when
human HF explants culturing, but only form spheroid
structures attached to the bulge area of HFs [14, 25].
Later, the group of M. Siber-Blum and other authors
reported the successful adaptation of the explant
method to the production of human NC-MSCs cultures
[15, 16, 22]. At the same time, several critical mo-
ments and shortcomings in the use of this technique
were revealed. The most critical and time-consuming
step in adult NC-MSCs obtaining is the establishment
of primary cultures [15, 16, 22]. The main difficulties
in obtaining primary cultures of human adult NC-MSCs
from HF explants are the detachment of HFs, the
absence of outgrowth of NC-MSCs and overgrowth
of keratinocytes [15, 16]. Also there are contradic-
tory data on NC-MSCs morphology (from spindle-like
to stellate) and markers expression [15, 16]. It can
be due to both the use of different culturing conditions
and the methods of extraction of HFs from a skin (dis-
sected vs plucked) and different skin regions. It should
be noted that the equivalence of the use of dissected
vs plucked human HFs for NC-MSCs isolation and
the identity of the resulting cell populations remain
an open question. There is an opinion that the bulge
region is damaged in plucked HFs. As a result, various
phenotypes of adult NC-MSCs derived from plucked
and dissected HF have been reported. So, in [15],
the NC-MSCs from plucked HFs were Sox10 nega-
tive, but positive for common used neural crest
markers, like Nestin, Slug, AP-2α and Sox9. Whereas
NC-MSCs the resulting from the dissected HFs were
Sox10+Sox2+Nestin+ by immunocytochemistry [16,
22]. There was also expression of Slug, Twist and
P75 genes detected by qPCR [16, 22].
In our study, to improve the reliability and effec-
tiveness of the procedure for NC-MSCs obtaining,
we modified the original protocol of M. Siber-Blum [11]
in the following points: 1) use of Dispase enzyme for
separation of the epidermis and facilitation of the HFs
dissection procedure; 2) use of a thin layer of collagen
hydrogel as an adhesive coating for the attachment
of HF explants; 3) maintenance of adhered explants
in the primary culture until the moment of subculturing.
In fine, these minor modifications have significantly
improved cell isolation and culturing protocol, which
will be discussed below.
So, we managed to significantly reduce the fre-
quency of HF explants detaching to less than 20%.
For comparison, when using the poly-D-lysine treated
plastic surface, more than 40% of explants of HFs [15]
are detached.
Regardless of the cell culture conditions used,
it has not yet been possible to obtain 100% outgrowth
of NC-MSCs from all adhered HFs and the reasons for
this remain unclear. A possible link can exist with the
growth phase of HF. So in [16], it is shown that more
often NC-MSCs emigrated from explanted HFs been
in early or late anagen growth stages. On the other
hand, in [17] it has shown the possibility of NC-MSCs
obtaining both from HFs in the anagen stage and
the telogen stage. In various studies, the outgrowth
of NC-MSCs was from 20% in [25] to 34% in [15]
or almost for 50% of explants [16]. Clewes et al. [16]
were noted that more often emigration of human
NC-MSCs was observed from the explants of HFs with
preserved dermal sheath — 45.3 ± 13.0%. NC-MSCs
did not emigrate from the separately cultured dermal
sheath of HFs and emigrated only from 6.3 ± 6.3% HFs
without dermal sheath. Thus, the role of the dermal
sheath is probably to preserve the bulge region unaf-
fected (intact, uninjured) during the extraction and
isolation of HFs.
Keratinocytes outgrowth from human HF explants
is a common problem [15, 16]. Interestingly, the emi-
gration of keratinocytes from the mouse bulge region
of whisker follicles is observed much later than the
emigration of NC-MSCs [11], or can be excluded alto-
gether by using a selective growth medium [26]. At the
same time, keratinocytes outgrowth is often observed
when culturing of rat bulge whisker follicle explants
(our unpublished observations) or human HF explants.
So, in [15] reported the keratinocytes outgrowth was
from 33–100% explanted HFs, which is comparable
to our data. Further progress in this direction may
be related to the development of a selective growth
medium, although according to our observations,
keratinocytes proliferate in the primary culture of ex-
planted HFs even in the absence of EGF in the growth
medium. It should be noted that the problem of con-
tamination of NC-MSCs cultures by keratinocytes with
further expansion can be successfully solved using the
technique of differential trypsinization, as it was made
in our presented work.
As for the phenotype of adult NC-MSCs obtained
during the large-scale expansion, it is comparable
to that described for cultures obtained from dis-
sected human HFs [16, 17, 22] and differs from that
described for cultures obtained from plucked human
HFs [15]. The reason for this can be both the HF isola-
tion methods and slightly different culture conditions.
A weak modification of growth conditions can either
influence the expression of neural crest markers
(like Sox9, Sox10 or p75), or give selective benefits
to different cell populations with neural crest origin,
Experimental Oncology 39, 171–180, 2017 (September) 179
which contains in HF. So, Sox10 and p75 are the first
markers that distinguish neural crest cells immedi-
ately after their induction from Sox2+ neuroepithelium
of neural tube [27]. And although these markers are
used for prospective isolation and/or for the confir-
mation of neural crest origin of cells from different
tissues of the adult organism, their expression is not
always observed in adult neural crest-derived cells,
especially in ectomesenchymal cells of the craniofa-
cial region [4, 5]. Thus, expression of Sox10 by neural
crest-derived cells at later stages of development
and in adulthood can be related to their broader
differentiation potential while retaining the ability
to acquire neuronal, glial and melanocyte fates [28,
29]. Whereas ectomesenchymal cells with differen-
tiation potential restricted to mesenchymal cell fate
can be Sox10 negative [4, 5]. The p75 function and
the heterogeneity of its expression in the populations
of adult neural crest-derived cells remain largely not
understood. A comparative study of the prolifera-
tion and differentiation potential of Sox10+p75+ and
Sox10+p75− cells is needed. Concerning the double
negative Sox10−p75− cells, we assume that they can
be progenitors with the development of potential
restricted to mesenchymal cell fate [30]. This is sup-
ported by the preservation of the expression of CD73,
CD90 and CD105 (markers of mesenchymal stem/
stromal cells) by Sox10−p75− cells.
An important point is that the NC-MSCs expanded
by our protocol are Sox2+. Sox2 is one of the main fac-
tors of pluripotency and a marker of neural stem/pro-
genitor cells of central nervous system. Although at the
time of induction, neural crest cells are Sox2 negative
[27, 30], its expression appears in them at later stages
of development and is associated with the preserva-
tion of multipotency and ability to neurogenesis [4, 5,
20, 30]. Detection of a small number of CD56+ cells
may indicate spontaneous neuronal differentiation
of NC-MSCs during expansion. It is important to note
that in addition to neurogenesis, Sox2+ NC-MSCs
play a key role in skin wound healing and in reparative
regeneration process regulation as shown in the ex-
periments with use of transgenic mouse lineages [21].
Also important findings in our study are the ex-
pression on the adult NC-MSCs of such receptors
for growth factors as CD140a (PDGFRα), CD140b
(PDGFRβ), CD349 (Frizzled-9) and CD166 (ALCAM).
Expression of CD140a and CD140b by NC-MSCs can
be associated with several key aspects of their biology,
such as differentiation into Schwann cells, or their ac-
tivation after tissue damage through platelet-released
PDGFs. The role of PDGF proteins in NC-MSCs biology
should also be studied in more details in the light of the
possible use for cell xeno-free expansion of a platelet
lysate which contains them in large quantities. Con-
cerning CD349 expression, this indicates the need
for Wnt-signaling studying in adult NC-MSCs biology.
It is known that in the early stages of development the
self-renewal of NC-MSCs is supported by the com-
bined action of Wnt and BMP signaling [31]. The role
of these factors in the proliferation, self-renewal and
differentiation of adult NC-MSCs is poorly understood.
CD166 expression may indicate the presence of an im-
munomodulatory properties in adult NC-MSCs. De-
tection of the minor population of HLA-DR+ cells can
also be associated with the immunological functions
of NC-MSCs. It is well known that bone marrow-de-
rived mesenchymal stem/stromal cells have the ability
for inducible expression of HLA-DR after exposition
to inflammatory signals. This aspect of adult NC-MSCs
biology is not actually studied.
Thereof, our study shows for the first time the
principal possibility of large-scale expansion of adult
NC-MSCs from HF with preservation of their pheno-
type and basic functional properties. The therapeutic
number of cells required for clinical use ranges from
40•106 to 200•106 and can be obtained during one
month of culturing. These terms theoretically allow
us to consider the possibility of developing autolo-
gous medicinal products based on human NC-MSCs
for the treatment of spinal cord injuries and traumatic
peripheral nerve defects not only in the chronic but
also in the subacute period. It is generally accepted
that reconstructive surgery of damaged spinal cord
and peripheral nerve cannot be carried out in the acute
period, and the most favorable results are obtained
when they are carried out in the subacute/early chronic
period in comparison to late chronic period. Previ-
ously, despite the positive results of adult NC-MSCs
application obtained in animal models of spinal cord
injury and peripheral nerve defect, some skepticism
is caused by the absence of protocols of obtaining
a therapeutic dose in a short period of time (no more
than 4–6 weeks). Next steps in this field should
be further improvement of the isolation technique
from HF, development of xeno-free conditions for adult
NC-MSCs expansion and conducting of randomized
clinical trials to assess the effectiveness of this cell
type in the treatment of various diseases and defects
of the neural crest-derived tissues and structures
in comparison with existing “gold standards” of surgi-
cal or therapeutic treatment for these conditions.
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Copyright © Experimental Oncology, 2017
|
| id | nasplib_isofts_kiev_ua-123456789-138539 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 1812-9269 |
| language | English |
| last_indexed | 2025-12-07T16:45:33Z |
| publishDate | 2017 |
| publisher | Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| record_format | dspace |
| spelling | Vasyliev, R.G. Rodnichenko, A.E. Gubar, O.S. Zlatska, A.V. Gordiienko, I.M. Novikova, S.N. Zubov, D.O. 2018-06-19T09:10:03Z 2018-06-19T09:10:03Z 2017 Large-scale expansion and characterization of human adult neural crest-derived multipotent stem cells from hair follicle for regenerative medicine applications / R.G. Vasyliev, A.E. Rodnichenko, O.S. Gubar, A.V. Zlatska, I.M. Gordiienko, S.N. Novikova, D.O. Zubov // Experimental Oncology. — 2017 — Т. 39, № 3. — С. 171–180. — Бібліогр.: 29 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/138539 Aim: The purpose of this work was to obtain, multiply and characterize the adult neural crest-derived multipotent stem cells from human hair follicle for their further clinical use. Materials and Methods: Adult neural crest-derived multipotent stem cells were obtained from human hair follicle by explant method and were expanded at large-scale up to a clinically significant number. The resulted cell cultures were examined by flow cytometry and immunocytochemical analysis. Their clonogenic potential, ability to self-renewal and directed multilineage differentiation were also investigated. Results: Cell cultures were obtained from explants of adult human hair follicles. Resulted cells according to morphological, phenotypic and functional criteria satisfied the definition of neural crest-derived multipotent stem cells. They had the phenotype Sox2⁺Sox10⁺Nestin⁺CD73⁺CD90⁺CD105⁺CD140a⁺CD 140b⁺CD146⁺CD166⁺CD271⁺CD349⁺ CD34⁻CD45⁻CD56⁻HLA⁻DR⁻, showed high clonogenic potential, ability to self-renewal and directed differentiation into the main derivatives of the neural crest: neurons, Schwann cells, adipocytes and osteoblasts. Conclusion: The possibility of a large-scale expansion of adult neural crest-derived multipotent stem cells up to 40–200·106 cells from minimal number of hair follicles with retention of their phenotype and functional properties are the significant step towards their translation into the clinical practice. en Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України Experimental Oncology Original contributions Large-scale expansion and characterization of human adult neural crest-derived multipotent stem cells from hair follicle for regenerative medicine applications Article published earlier |
| spellingShingle | Large-scale expansion and characterization of human adult neural crest-derived multipotent stem cells from hair follicle for regenerative medicine applications Vasyliev, R.G. Rodnichenko, A.E. Gubar, O.S. Zlatska, A.V. Gordiienko, I.M. Novikova, S.N. Zubov, D.O. Original contributions |
| title | Large-scale expansion and characterization of human adult neural crest-derived multipotent stem cells from hair follicle for regenerative medicine applications |
| title_full | Large-scale expansion and characterization of human adult neural crest-derived multipotent stem cells from hair follicle for regenerative medicine applications |
| title_fullStr | Large-scale expansion and characterization of human adult neural crest-derived multipotent stem cells from hair follicle for regenerative medicine applications |
| title_full_unstemmed | Large-scale expansion and characterization of human adult neural crest-derived multipotent stem cells from hair follicle for regenerative medicine applications |
| title_short | Large-scale expansion and characterization of human adult neural crest-derived multipotent stem cells from hair follicle for regenerative medicine applications |
| title_sort | large-scale expansion and characterization of human adult neural crest-derived multipotent stem cells from hair follicle for regenerative medicine applications |
| topic | Original contributions |
| topic_facet | Original contributions |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/138539 |
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