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Differences in Growth Response of Human Hair Follicle Mesenchymal Stem Cells to Herbal Extracts and a Growth Factor

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Background: One of the key questions in biochemistry is why cell becomes aged and what are the involved factors? Why cell growth is stopped after some divisions and cells become senescent? This occurs in a greater frame in the whole body and cells dye after a while. Androgenetic alopecia (AGA) is characterized by a loss or decrease in hair follicle size, which could be related to the loss of hair follicle stem cells. Therefore, it is of great importance to develop novel therapies to increase hair follicle stem cells viability and proliferation. Objective: In this study, we examined the effects of bFGF and aqueous Rosemary leaf and Marshmallow root extracts on human hair follicle mesenchymal stem cells (hHF-MSCs) proliferation in order to identify their potential for hair growth. Methods: hHF-MSCs were isolated from hair follicle tissues and their mesenchymal nature confirmed by detecting cell surface antigens via flow cytometry. Bromodeoxyuridine (Brdu) incorporation assay was used to study the cell proliferation effect of herbal extracts in hHF-MSCs. Results: Human hair follicle-derived mesenchymal stem cells (hHF -MSCs) were obtained by organ culture. They exhibited surface markers of mesenchymal stem cells as shown by positive staining for CD44, CD90 and CD105. Herbal extracts and bFGF were found to induce significant proliferation of human hHF-MSCs at concentrations ranging from 10 to 20 µl/ml and 15 to 25µl/ml. 78 Conclusion: These results suggest that herbal extract may produce positive effects on the hair growth promotion of hHF-MSCs and suggesting that herbal extracts may be a good candidate for helping hair growth promotion.
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Volume 17, No. 65, Winter 2018
Journal of Medicinal Plants
Differences in Growth Response of Human Hair Follicle Mesenchymal
Stem Cells to Herbal Extracts and a Growth Factor
Ahmadi Ashtiani HR
(Ph.D.)1
*, Salehinia F
(M.Sc.)2
, Rastegar H
(Ph.D.)3
, Allameh AA
(Ph.D.)4
,
Rezazadeh Sh
(Ph.D.)3,5
1- Department of Basic Sciences, Faculty of Pharmacy, Pharmaceutical Sciences
Branch, Islamic Azad University, Tehran - Iran (IAUPS)
2- Department of Biology, Payame Noor University, Tehran, Iran
3- Cosmetic Products Research Center, Iranian FDA, MOH & Medical Education,
Tehran, Iran
4- Department of Clinical Biochemistry, School of Medicine, Tarbiat Modarres
University, Tehran, Iran
5- Medicinal Plants Research Center, Institute of Medicinal Plants, ACECR, Karaj, Iran
*
Corresponding author: Islamic Azad University, Faculty of Pharmacy, Yakhchal
St., Shariati Ave., Tehran, Iran
Tel: +98-912-0293946
E-mail: ahmadihr@yahoo.com
Received: 14 July 2016 Accepted: 28 Oct. 2017
Abstract
Background:
One of the key questions in biochemistry is why cell becomes aged and what are
the involved factors? Why cell growth is stopped after some divisions and cells become senescent?
This occurs in a greater frame in the whole body and cells dye after a while. Androgenetic
alopecia (AGA) is characterized by a loss or decrease in hair follicle size, which could be related to
the loss of hair follicle stem cells. Therefore, it is of great importance to develop novel therapies to
increase hair follicle stem cells viability and proliferation.
Objective:
In this study, we examined the effects of bFGF and aqueous Rosemary leaf and
Marshmallow root extracts on human hair follicle mesenchymal stem cells (hHF-MSCs)
proliferation in order to identify their potential for hair growth.
Methods:
hHF-MSCs were isolated from hair follicle tissues and their mesenchymal nature
confirmed by detecting cell surface antigens via flow cytometry. Bromodeoxyuridine (Brdu)
incorporation assay was used to study the cell proliferation effect of herbal extracts in hHF-MSCs.
Results:
Human hair follicle-derived mesenchymal stem cells (hHF -MSCs) were obtained by
organ culture. They exhibited surface markers of mesenchymal stem cells as shown by positive
staining for CD44, CD90 and CD105. Herbal extracts and bFGF were found to induce significant
proliferation of human hHF-MSCs at concentrations ranging from 10 to 20 µl/ml and 15 to
25µl/ml.
Conclusion:
These results suggest that herbal extract may produce positive effects on the hair
growth promotion of hHF-MSCs and suggesting that herbal extracts may be a good candidate for
helping hair growth promotion.
Keywords:
Hair follicle, Herbal extracts, Proliferation, Stem cell
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Journal of Medicinal Plants, Volume 17,
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Differences in Growth …
Introduction
Hair follicles are renewable organs; they
undergo repeated cycles of growth (anagen),
regression (catagen), and rest (telogen)
throughout the life of mammals [1]. It has
been reported that there is an essential role for
the follicular dermal papilla and its related
dermal sheath cells in hair follicle
development and regeneration [2]. At present,
the majority of skin biology researches are
focused on epidermal stem cells. However, the
characteristics of MSCs also can be observed
in hair follicle dermal cells [3, 4]. Pluripotent
stem cells (PSCs) including human hair
follicle stem cells have been demonstrated to
be one of the best candidates for the generation
of other cell lines [2].
The hair follicle stem cells (HFSCs)
locating at the bulge region of hair follicle can
be easily isolated; they possess the distinct
characteristics of the adult stem cells, such as
self-renewing, high proliferative potential [5,
6]. Therefore, hair follicle stem cells appear to
be highly appropriate seed cells for tissue
engineering and clinical application. Self-
renewal and multilineage differentiation are
other characteristics of stem cells. Since
numerous undifferentiated stem cells are
required for clinical applications, several
laboratories have supplemented their
expansion medium with growth factors to
accelerate stem cell proliferation [7].
In Androgenetic alopecia (AGA), large
terminal follicles diminish in size with time,
and the resulting miniaturized follicle
produces eventually microscopic velus hair
[8]. The number of men and women who
suffer from AGA is increasing, despite the
development of several medical treatments [9].
Therefore, it is of great importance to develop
novel therapies to prevent hair loss and to
enhance hair growth. Multipotent adult stem
cells have many potential therapeutic
applications. One of these therapies is
applying herbal extracts which are mentioned
in Iranian traditional medicine such as
Rosemarinus officinalis that improves
microcirculation around hair follicle also has
antioxidant properties, Altheae officinalis has
antioxidant and anti-inflammatory properties
so can relieve the micro-inflammation around
hair follicle which are the result of
environmental stress and exogenous and
endogenous free radicals. Recent studies
suggest that hair follicles are a source of easily
accessible multipotent stem cells. Hair follicle
stem cells could have important therapeutic
applications [7].
Based on these lines, this study was
planned to investigate the effects of some
herbal extracts that have been used
traditionally in oriental medicine for treating
hair loss on hair follicle stem cells
proliferation as well as its molecular
mechanism.
Material and methods
Isolation of hair follicles stem cell and cell
culture establishment
Hair follicles were isolated from the
occipital scalp skin of three male individuals
(age range 38–54 years, median 43 years) by
plucking after full consent and ethical approval
according to the methods of Yimei Wang [10].
We conformed to the Declaration of Helsinki
Principle guidelines. Hair follicle root tissues
36
Ahmadi Ashtiani & et al.
were obtained by sterilized ophthalmic
scissors and washed 3 times in PBS containing
penicillin and streptomycin 1%, then placed
into 96-well plates, cultured with hHF-MSCs
medium: Dulbecco’s modified Eagle’s
medium (DMEM)/Ham’s F-12 medium
(DMEM/F12), 10 % fetal bovine serum, 2
ng/ml basic fibroblast growth factor (bFGF)
for 7 days. The wells populated with cells
originating from the dermal sheath or papilla
and which had the morphological appearance
of mesenchymal cells were selected, pooled
and expanded. Expanded cells were cultured in
DMEM/F-12 containing 10% FBS and 1%
antibiotic/anti-mycotic and supplemented with
2 ng/ml bFGF. Prior to the proliferation assay,
the cells were cultured in stromal medium
(DMEM/F-12, 10% FBS) for 7 days.
Immunophenotyping of human hair follicle
stem cell by flow cytometry
Immunophenotyping of hHF-MSCs was
carried out as previously described [11]. hHF-
MSCs were detached from the tissue culture
plate with trypsin–EDTA and counted. The
cells (2 × 10
5
) were divided into aliquots and
centrifuged at 104 g for 5 min at RT. The
pellet was resuspended in human serum and
kept on ice for 30 min. After centrifugation at
104 g for 5 min, the pellet was suspended in
3% human serum albumin (HSA)/PBS and
incubated with appropriate antibodies
including Fluorescent Isothiocyanate (FITC)-
conjugated anti-CD44, anti-CD90, anti-
CD105, anti-HLA-DR (Biolegend, USA), anti-
CD45, anti-CD31, anti-CD73 (BD, USA) and
anti-CD15 (Abcam, MA) for 1 h in ice,
washed twice in PBS, and centrifuged for 5
min. The cells were resuspended in 100 µL of
PBS and studied by a Coulter Epics-XL flow
cytometer (Beckman Coulter, Fullerton, CA,
USA). An isotype control with FITC or PE
labeling was included in each experiment, and
specific staining was measured from the cross
point of the isotype with the specific antibody
graph. Histograms were created with Win
MDI 2.8 software (Scripps Institute, La Jolla,
CA, USA).
Plant Material
The root of Althaea officinalis and leaves of
Rosmarinus officinalis were procured from
local market and identified by comparing with
standard herbarium specimens available in
Islamic Azad University, Pharmaceutical
Sciences Branch, Tehran, Iran. Voucher
numbers: Rosemarinus officinalis L. is 250-
PMP/A and Altheae officinalis is 312-PMP/A.
These parts of plant are crushed in mixer
and passed through the sieve number 80. The
various powder drugs were subjected to
pharmacognostic studies for confirmation.
The herbs used in the present study for
making herbal extracts were pre-washed,
dried, crushed, grinded and passed through
100 mesh stainless steel sieves after 72 hours
of maceration in 70°C (Indirect heat) to obtain
a homogenous sample and water was used as
base. The extracts were prepared utilizing this
methodology; the extracts were filtered
through muslin cloth then through filter paper
and milipore under vacuum. The extracts were
used immediately.
To standardize the herbal extracts thin layer
chromatography and paper chromatography
were used various mixtures of solvent systems
37
Journal of Medicinal Plants, Volume 17,
No. 65, Winter 2018
Differences in Growth …
were performed to verify the exact identity of
the components discovered in the qualitative
chemical tests.
Herbal Extracts’ Standardization
To standardize the herbal extracts thin layer
chromatography was used and performed to
verify the exact identity of the components
discovered in the qualitative chemical tests.
For this aim the chromatography layers were
prepared at first. The absorbent powder with
volatile solvent such as acetone was deposited
on a thin layer of a glass plate. After its
dryness, a small drop of herbal extract sample
was poured at the lowest area of column and
allowed it to separate the various layers.
Considering the active ingredients of
Rosemarinus officinalis are rosemarinic and
caffeic acid and the active ingredient of
Altheae officinalis is flavonoids thus the
samples were compared with these
standardized pure materials.
Graphs Preparation
Graphs are prepared based on average of
living cells in different concentrations of three
groups: aqueous extract of leaf of rosemarinus
officinalis, aqueous extract of root of altheae
officinalis and the mix of two herbs as the
third group after 48 hours. Considering that the
Kolmogorov-Smirnov test confirmed the data
of different concentrations of three groups are
normal (P>0.05), to compare the different
concentrations of various groups, One-way
ANOVA and Tukey's post hoc tests are used.
Determination of Herbal Extracts Lethal
Concentration 50
The herbs used in the present study for
making herbal extracts were dried, crushed
and passed through 80 mesh stainless steel
sieves and water was used as base. The
extracts was prepared described previously [9].
Treatments were designed in 6 groups 3 times
5, 10, 20, 30, 50 and 100µl/ml for Rosmarinus
officinalis leaf extract and 5, 15, 25, 50 and
100 µl/ml for Althaea officinalis root extract.
Preparation of Combined Drug Herbal
Extracts Formulation
After selection of method for preparation,
multi ingredients of effective concentrations
based on the preliminary physical and
biological screening was prepared. The
method selected was direct binge method and
six different formulations having
concentrations 5, 15, 25, 50, 100 and 200
µl/ml of extracts were prepared for maximum
activity (Table 1).
Table 1- Selection of concentration of herbal extracts
Herbal extracts LC50 (µl/ml) Optimum dosages*
(µl/ml)
Rosemarinus officinalis leaf extract 30 20 10
Althaea officinalis root extract 50 25 15
Rosemarinus officinalis leaf extract+ Althaea officinalis root extract 25 15 5
* The best minimum and maximum dosages which induce maximum viability for MSCs
38
Ahmadi Ashtiani & et al.
Brdu cell proliferation assay
Cell proliferation was estimated in hHF-MSCs
culture at different concentrations (10 and 20
µl/ml of Rosmarinus officinalis leaf extract, 15
and 25 µl/ml of Althaea officinalis, 5 and 15
µl/ml of combination) of herbal extracts using a
colorimetric bromodeoxyuridine (Brdu) kit
(Roche Diagnostics, East Sussex, UK) according
to the previous study [12]. Briefly, cells labeled
with 10 µM Brdu for 2 hrs at RT were fixed and
made permeable with the FixDenat solution for
30 mins, then incubated with monoclonal anti-
Brdu peroxidase-conjugated antibody for 90 min.
The cells were then washed three times with wash
buffer, and then peroxidase activity was measured
using tetramethyl-benzidine as substrate. The
reaction was terminated by adding 25 µl of 1M
H2SO4, and absorbance at 450 nm was recorded
in an ELISA plate reader. To study the rate of cell
proliferation in the Brdu assay, the number of
cells was adjusted to 20,000 to obtain an
acceptable optical density (OD) in treatment
groups.
Results
Isolation and Characterization of hHF-MSCs
The hHFM-SCs, resembling typical
fibroblast morphology, migrated out from the
hair follicle root tissue and adhered to the
surface of the culture plate (Figure 1). The
fibroblast-like cells at passage 3 were shown in
Figure 1.
Immunophenotyping of hHF-MSCs:
Flow cytometry analyses showed that the
majority of hHFMSCs expressed MSC markers
CD44, CD90 and CD105 (25%, 30%, 29% resp.)
(Figure 2).
Herbal extracts induced the proliferation of
human hair follicle mesenchymal stem cells
The effect of herbal extracts on the
hHFMSCs proliferation was examined.
hHFMSCs were treated by various
concentrations of herbal extracts and bFGF for
14 days and cell proliferation was measured by
BrdU methods at days 0, 2, 4 and 14. BrdU
assays showed a concentration dependent
increased in cell growth activity in the
hHFMSCs following exposure to Altheae
officinalis extract but not Rosmarinus
officinalis extract. As it is observed in Figure
3, proliferation promoting effects of
Rosmarinus officinalis leaf extract on
hHFMSCs start at 10 µl/ml and increased by
Figure 1- Isolation and characterization of human hair follicle mesenchymal stem cells (hHF-MSCs).
The hHF-
MSCs, resembling typical fibroblast-like cells
39
Journal of Medicinal Plants, Volume 17,
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Differences in Growth …
Figure 2- Flow cytometric analysis of cell surface markers on hHF-MSCs cells.
2 × 10
5
cells were incubated with
primary antibodies against CD29, CD73, CD105, CD90, CD106 and CD44 respectively, followed by incubation with a
secondary FITC-labeled antibody. Controls were incubated with secondary antibody only. Percentages indicate the fraction
of cells that stained positive.
Figure 3- Measurement the rate of human hair follicle mesenchymal stem cells proliferation in minimum and
maximum dosages of Rosemarinus officinalis aqueous extract at days 0, 2, 7 and 14 of treatment in DMEM
F12/FBS 10% and penicillin/streptomycin 1% by Brdu assay
: P <0.01 vs. Control at the same day
: P <0.01 vs. Minimum dosage at the same day
*
*
**
*
*
**
**
**
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2
2.1
01020
OD
Concentration(µl/ml)
Day0
Day2
Day7
Day14
40
Ahmadi Ashtiani & et al.
*
*
**
*
*
**
*
*
**
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
0515
OD
Concentration(µl/ml)
Day0
Day2
Day7
Day14
20 µl/ml but then decreased at day 14 in 20
µl/ml (from 1.64, 1.68 and 1.47% in 10µl/ml
to 2.08, 2.02 and 1.69% in 20 µl/ml at day 2, 7
and 14 vs. control respectively; P < 0.01, one
way ANOVA); also as observed in Figure 4,
proliferation promoting effects of Althaea
officinalis root extract on hHFMSCs start at 15
and increased by 25 µl/ml (from 1.3%, 1.4%,
1.28% in 15 µl/ml to 1.8%, 1.76%, 1.6% in 25
µl/ml at day 2, 7 and 14 vs. control
respectively; P < 0.01, one way
ANOVA);eventually as observed in Figure 5,
Figure 4-
Measurement the rate of human hair follicle mesenchymal stem cells proliferation in minimum and
maximum dosages of Altheae officinalis aqueous extract at days 0, 2, 7 and 14 of treatment in DMEM F12/FBS
10% and penicillin/streptomycin 1% by Brdu assay.
: P<0.01 vs. Control at the same day
: P<0.01 vs. Minimum dosage at the same day
Figure 5- Measurement the rate of human hair follicle mesenchymal stem cells proliferation in minimum and
maximum dosages of blend of Rosemarinus officinalis and Altheae officinalis aqueous extract at days 0, 2, 7 and
14 of treatment in DMEM F12/FBS 10% and penicillin/streptomycin 1% by Brdu assay
: P<0.01 vs. Control at the same day, : P<0.01 vs. Minimum dosage at the same day
*
*
**
*
*
**
*
*
**
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
01525
OD
Concentration(µl/ml)
Day0
Day2
Day7
Day14
41
Journal of Medicinal Plants, Volume 17,
No. 65, Winter 2018
Differences in Growth …
proliferation promoting effects of combination
of both herbal extracts on hHFMSCs start at 5
and increased by 15 µl/ml (from 1.6%, 1.19%,
1.55% at 5 µl/ml to 2.02%, 2.05%, 1.68% in
15µl/ml at day 2, 7 and 14 vs. control
respectively; P < 0.01, one way ANOVA).
Discussion
During the growth of a hair, the follicle
under-goes changes from an actively growing
phase (anagen), to a remodeling phase
(catagen), and finally to a quiescent phase
(telogen), only to start growing again. One
most key element that control hair follicle
cycling is the follicular stem cells [13]. Recent
studies demonstrated that alopecia is
associated with destruction of hair follicle
stem cells and hair loss [14]. The identification
and characterization of stem cells is currently
one of the most investigated areas of
biological and biomedical research.
Multipotent MSCs were originally found in
the bone marrow, and were considered to be
the inherent stem cells at this location [15].
Later, these MSCs or MSC-like cells have
been separated from, and identified in various
tissues, including white adipose tissue,
pancreas, skin, and the umbilical cord blood
[16–19]. In recent years, studies have shown
the presence of MSCs in the dermal sheath and
dermal papillae of the hair follicle: In 2006, a
comparative study investigated the properties
of follicular dermal stem cells from whisker
hairs of Wistar rats and bone marrow MSCs
were isolated from femora of the same animals
[20]. The results from that study showed that
dermal stem cells from hair follicles have a
similar morphology and population doubling
time, and express the same cell-surface
markers as the MSCs.
To our knowledge, the proliferation effects
of herbal extracts in the hair follicle stem cell
have not been previously elucidated. We
hypothesized that the hair loss seen in AGA
may result from loss of hair follicle stem cells.
Based on these lines, this study investigated
the effects of herbal extracts for treating hair
loss in the hair follicle stem cells. In this study,
we established a hair follicle stem cell culture
model in vitro to clarify the effects of herbal
extracts on the hair follicle growth. Our results
are consistent with several previous studies that
have demonstrated the multipotency of rat or
human skin dermal fibroblasts [21-25]. It has
been established that the bulge area of the hair
follicle is an enriched source of epidermal stem
cells [26-29]. The present study further supports
these data by showing that HF-MSCs express
CD44, CD73, CD90 and CD105 which are the
surface markers that characterize MSCs.
In this study, herbal extracts were found to
induce significant proliferation of human hair
follicle stem cell at concentrations ranging
from 5 to 25µl/ml.
In a study Roh SS et.al demonstrated that
Asiasari radix extract increased the
proliferation of both HaCaT and human DP
cells in vitro [31]. Hay IC et.al have been
investigated the efficacy of herbal extracts in
the treatment of patients with alopecia and
their results demonstrated that Nineteen (44%)
of 43 patients in the active group showed
improvement compared with 6 (15%) of 41
patients in the control group [32]. In another
study, Bureau JP et al. demonstrated that the
blend of Pimenta racemosa, Myrtus
42
Ahmadi Ashtiani & et al.
communis, Cedrus atlantica, Laurus nobilis,
Pogostemon patchouli, Rosmarinus officinalis,
Salvia officinalis, S. sclarea, Thymus
satureioides, Cananga odorata increased hair
density and the ratio of anagen hair per total
hair in the double-blind randomized study vs.
placebo in healthy male and female volunteers
[33]. Although minoxidil, finasteride, and
dutasteride including other synthetic
therapeutic agents are mostly used for alopecia
treatment, their adverse effects encourage
sorting of alternative efficient treatment agents
with a limited side effect particularly herbs.
Thus, alternative herb medicine has attracted
interest for hair loss treatment. Natural-derived
compounds are able to prevent many diseases
such as alopecia. Roh SS et.al was shown that
Sophora flavescens is oriental medicine for
hair loss treatment, which proven on hair
growth promoting [30].
The effect of herbal extract on cancerous
cell proliferation is studied for years and the
results on DMBA induced cancerous cells
showed that flavonoids of Rosemarinus
officinalis and Altheae officinalis are able to
activate PPARɣ which leads to inhibition of
COX-2 over expression (that prevents
apoptosis and induce cell proliferation);
rosemarinic acid that exist in Rosemarinus
officinais extract is able to do it as well by
decreasing of COX-2 inhibition [34] but in
another study, a combination of ethanolic
extract of some herbs such as Rosemarinic
acid and Altheae officinalis an increase in
dermal pailla cell proliferation of human hair
follicle was observed. While in another study
showed that ethanolic extract of Rosemarinus
officinalis and Altheae officinalis have positive
effect on viability and proliferation of dermal
papillae cells of human hair follicle; based on
this study these herbal extracts have significant
effects on expression of CyclinD1, Cdk4, Erk,
Akt, Bcl-2 and Bax proteins in cel culture of
human dermal papillae; It is suggested that
activation of Akt and Erk pathways lead to this
or maybe it is due to increase in expression of
Cdk4 and CyclinD1 [35] which is the
checkpoint of G1/S as the regulator of cell
cycle [36]; the role of signaling pathway of
Akt on mitogenesis and cell growth is
previously showed [37] but maybe the
antioxidant content of such herbal extracts
helps to save the cellular energy therefor cell
can focus on its vital activity such as
proliferation and mitosis, gene expression and
transcription instead of fighting to free radicals
while it is previously showed that Akt has a
key role on cell viability signals [38, 39].
Conclusion
Stem cell was isolated from human hair
follicle tissues by noninvasive means. These
results suggest that herbal extract may produce
positive effects on the hair growth promotion
Overall; we have demonstrated the potential
hair growth promoting effect of herbal
extracts, suggesting that herbal extracts may
be a good candidate for hair follicle stem cells
proliferation and helping hair growth
promotion.
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Journal of Medicinal Plants, Volume 17,
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Differences in Growth …
References
1. Hoffman RM. The hair follicle as a gene
therapy target. Nature Biotechnol. 2000; 18:
20 - 1.
2. Ohyama M, Zheng Y, Paus R and Stemm
K.S. The mesenchymal component of hair
follicle neogenesis: background, methods and
molecular characterization. Experimental
Dermatol. 2009; 19: 89 - 99.
3. Richardson G.D., Arnott E.C., Whitehouse
C.J., Lawrence C.M., Hole N. and et al.
Cultured cells from the adult human hair
follicle demis can be directed toward
adipogenic and osteogenic differentiation. The
Journal of Investigative Dermatol. 2005; 124:
1090 - 1091.
4. Reynolds A.J., Lawrence C., Cserhalmi-
Friedman P.B., Christiano A.M. and Jahoda,
C.A. Trans-gender induction of hair follicles.
Nature 1999; 402: 33 - 34.
5. Hsu YC, Pasolli HA and Fuchs E.
Dynamics between stem cells, niche, and
progeny in the hair follicle. Cell 2011; 144:
92-105.
6. Tanimura S, Tadokoro Y, Inomata K, Binh
NT, Nishie W, Yamazaki S and et al. Hair
follicle stem cells provide a functional niche
for melanocyte stem cells. Cell Stem Cell
2011; 8: 177-187.
7. Zhang X, Wang Y, Gao Y, Liu X, Bai T,
Li M, Li L, Chi G, Xu H, Liu F, Liu JY and Li
Y. Maintenance of high proliferation and
multipotent potential of human hair follicle-
derived mesenchymal stem cells by growth
factors. Int. J. Mol. Med. 2013 Apr; 31 (4):
913-21.
8. Paus R and Cotsarelis G. The biology of
hair follicles. N. Engl. J. Med. 1999; 341 (7):
491 - 497.
9. Rastegar H, Ahmadi Ashtiani H, Aghaei
M, Ehsani A and Barikbin B. Combination of
herbal extracts and platelet-rich plasma
induced dermal papilla cell proliferation:
involvement of ERK and Akt pathways. J.
Cosmet. Dermatol. 2013 Jun; 12 (2): 116-22.
10. Wang Y, Liu J, Tan X, Li G, Gao Y, Liu
X, Zhang L and Li Y. Induced pluripotent
stem cells from human hair follicle
mesenchymal stem cells. Stem Cell Rev. 2013
Aug; 9 (4): 451-60.
11. Ahmadi-Ashtiani H, Allameh A, Rastegar
H, Soleimani M and Barkhordari E. Inhibition
of cyclooxygenase-2 and inducible nitric oxide
synthase by silymarin in proliferating
mesenchymal stem cells: comparison with
glutathione modifiers. J. Nat. Med. 2012 Jan;
66 (1): 85-94
12. Allameh A, Ahmadi-Ashtiani H, Emami
Aleagha MS and Rastegar H. The metabolic
function of hepatocytes differentiated from
human mesenchymal stem cells is inversely
related to cellular glutathione levels. Cell
Biochem. Funct. 2014 Mar; 32 (2): 194-200
13. Lavker RM, Sun TT, Oshima H,
Barrandon Y, Akiyama M, Ferraris C,
Chevalier G, Favier B, Jahoda CA, Dhouailly
D, Panteleyev AA and Christiano AM. Hair
follicle stem cells. J. Investig Dermatol. Symp.
Proc. 2003 Jun; 8 (1): 28-38.
14. Garza LA, Yang CC, Zhao T, Blatt HB,
Lee M, He H, Stanton DC, Carrasco L, Spiegel
JH, Tobias JW and Cotsarelis G. Bald scalp in
men with androgenetic alopecia retains hair
follicle stem cells but lacks CD200-rich and
CD34-positive hair follicle progenitor cells. J.
44
Ahmadi Ashtiani & et al.
Clin. Invest. 2011 Feb; 121 (2): 613-22.
15. Uccelli A., Moretta L. and Pistoia V.
Mesenchymal stem cells in health and disease.
Nature Reviews Immunology 2008; 8: 726 -
736.
16. Zuk P.A., Zhu M., Ashjian P., De Ugarte
D.A., Huang J.I. and et al. Human adipose
tissue is a source of multipotent stem cells.
Molecular Biology of the Cell 2002; 13: 4279 -
4295.
17. Baertschiger R.M., Bosco D., Morel P.,
Serre-Beinier V., Berney T. and et al.
Mesenchymal stem cells derived from human
exocrine pancreas express transcription factors
implicated in beta-cell development. Pancreas
2008; 37: 75 - 84.
18. Metcalfe A.D. and Ferguson M.W. Skin
stem and pro-genitor cells: using regeneration
as a tissue-engineering strategy. Cellular and
Molecular Life Sciences 2008; 65: 24 - 32.
19. Markov V., Kusumi K., Tadesse M.G.,
William D. A., Hall D. M. and et al. Identification
of cord blood-derived mesenchy-mal
stem/stromal cell populations with distinct growth
kinetics, differentiation potentials, and gene
expression profiles. Stem Cells and Development
2007; 16: 53 - 73.
20. Hoogduijn M.J., Gorjup E. and Genever
P.G. Comparative characterization of hair
follicle dermal stem cells and bone marrow
mesenchymal stem cells. Stem Cell and
Development 2006; 15: 49 - 60.
21. Toma JG, Akhavan M, Fernandes KJ,
Barnabé-Heider F, Sadikot A, Kaplan DR and
Miller FD. Isolation of multipotent adult stem
cells from the dermis of mammalian skin. Nat.
Cell Biol. 2001; 3: 778-784.
22. Fernandes KJ, McKenzie IA, Mill P and et
al: A dermal niche for multipotent adult skin-
derived precursor cells. Nat. Cell Biol. 2004;
6: 1082-1093.
23. Toma JG, McKenzie IA, Bagli D and
Miller FD. Isolation and characterization of
multipotent skin-derived precursors from
human skin. Stem Cells 2005; 23: 727-737.
24. Chen FG, Zhang WJ, Bi D, Liu W, Wei X,
Chen FF, Zhu L, Cui L and Cao Y. Clonal
analysis of nestin(-) vimentin(+) multi-potent
fibroblasts isolated from human dermis. J. Cell
Sci. 2007; 120: 2875-2883.
25. Lorenz K, Sicker M, Schmelzer E, Rupf T,
Salvetter J, Schulz-Siegmund M and Bader A.
Multilineage differentiation potential of human
dermal skin-derived fibroblasts. Exp.
Dermatol. 2008; 17: 925-932.
26. Cotsarelis G, Sun TT and Lavker RM.
Label-retaining cells reside in the bulge area of
pilosebaceous unit: implications for follicular
stem cells, hair cycle, and skin carcinogenesis.
Cell 1990; 61: 1329-1337.
27. Morris RJ and Potten CS. Highly persistent
label-retaining cells in the hair follicles of mice
and their fate following induction of anagen. J.
Invest. Dermatol. 1999; 112: 470-475.
28. Tumbar T, Guasch G, Greco V, Blanpain
C, Lowry WE, Rendl M and Fuchs E:
Defining the epithelial stem cell niche in skin.
Science 2004; 303: 359-363.
29. Morris RJ, Liu Y, Marles L, Yang Z,
Trempus C, Li S, Lin JS, Sawicki JA and
Cotsarelis G. Capturing and profiling adult
hair follicle stem cells. Nat. Biotechnol. 2004;
22: 411-417.
30. Roh SS, Kim CD, Lee MHet al. The hair
45
Journal of Medicinal Plants, Volume 17,
No. 65, Winter 2018
Differences in Growth …
growth promoting effect of Sophora flavescens
extract and its molecular regulation. J.
Dermatol. Sci. 2002; 30: 43 - 9.
31. Rho SS, Park SJ, Hwang SL, Lee MH,
Kim CD, Lee IH, Chang SY and Rang MJ.
The hair growth promoting effect of Asiasari
radix extract and its molecular regulation. J.
Dermatol. Sci. 2005; 38 (2): 89-97.
32. Hay IC, Jamieson M and Ormerod AD.
Randomized trial of aromatherapy: successful
treatment of alopecia areata. Arch. Dermatol.
1998; 134: 1349 - 52.
33. Bureau JP, Ginouves P, Guilbaud J and
Roux ME. Essential oils and low-intensity
electromagnetic pulses in the treatment of
androgen-dependent alopecia. Adv. Ther.
2003; 20: 220 - 9.
34. Hamta A and Parvini P. Study of
Cytotoxic Effects of Taxol and Rosemary
Extracts on Cancerous Cells Derived From
DMBA-induced Breast Cancer in SD Rats,
Journal of Cell & Tissue (JCT), Summer 2011;
2 (2): 117-126.
35. Rastegar H, Ahmadi Ashtiani H.R, Aghaei
M, Barikbin B and Ehsani A.H. Herbal etracts
induce dermal papilla cell proliferation of
human hair follicles, Annals of Dermatol. 2015
Dec.; 27 (6): 667-675.
36. Jin S, Mazzacurati L, Zhu X, Tong T,
Song Y, Shujuan S and et al. Gadd45a
contributes to p53 stabilization in response to
DNA damage. Oncogene 2003; 22: 8536-
8540.
37. Robinson MJ and Cobb MH. Mitogen-
activated protein kinase pathways, Cure. Opin.
Cell Boil. 1997; 9: 180-186.
38. Ahmad S, Singh N and Glazar RI. Role of
AKT1 in 17betaesteradiol-and insulin-like
growth factor I (IGF-I)-dependent proliferation
and prevention of apoptosis in MCF-7 breast
carcinoma cells. Biochem. Pharmacol. 1999;
58: 425-430.
39. Tang Y, Zhou H, Chen A, Pittman RN and
Field J. The AKT proto-oncogene links Ras to
Pak and cell survival signals. J. Biol. Chem.
2000; 275: 9106-9109.
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... In addition to genetic factors, alopecia is caused by excessive stress, malnutrition, hormonal imbalances, and aging and is accompanied by a reduction in hair follicle size and anagen follicles [28][29][30]. Currently, many studies have been conducted using natural materials, such as herbal medicines and biopharmaceuticals, to identify therapeutic compounds capable of promoting hair growth and reducing hair loss [31][32][33][34]. While the activity of glutathione and SOD etc. in the blood of hair loss patients has been found to be significantly low in hair loss patients, the levels of thiobarbituric acid reactive substances (TBARS) are significantly higher than those of patients without hair loss [34]. ...
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This study investigated the hair regeneration promotion and hair loss prevention properties of Nelumbinis Semen (NS) extract in vitro and in vivo. The effect of NS on the proliferation and migration of human dermal papilla cells (hDPCs) was measured in vitro via CCK-8 and scratch migration assays, after which the antioxidant activity of NS was also quantified. NS extracts were then applied to the back of 7-week-old C57BL/6 mice for 3 weeks to monitor hair growth patterns and hair follicle (HF) histology. The mice were divided into three groups: negative control group (NC; DMSO), positive control group (PC; 3% minoxidil), and experimental group (NS extract 1,000 ppm). Moreover, to study the molecular mechanisms by which NS extract regenerates hair growth, real-time PCR was used to analyze factors related to the hair growth cycle. The NS extracts were found to possess high antioxidant properties due to their high flavonoid contents and electron-donating ability. Moreover, NS extracts enhanced hDPC proliferation and migration in a concentration-dependent manner (15.63–125 ppm). The hair growth index and growth area of the NS group (2.81 score, 81%) on day 14 were higher than those of the PC group (2.65 score, 68%) (p
... ese properties are crucial in triggering proliferation of hair follicle cells leading to hair growth. Studies by [31] on hair growth promoting e ects of antioxidants and anti-in ammatory extracts of Rosemarinus o cinalis and Altheae o cinalis supported the idea. e mechanism involves follicle stem cells resuscitation by the cleansing removal of microin ammations which emerge from stress and exposure to free radicals. ...
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