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Minoxidil Promotes Hair Growth through Stimulation of Growth Factor Release from Adipose-Derived Stem Cells

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Minoxidil directly promotes hair growth via the stimulation of dermal papilla (DP) and epithelial cells. Alternatively, there is little evidence for indirect promotion of hair growth via stimulation of adipose-derived stem cells (ASCs). We investigated whether minoxidil stimulates ASCs and if increased growth factor secretion by ASCs facilitates minoxidil-induced hair growth. Telogen-to-anagen induction was examined in mice. Cultured DP cells and vibrissae hair follicle organ cultures were used to further examine the underlying mechanisms. Subcutaneous injection of minoxidil-treated ASCs accelerated telogen-to-anagen transition in mice, and increased hair weight at day 14 post-injection. Minoxidil did not alter ASC proliferation, but increased migration and tube formation. Minoxidil also increased the secretion of growth factors from ASCs, including chemokine (C-X-C motif) ligand 1 (CXCL1), platelet-derived endothelial cell growth factor (PD-ECGF), and platelet-derived growth factor-C (PDGF-C). Minoxidil increased extracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylation, and concomitant upregulation ofPD-ECGFandPDGF-CmRNA levels were attenuated by an ERK inhibitor. Subcutaneous injection of CXCL1, PD-ECGF, or PDGF-C enhanced anagen induction in mice, and both CXCL1 and PDGF-C increased hair length in ex vivo organ culture. Treatment with CXCL1, PD-ECGF, or PDGF-C also increased the proliferation index in DP cells. Finally, topical application of CXCL1, PD-ECGF, or PDGF-C with 2% minoxidil enhanced anagen induction when compared to minoxidil alone. Minoxidil stimulates ASC motility and increases paracrine growth factor signaling. Minoxidil-stimulated secretion of growth factors by ASCs may enhance hair growth by promoting DP proliferation. Therefore, minoxidil can be used as an ASC preconditioning agent for hair regeneration.
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International Journal of
Molecular Sciences
Article
Minoxidil Promotes Hair Growth through
Stimulation of Growth Factor Release from
Adipose-Derived Stem Cells
Nahyun Choi 1,2, Soyoung Shin 3, Sun U. Song 4, * and Jong-Hyuk Sung 1, 2, *
1College of Pharmacy, Yonsei University, Incheon 21983, Korea; nh147837@gmail.com
2STEMORE Co., Ltd., Incheon 21983, Korea
3College of Pharmacy, Wonkwang University, Iksan 54538, Jeonbuk, Korea; shins@wku.ac.kr
4Translational Research Center and Inha Research Institute for Medical Sciences,
Inha University School of Medicine, Incheon 21983, Korea
*Correspondence: sunuksong@inha.ac.kr (S.U.S.); brian99@empal.com (J.-H.S.);
Tel.: +82-32-890-2460 (S.U.S.); +82-32-749-4506 (J.-H.S.)
Received: 12 February 2018; Accepted: 26 February 2018; Published: 28 February 2018
Abstract:
Minoxidil directly promotes hair growth via the stimulation of dermal papilla (DP) and
epithelial cells. Alternatively, there is little evidence for indirect promotion of hair growth via
stimulation of adipose-derived stem cells (ASCs). We investigated whether minoxidil stimulates
ASCs and if increased growth factor secretion by ASCs facilitates minoxidil-induced hair growth.
Telogen-to-anagen induction was examined in mice. Cultured DP cells and vibrissae hair follicle
organ cultures were used to further examine the underlying mechanisms. Subcutaneous injection
of minoxidil-treated ASCs accelerated telogen-to-anagen transition in mice, and increased hair
weight at day 14 post-injection. Minoxidil did not alter ASC proliferation, but increased migration
and tube formation. Minoxidil also increased the secretion of growth factors from ASCs, including
chemokine (C-X-C motif) ligand 1 (CXCL1), platelet-derived endothelial cell growth factor (PD-ECGF),
and platelet-derived growth factor-C (PDGF-C). Minoxidil increased extracellular signal–regulated
kinases 1/2 (ERK1/2) phosphorylation, and concomitant upregulation of PD-ECGF and PDGF-C
mRNA levels were attenuated by an ERK inhibitor. Subcutaneous injection of CXCL1, PD-ECGF,
or PDGF-C enhanced anagen induction in mice, and both CXCL1 and PDGF-C increased hair length in
ex vivo organ culture. Treatment with CXCL1, PD-ECGF, or PDGF-C also increased the proliferation
index in DP cells. Finally, topical application of CXCL1, PD-ECGF, or PDGF-C with 2% minoxidil
enhanced anagen induction when compared to minoxidil alone. Minoxidil stimulates ASC motility
and increases paracrine growth factor signaling. Minoxidil-stimulated secretion of growth factors by
ASCs may enhance hair growth by promoting DP proliferation. Therefore, minoxidil can be used as
an ASC preconditioning agent for hair regeneration.
Keywords: minoxidil; adipose-derived stem cells; hair growth; CXCL1; PD-ECGF; PDGF-C
1. Introduction
Adipose-derived stem cells (ASCs) have stimulatory effects on dermal papilla (DP) cells to
promote hair-growth [
1
6
]. For example, ASCs secrete multiple growth factors, such as vascular
endothelial growth factor (VEGF) and basic fibroblast growth factors (bFGF), which can increase the
proliferation of DP cells [
1
]. Festa et al. showed that adipocyte lineage cells drive hair cycling by
contributing to the skin stem cell niche, and suggested that platelet-derived growth factor-A (PDGF-A)
expression by immature adipocytes regulates follicular stem cell activity [
2
]. Grafting of ASC-enriched
adipose tissue (i.e., by injection of the stromal vascular fraction of lipoaspirate) has shown promise as
Int. J. Mol. Sci. 2018,19, 691; doi:10.3390/ijms19030691 www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2018,19, 691 2 of 15
an alternative approach to treating baldness in men and women [
7
]. We have identified stimulators
that enhance the hair-regenerative potential of ASCs
in vivo
. For instance, vitamin C and low-dose
ultraviolet B (UVB) increased secretion of hair growth-promoting factors by ASCs and induced anagen
in animal models [
3
,
4
]. Of the potential stimulators tested, we found that platelet-derived growth
factor-D (PDGF-D) exhibited the strongest effects on ASCs and increased secretion of growth factors
via mitogen-activated protein kinase (MAPK) pathways [
5
]. In addition, CAP-18 for cathelicidin
antimicrobial peptide (LL-37) increased the secretion of growth factors and the hair-regenerative
efficacy of ASCs via early growth response 1 (ERG1) protein and the MAPK pathway [8].
Minoxidil was first developed to treat male- and female-pattern alopecia. In addition to
vasodilation, there is strong evidence that minoxidil directly promotes hair growth via the stimulation
of DP and epithelial cells [
9
14
]. Minoxidil stimulated mouse vibrissae follicles in organ culture
and induced proliferation of hair epithelial cells near the follicle base [
9
]. Further, minoxidil and its
derivatives showed cytoprotective activity
in vivo
and increased prostaglandin E2 (PGE2) production
by human DP fibroblasts [
11
]. In cultured DP cells, minoxidil-induced hair growth was mediated by
adenosine receptors [12]. Minoxidil also promoted the survival of human DP cells by activating both
the ERK and protein kinase B (Akt) pathways, and prevented apoptotic cell death by increasing the ratio
of Bcl-2/Bax [
15
]. Moreover, minoxidil activated the
β
-catenin pathway in human DP cells, suggesting
a possible mechanism for its anagen prolongation effect [
13
]. Minoxidil suppressed androgen receptor
(AR)-mediated functions by decreasing AR transcriptional activity in reporter assays and reducing
expression of AR targets at the protein level [
16
]. Otomo summarized the primary mechanisms of
minoxidil action as (a) induction of growth factors in DP cells, such as VEGF, hepatocyte growth factor
(HGF), and insulin-like growth factor-1 (IGF-1); (b) inhibition of TGF-
β
-induced apoptosis of hair
matrix cells; and, (c) increase of blood flow by dilating hair follicle arteries [
14
]. However, there is little
evidence that minoxidil can indirectly promote hair growth via ASCs, even though ASCs contribute to
the stem cell niche for hair follicles and exert stimulatory effects on hair cycle progression. Therefore,
in the present study, we examined possible indirect hair growth-promoting effects of minoxidil via
ASCs. Specifically, we investigated whether minoxidil stimulates growth factor secretion by ASCs to
enhance follicular cell activity and hair growth.
2. Results
2.1. Minoxidil-Pretreated Adipose-Derived Stem Cells (ASCs) Promote Hair Growth In Vivo
ASCs are known to stimulate hair growth [
1
6
]. We found that injection of naïve (untreated)
human ASCs increased telogen-to-anagen induction in mice only slightly following subcutaneous
injection, while ASCs pretreated with minoxidil induced robust hair growth (Figure 1A,B). To examine
the effect of ASCs pretreated with minoxidil on hair follicle, we performed hematoxylin and eosin (HE)
staining and immunofluorescence staining for Ki67, which is a proliferating cell marker in DP. The skin
section of ASC
MXD
-treated mice showed higher number of mature hair follicle compared to vehicle-
or ASC
Ctrl
-treated mice (Figure 1C). In addition, most hair follicles of ASC
MXD
-treated mice showed
DP with Ki67
+
cells contrary to vehicle- or ASC
Ctrl
-treated mice (Figure 1D). This result suggests that
minoxidil can promote telogen to anagen induction, thereby promoting hair growth.
Int. J. Mol. Sci. 2018,19, 691 3 of 15
Int. J. Mol. Sci. 2018, 19, x 3 of 15
Figure 1. Adipose-derived stem cells (ASCs) pretreated with minoxidil promote hair growth in vivo.
Minoxidil-treated ASCs or untreated ASCs were injected into the dorsal skin of shaved mice.
Photograph was taken (A), and hair weight measured (B) 14 days later; (C) Skin section was analyzed
by HE staining and the number of mature hair follicle was measured; (D) The hair follicle with Ki67+
cells in DP was shown by immunostaining. Asterisks indicate hair follicles with Ki67+ DP cells. ** p <
0.01, *** p < 0.001. n = 7 or 8 mice per group. All error bars indicate SEM.
2.2. Minoxidil can Induce Migration of ASCs
To examine whether minoxidil affects ASC proliferation, we determined the live cell number
over two days and seven days of minoxidil treatment. Minoxidil had no effect on ASC proliferation
under either condition, even at the highest dose (Figure 2A,B). To explore whether minoxidil affects
ASC migration, we conducted scratch and transwell migration assays. Minoxidil at 20 and 50 µM
dose-dependently increased ASC migration into both the scratch wound assay (Figure 2C) and
transwell migration assay (Figure 2D). To determine whether the effect of minoxidil on specific to the
ASCs, we examined the effect of minoxidil on growth and migration of dermal fibroblast cells.
Minoxidil did not induce cell growth either migration (Figure S1). Initially, topically applied
minoxidil was believed to stimulate hair growth by indirect actions, such as vasodilatation and
increased blood ow to the DP [10,14]. Therefore, we examined whether minoxidil affects blood
vessel formation by ASCs using an in vitro tube formation assay. Indeed, minoxidil dose-
dependently increased the number of nascent tubes after 12–16 h (Figure 2E) and the expression level
of endothelial cell markers including tyrosine kinase with immunoglobulin-like and EGF-like
domains 1(TIE1), vascular endothelial growth factor receptor 1 (VEGFR1), VEGFR2 and endothelin
receptor type B (EDNRB) (Figure 2F). Collectively, these results suggest that minoxidil may promote
hair growth by enhancing ASC migration and ASC-dependent angiogenesis.
Figure 1.
Adipose-derived stem cells (ASCs) pretreated with minoxidil promote hair growth
in vivo
. Minoxidil-treated ASCs or untreated ASCs were injected into the dorsal skin of shaved
mice. Photograph was taken (
A
), and hair weight measured (
B
) 14 days later; (
C
) Skin section was
analyzed by HE staining and the number of mature hair follicle was measured; (
D
) The hair follicle
with Ki67
+
cells in DP was shown by immunostaining. Asterisks indicate hair follicles with Ki67
+
DP
cells. ** p< 0.01, *** p< 0.001. n= 7 or 8 mice per group. All error bars indicate SEM.
2.2. Minoxidil Can Induce Migration of ASCs
To examine whether minoxidil affects ASC proliferation, we determined the live cell number
over two days and seven days of minoxidil treatment. Minoxidil had no effect on ASC proliferation
under either condition, even at the highest dose (Figure 2A,B). To explore whether minoxidil affects
ASC migration, we conducted scratch and transwell migration assays. Minoxidil at 20 and 50
µ
M
dose-dependently increased ASC migration into both the scratch wound assay (Figure 2C) and
transwell migration assay (Figure 2D). To determine whether the effect of minoxidil on specific to
the ASCs, we examined the effect of minoxidil on growth and migration of dermal fibroblast cells.
Minoxidil did not induce cell growth either migration (Figure S1). Initially, topically applied minoxidil
was believed to stimulate hair growth by indirect actions, such as vasodilatation and increased blood
flow to the DP [
10
,
14
]. Therefore, we examined whether minoxidil affects blood vessel formation
by ASCs using an
in vitro
tube formation assay. Indeed, minoxidil dose-dependently increased
the number of nascent tubes after 12–16 h (Figure 2E) and the expression level of endothelial cell
markers including tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE1), vascular
endothelial growth factor receptor 1 (VEGFR1), VEGFR2 and endothelin receptor type B (EDNRB)
(Figure 2F). Collectively, these results suggest that minoxidil may promote hair growth by enhancing
ASC migration and ASC-dependent angiogenesis.
Int. J. Mol. Sci. 2018,19, 691 4 of 15
Int. J. Mol. Sci. 2018, 19, x 4 of 15
Figure 2. Minoxidil promotes ASC migration and tube formation but not proliferation. (A,B) No effect
of minoxidil on proliferation of ASCs. (C,D) Minoxidil enhances ASC migration as evidenced by both
scratch migration assay (C) and transwell migration assay (D); (E) Tube formation assay showing
enhanced formation by minoxidil-treated ASCs. (F) Relative mRNA expression levels of endothelial
cell markers including TIE1, VEGFR1, VEGFR2, and EDNRB genes were increased in tubes from
minoxidil-treated ASCs. * p < 0.05, ** p < 0.01, *** p < 0.001. Three independent experiments were
conducted per data point. All error bars indicate SEM.
2.3. CXCL1, ECGF, and PDGF-C Induce Hair Growth
It has been reported that growth factors secreted by ASCs, such as VEGF, fibroblast growth
factor-1 (FGF1), bFGF, and PDGF-A, regulate hair follicular stem cell activity and induce the anagen
phase of the hair cycle in vivo [14]. We speculated that minoxidil may promote hair growth indirectly
by enhancing growth factor release from ASCs. To explore this possibility, we compared expression
patterns between untreated naïve and minoxidil-treated ASCs by qPCR array. Minoxidil upregulated
Figure 2.
Minoxidil promotes ASC migration and tube formation but not proliferation. (
A
,
B
) No effect
of minoxidil on proliferation of ASCs. (
C
,
D
) Minoxidil enhances ASC migration as evidenced by both
scratch migration assay (
C
) and transwell migration assay (
D
); (
E
) Tube formation assay showing
enhanced formation by minoxidil-treated ASCs. (
F
) Relative mRNA expression levels of endothelial
cell markers including TIE1, VEGFR1, VEGFR2, and EDNRB genes were increased in tubes from
minoxidil-treated ASCs. * p< 0.05, ** p< 0.01, *** p< 0.001. Three independent experiments were
conducted per data point. All error bars indicate SEM.
2.3. CXCL1, ECGF, and PDGF-C Induce Hair Growth
It has been reported that growth factors secreted by ASCs, such as VEGF, fibroblast growth
factor-1 (FGF1), bFGF, and PDGF-A, regulate hair follicular stem cell activity and induce the anagen
phase of the hair cycle
in vivo
[
14
]. We speculated that minoxidil may promote hair growth indirectly
by enhancing growth factor release from ASCs. To explore this possibility, we compared expression
patterns between untreated naïve and minoxidil-treated ASCs by qPCR array. Minoxidil upregulated
the expression of PD-ECGF over six-fold when compared to untreated ASCs (Figure S2), and this result
was confirmed (Figure 3A). Moreover, minoxidil also upregulated the expression levels of CXCL1 and
PDGF-C (Figure 3A).
Int. J. Mol. Sci. 2018,19, 691 5 of 15
Int. J. Mol. Sci. 2018, 19, x 5 of 15
the expression of PD-ECGF over six-fold when compared to untreated ASCs (Figure S2), and this
result was confirmed (Figure 3A). Moreover, minoxidil also upregulated the expression levels of
CXCL1 and PDGF-C (Figure 3A).
Figure 3. Minoxidil upregulates expression of hair-growth promoting factors CXCL1, PD-ECGF, and
PDGF-C in ASCs. (A) Relative mRNA expression levels of growth factors including CXCL1, PD-ECGF,
and PDGF-C were measured in minoxidil-treated ASCs; (BD) Human recombinant CXCL1, PD-
ECGF, and PDGF-C proteins each facilitated hair growth and increased the number of mature hair
follicle after injection into the dorsal skin of shaved mice as revealed by hair weight at 2 weeks post-
injection. n = 6–8 mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001. (E) CXCL1, PD-ECGF, and PDGF-
C proteins each facilitate mouse vibrissal hair follicle growth ex vivo. ** p < 0.01. n = 10 samples per
treatment group. All error bars indicate SEM.
Figure 3.
Minoxidil upregulates expression of hair-growth promoting factors CXCL1, PD-ECGF,
and PDGF-C in ASCs. (
A
) Relative mRNA expression levels of growth factors including CXCL1,
PD-ECGF, and PDGF-C were measured in minoxidil-treated ASCs; (
B
D
) Human recombinant
CXCL1, PD-ECGF, and PDGF-C proteins each facilitated hair growth and increased the number
of mature hair follicle after injection into the dorsal skin of shaved mice as revealed by hair weight
at 2 weeks post-injection. n= 6–8 mice per group. * p< 0.05, ** p< 0.01, *** p< 0.001. (
E
) CXCL1,
PD-ECGF, and PDGF-C proteins each facilitate mouse vibrissal hair follicle growth ex vivo. ** p< 0.01.
n= 10 samples per treatment group. All error bars indicate SEM.
To investigate whether CXCL1, PD-ECGF, and PDGF-C can induce the anagen phase of the
hair cycle
in vivo
, we injected recombinant human CXCL1, PD-ECGF, or PDGF-C protein into the
subcutaneous dermis of shaved mice. All three factors significantly induced the anagen phase of
the hair cycle
in vivo
(Figure 3B,C) and increased the number of mature hair follicle (Figure 3D),
suggesting that minoxidil may induce anagen by triggering CXCL1, PD-ECGF, or PDGF-C release
from ASCs. Moreover, treatment with CXCL1 or PDGF-C, but not PD-ECGF, also increased the length
Int. J. Mol. Sci. 2018,19, 691 6 of 15
of isolated mouse vibrissal hair follicles in organ culture (Figure 3E). These results strongly suggest
that minoxidil promotes hair growth through growth factor release from ASCs.
2.4. Minoxidil Regulates Expression of PD-ECGF and PDGF-C in ASCs through the ERK Pathway
It has been reported that the MAPK pathway regulates expression of growth factors in ASCs,
including vascular endothelial growth factor A (VEGFA) and FGF1 [
5
,
8
]. Therefore, we examined
whether minoxidil regulates the expression of CXCL1, PD-ECGF, or PDGF-C through the MAPK
pathway. Indeed, minoxidil dose-dependently upregulated phospho-ERK expression, a response
suppressed by the specific mitogen-activated protein kinase kinase (MEK) inhibitor PD98059
(Figure 4A). Further, PD98059 reversed minoxidil-induced upregulation of PD-ECGF and PDGF-C
in ASCs (Figure 4B). Alternatively, minoxidil-induced upregulation of CXCL1 was not affected by
PD98059 (Figure 4B), suggesting that minoxidil upregulates growth factor expression in ASCs through
multiple pathways, including the MAPK pathway.
Figure 4.
Minoxidil upregulates expression of PD-ECGF and PDGF-C through the ERK pathway in
ASCs. (
A
) The MEK inhibitor PD98059 reversed minoxidil-induced ERK phosphorylation; (
B
) PD98059
also suppressed minoxidil-induced upregulation of PD-ECGF and PDGF-C expression by ASCs.
Three independent experiments were carried out per data point. * p< 0.05, ** p< 0.01,
*** p< 0.001
.
All the error bars indicate SEM.
2.5. ASC Growth Factors CXCL1, PD-ECGF, and PDGF-C Induce DP Cell Proliferation
It has been reported that minoxidil stimulates the growth of human hairs by prolonging anagen
through proliferative and anti-apoptotic effects on DP cells [
13
,
15
]. We therefore directly examined
whether CXCL1, PD-ECGF, or PDGF-C increase DP cell proliferation, and indeed, all three factors when
Int. J. Mol. Sci. 2018,19, 691 7 of 15
applied separately dose-dependently increased cultured DP cell proliferation (Figure 5A). To confirm
this observation is specific to DP cells, we examined whether CXCL1, PD-ECGF, or PDGF-C increase
the proliferation of human dermal fibroblast cells. All three proteins did not induce the proliferation of
fibroblast contrary to increase in DP cells (Figure S3). Further, all three factors enhanced the number of
DP cells in S-phase as evidenced by 5-bromo-2
0
-deoxyuridine (BrdU) labeling. Moreover, treatment of
CXCL1, PD-ECGF, or PDGF-C in DP cells also dose-dependently increased the percentage of BrdU
+
cells, which is proliferation index (Figure 5B,C). These results further suggest that minoxidil may
enhance DP proliferation indirectly by inducing release of growth factors, such as CXCL1, PD-ECGF,
and PDGF-C from ASC.
Int. J. Mol. Sci. 2018, 19, x 7 of 15
2.5. ASC Growth Factors CXCL1, PD-ECGF, and PDGF-C Induce DP Cell Proliferation
It has been reported that minoxidil stimulates the growth of human hairs by prolonging anagen
through proliferative and anti-apoptotic effects on DP cells [13,15]. We therefore directly examined
whether CXCL1, PD-ECGF, or PDGF-C increase DP cell proliferation, and indeed, all three factors
when applied separately dose-dependently increased cultured DP cell proliferation (Figure 5A). To
confirm this observation is specific to DP cells, we examined whether CXCL1, PD-ECGF, or PDGF-C
increase the proliferation of human dermal fibroblast cells. All three proteins did not induce the
proliferation of fibroblast contrary to increase in DP cells (Figure S3). Further, all three factors
enhanced the number of DP cells in S-phase as evidenced by 5-bromo-2-deoxyuridine (BrdU)
labeling. Moreover, treatment of CXCL1, PD-ECGF, or PDGF-C in DP cells also dose-dependently
increased the percentage of BrdU+ cells, which is proliferation index (Figure 5B,C). These results
further suggest that minoxidil may enhance DP proliferation indirectly by inducing release of growth
factors, such as CXCL1, PD-ECGF, and PDGF-C from ASC.
Figure 5. CXCL1, PD-ECGF, and PDGF-C induce proliferation of DP cells. (A) Cell growth was
measured after treatment of CXCL1, PD-ECGF, or PDGF-C protein in DP cells for 3 days. Three
independent experiments were conducted. * p < 0.05. (B,C) Proliferation index (% of BrdU+ cells; green)
after treatment of CXCL1, PD-ECGF, or PDGF-C protein in DP cells. Three independent experiments
were carried out per data point. ** p < 0.01, *** p < 0.001. All error bars indicate SEM.
2.6. Application of CXCL1, PD-ECGF, or PDGF-C Acts Synergistically with Minoxidil to Induce Hair
Growth
Our results suggest that upregulation of CXCL1, PD-ECGF, or PDGF-C by minoxidil stimulates
DP cell proliferation, resulting in hair growth. Minoxidil has been widely used to treat androgenetic
alopecia. Therefore, to investigate whether application of CXCL1, PD-ECGF, or PDGF-C acts
synergistically with 2% minoxidil to induce the anagen phase of the hair cycle, we compared hair
growth on the dorsal skin among shaved mice that were treated with 2% minoxidil alone or minoxidil
plus either CXCL1, PD-ECGF, or PDGF-C for 14 days. Co-administration of each protein increased
Figure 5.
CXCL1, PD-ECGF, and PDGF-C induce proliferation of DP cells. (
A
) Cell growth
was measured after treatment of CXCL1, PD-ECGF, or PDGF-C protein in DP cells for 3 days.
Three independent experiments were conducted. * p< 0.05. (
B
,
C
) Proliferation index (% of BrdU
+
cells; green) after treatment of CXCL1, PD-ECGF, or PDGF-C protein in DP cells. Three independent
experiments were carried out per data point. ** p< 0.01, *** p< 0.001. All error bars indicate SEM.
2.6. Application of CXCL1, PD-ECGF, or PDGF-C Acts Synergistically with Minoxidil to Induce Hair Growth
Our results suggest that upregulation of CXCL1, PD-ECGF, or PDGF-C by minoxidil stimulates
DP cell proliferation, resulting in hair growth. Minoxidil has been widely used to treat androgenetic
alopecia. Therefore, to investigate whether application of CXCL1, PD-ECGF, or PDGF-C acts
synergistically with 2% minoxidil to induce the anagen phase of the hair cycle, we compared hair
growth on the dorsal skin among shaved mice that were treated with 2% minoxidil alone or minoxidil
plus either CXCL1, PD-ECGF, or PDGF-C for 14 days. Co-administration of each protein increased
hair weight when compared to 2% minoxidil alone (Figure 6A,B), suggesting that the addition of these
proteins may enhance the efficacy of minoxidil.
Int. J. Mol. Sci. 2018,19, 691 8 of 15
Int. J. Mol. Sci. 2018, 19, x 8 of 15
hair weight when compared to 2% minoxidil alone (Figure 6A,B), suggesting that the addition of
these proteins may enhance the efficacy of minoxidil.
Figure 6. Application of CXCL1, PD-ECGF, or PDGF-C enhances the hair growth-promoting effect of
minoxidil (2%) when co-applied on the dorsal skin of shaved mice. (A) Photograph was taken and
hair weight was measured 14 days later for CXCL1 application. ** p < 0.01. n = 6 mice per group; (B)
Photograph was taken and hair weight was measured 14 days later for PD-ECGF or PDGF-C
application. * p < 0.05, ** p < 0.01. n.s. indicates not significant. n = 5 or 6 mice per group. All of the
error bars indicate SEM.
3. Discussion
Minoxidil promotes hair growth directly by stimulating DP and epithelial cells, but previous
studies provided little or no evidence for indirect hair growth-promoting effects through the
stimulation of ASCs. Therefore, we investigated whether minoxidil stimulates ASCs and enhances
hair growth through growth factor release. We first demonstrated that subcutaneous injection of
minoxidil-treated ASCs accelerated telogen-to-anagen transition in C3H/HeJ mice and increased hair
weight after two weeks. Although minoxidil did not alter the proliferation of ASCs, it did increase
migration, tube formation, and secretion of growth factors, notably CXCL1, PD-ECGF, or PDGF-C
(the later two through ERK activation). Further, each of these growth factors enhanced DP cell
proliferation in vitro and anagen induction in mice, while CXCL1 and PDGF-C also increased the
length of isolated mouse vibrissal hair follicles in organ culture. Moreover, these hair growth-
promoting effects were synergistic with 2% minoxidil. Collectively, these results indicate that
minoxidil stimulates hair growth in part through stimulation of CXCL1, PD-ECGF, or PDGF-C
release from ASCs (Figure 7).
Figure 6.
Application of CXCL1, PD-ECGF, or PDGF-C enhances the hair growth-promoting effect of
minoxidil (2%) when co-applied on the dorsal skin of shaved mice. (
A
) Photograph was taken and
hair weight was measured 14 days later for CXCL1 application. ** p< 0.01. n= 6 mice per group;
(
B
) Photograph was taken and hair weight was measured 14 days later for PD-ECGF or PDGF-C
application. * p< 0.05, ** p< 0.01. n.s. indicates not significant. n= 5 or 6 mice per group. All of the
error bars indicate SEM.
3. Discussion
Minoxidil promotes hair growth directly by stimulating DP and epithelial cells, but previous
studies provided little or no evidence for indirect hair growth-promoting effects through the stimulation
of ASCs. Therefore, we investigated whether minoxidil stimulates ASCs and enhances hair growth
through growth factor release. We first demonstrated that subcutaneous injection of minoxidil-treated
ASCs accelerated telogen-to-anagen transition in C3H/HeJ mice and increased hair weight after
two weeks. Although minoxidil did not alter the proliferation of ASCs, it did increase migration,
tube formation, and secretion of growth factors, notably CXCL1, PD-ECGF, or PDGF-C (the later two
through ERK activation). Further, each of these growth factors enhanced DP cell proliferation
in vitro
and anagen induction in mice, while CXCL1 and PDGF-C also increased the length of isolated mouse
vibrissal hair follicles in organ culture. Moreover, these hair growth-promoting effects were synergistic
with 2% minoxidil. Collectively, these results indicate that minoxidil stimulates hair growth in part
through stimulation of CXCL1, PD-ECGF, or PDGF-C release from ASCs (Figure 7).
Int. J. Mol. Sci. 2018,19, 691 9 of 15
Int. J. Mol. Sci. 2018, 19, x 9 of 15
Figure 7. Minoxidil promotes the proliferation of DP cells and hair growth through stimulation of
growth factor release from adipose-derived stem cells. Minoxidil stimulates the release of growth
factors including PD-ECGF, PDGF-C, and CXCL1 from ASCs via ERK and the other pathway,
respectively, thereby promoting of DP cells proliferation and hair growth.
Chemokine (C-X-C motif) ligand 1 is a secreted growth factor that signals through the G-protein
coupled CXC receptor 2, while CXCL1 is known to act as a potent chemoattractant for neutrophils
during inflammation [17–19]. However, it has not been linked to hair regeneration. PD-ECGF
promotes angiogenesis in vivo and stimulates the in vitro growth of multiple endothelial cell types
[20–22]. It has a highly restricted target cell specificity, acting only on endothelial cells. Of interest,
PD-ECGF was one of the top three genes that were upregulated in ASCs by minoxidil according to
qPCR arrays. Moreover, our work in vitro and in vivo revealed that minoxidil-induced PD-ECGF
release from ASCs, resulting in DP proliferation and hair growth. On the contrary, it has been well-
known that PDGF signaling in the dermis and in dermal condensates is dispensable for hair follicle
induction and formation [23]. For example, PDGF-A secreted from ASCs regulates follicular stem cell
activity [2]. PDGF-A and -B are involved in the induction and maintenance of the anagen phase in
Figure 7.
Minoxidil promotes the proliferation of DP cells and hair growth through stimulation of
growth factor release from adipose-derived stem cells. Minoxidil stimulates the release of growth factors
including PD-ECGF, PDGF-C, and CXCL1 from ASCs via ERK and the other pathway, respectively,
thereby promoting of DP cells proliferation and hair growth.
Chemokine (C-X-C motif) ligand 1 is a secreted growth factor that signals through the G-protein
coupled CXC receptor 2, while CXCL1 is known to act as a potent chemoattractant for neutrophils
during inflammation [
17
19
]. However, it has not been linked to hair regeneration. PD-ECGF promotes
angiogenesis
in vivo
and stimulates the
in vitro
growth of multiple endothelial cell types [
20
22
]. It has
a highly restricted target cell specificity, acting only on endothelial cells. Of interest, PD-ECGF was
one of the top three genes that were upregulated in ASCs by minoxidil according to qPCR arrays.
Moreover, our work
in vitro
and
in vivo
revealed that minoxidil-induced PD-ECGF release from
ASCs, resulting in DP proliferation and hair growth. On the contrary, it has been well-known that
PDGF signaling in the dermis and in dermal condensates is dispensable for hair follicle induction and
formation [
23
]. For example, PDGF-A secreted from ASCs regulates follicular stem cell activity [
2
].
Int. J. Mol. Sci. 2018,19, 691 10 of 15
PDGF-A and -B are involved in the induction and maintenance of the anagen phase in the mouse
hair cycle [
24
]. Our work revealed that PDGF-C also functions in hair growth by upregulating DP
proliferation. Although these results might be expected because many growth factors are already
known to promote hair cycling, upregulation by minoxidil is meaningful for further usability of this
agent to treat androgenetic alopecia. Indeed, the co-application of PDGF-C, CXCL1, or PD-ECGF with
2% minoxidil synergistically increased hair growth (Figure 6).
It has been reported that PDGF-D treatment induces growth factor secretion from ASCs via
phospho-activation of ERK [
5
,
8
]. Meldrum’s group also reported that preconditioning (i.e., by hypoxia
or transforming growth factor) induced the secretion of numerous growth factors from mesenchymal
stem cells through the MAPK pathways [
25
28
]. Similarly, our work revealed that minoxidil induced
PD-ECGF and PDGF-C release from ASCs via ERK phosphorylation (Figure 4). Therefore, it is
reasonable to assume that growth factor secretion by ASCs is primarily regulated by the MAPK
pathway. However, it appears that CXCL1 is induced via an ERK-independent pathway, suggesting
that minoxidil may activate multiple signaling pathways in ASCs. Further investigation is required to
identify the pathway mediating minoxidil-induced CXCL1 release.
Minoxidil reportedly increased the proliferation of DP cells
in vitro
. For example, minoxidil
promoted the survival of human DP cells by activating both ERK and Akt pathways, and prevented cell
death by increasing the ratio of Bcl-2/Bax [
15
]. In addition, minoxidil plus all-trans retinoic acid (ATRA)
additively promoted hair growth in human hair follicle culture [
29
]. A combination of minoxidil with
ATRA elevated phosphorylated ERK, phosphorylated Akt in DP cells and keratinocytes [
29
]. However,
minoxidil did not increase the proliferation in ASCs, which indicates that proliferative effect of
minoxidil is cell type dependent.
ACSs are considered an important component of other stem cell niches. Indeed, it has been shown
that adipocyte lineage cells are part of the skin stem cell niche that drives hair cycling, and it was
suggested that PDGF-A expression by immature adipocytes is a key regulator of follicular stem cell
activity [
2
]. Although we did not investigate the effects of minoxidil or ASC-released growth factors
on DP stem cells, ASC-released growth factors (specifically CXCL1, PD-ECGF and PDGF-C) induced
DP cell proliferation, consistent with a pivotal role in the regulation of DP stem cells. To determine
whether the effect of minoxidil on specific to the ASCs, minoxidil didn’t induce cell growth either
migration of dermal fibroblast cells (Figure S1).
We previously reported that preconditioned ASCs by growth factor, such as PDGF-D, enhance
hair generative potential in tellogen to anagen induction model [
5
]. The purpose of this model is to
induce anagen phase more quickly after we injected ASCs by paracrine effect. Anagen hair induction
was not limited to the injection site. Instead, darkening of the skin or hair appeared across all areas
of the back. We showed increased hair weight in ASC
MXD
-treated mice, which indicates the increase
of hair length as well as hair number. The measurement of hair weight as an evaluation for hair
growth is not enough to conclude increased telogen-to-anagen induction. Therefore, we examined
hair follicle and proliferating DP cells in sectioned skin. The skin section of ASC
MXD
-treated mice
showed higher number of mature hair follicle compared to vehicle- or ASC
Ctrl
-treated mice (Figure 1C).
In addition, most hair follicles of ASC
MXD
-treated mice showed DP with Ki67
+
cells contrary to
vehicle- or ASC
Ctrl
-treated mice (Figure 1D). This result suggests that minoxidil can promote telogen
to anagen induction, thereby promoting hair growth. Although the measurement of hair weight is not
enough to examine the growth of hair follicle and DP proliferation, this method is easy to monitor hair
growth quickly.
In summary, subcutaneous injection of minoxidil-treated ASCs accelerated the telogen-to-anagen
transition in mice, and direct minoxidil treatment increased migration, tube formation, and growth
factor secretion by ASCs. The most strongly upregulated growth factors, CXCL1, PD-ECGF and
PDGF-C, individually enhanced anagen induction in mice, while CXCL1 and PDGF-C also increased
the length of isolated mouse vibrissal hair follicles in organ culture. Therefore, minoxidil can be used
as a novel ASC preconditioning agent for hair regeneration.
Int. J. Mol. Sci. 2018,19, 691 11 of 15
4. Materials and Methods
4.1. Cell Culture
Human adipocyte-derived stem cells (ASCs) were isolated via liposuction of subcutaneous
fat as described previously [
30
,
31
]. Briefly, fat was washed with phosphate-buffered saline
(
PBS),
added 0.075% collagenase and incubated for 45 min at 37
C with gently shaking. The pellet after
centrifugation was filtered with 100
µ
m nylon mesh, gathered again after centrifugation. Then,
cells were cultured with essential medium, including
α
-minimum essential media (MEM) (Hyclone,
Logan, UT, USA), 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) and 1% anti-antibiotics (Gibco)
until passage 3. Then, medium was changed to minimum essential medium including
α
-MEM,
10% fetal bovine serum, and 1% penicillin/streptomycin (Gibco) at four passages. ASCs were
used at passages 5–7 for all of the experiments. Characterization of ASCs was performed using
flow cytometry. ASCs were positive for CD44, CD73, CD90, CD105, human leukocyte antigen-I
(HLA-I), and podocalyxin (PODXL), but were negative for hematopoietic markers such as CD34 and
CD45
[32,33]
. Multipotent differentiation potential was examined as described previously [
34
,
35
],
and ASCs could be differentiated into adipocytes, osteocytes, and chondrocytes. We purchased human
DP cells from PromoCell (#C-12071, PromoCell, Logan, Heidelberg, Germany). Human DP cells were
cultured in Follicle DP Cell Medium (C-26505; PromoCell) with SupplementMix (C-39625; PromoCell)
and 0.1% anti-antibiotics (Gibco). DP cells were used at passages 3, 4. We also purchased human
dermal fibroblast cells from PromoCell (#C-12302). Dermal fibroblasts were cultured in dulbecco
modified eagle medium (DMEM)/high glucose (Hyclone) with 10% fetal bovine serum and 1%
penicillin/streptomycin, and were used at passages 8–10. All of the cells were maintained at 37
C in a
humidified 5% CO2incubator (Theremo Fisher scientific, Waltham, MA, USA).
4.2. Cell Growth Assay
For measuring the effects of minoxidil on ASC proliferation, cells were seeded in 6-well plates at
1×104cells/well
, treated with minoxidil (0.1–100
µ
M), and incubated for two days in an InCu-saFe
CO
2
incubator (Panasonic, Kadoma, Osaka, Japan). Live cell number was determined using the
IncuCyte zoom2014A live cell analysis system (Panasonic, Kadoma, Osaka, Japan) [
5
]. Alternatively,
ASCs were seeded in 12-well plates at 5000 cells/well, treated with minoxidil (20, 50, or 100
µ
M),
and incubated for seven days. Cells were then trypsinized, stained with Trypan Blue (Sigma-Aldrich,
St. Louis, MO, USA), and counted each day using a hemocytometer hemocytometer. To assess
the effects of growth factors secreted by ASCs on DP cell proliferation, 1
×
10
4
DP cells were
seeded in 12-well plates and treated with the indicated synthesized peptides for up to three days.
Cells were trypsinized and stained with Trypan Blue. Viable cell number was counted each day using
a hemocytometer.
4.3. Scratch Migration Assay
Cells were seeded into 6-well plates and cultured to confluence. A sterile 1 mL pipette tip
was used to scratch the cell monolayer. Cultures were then washed with PBS to removed deplated
cells and cultured with minoxidil in serum-free medium for four days. Migration of cells into the
scratched area (wound closure) was visualized using a ZEISS Observer D1 microscope (Carl ZEISS,
Oberkochen, Germany). Multiple images were acquired per well and average cell counts within the
wound monitored over 4 days.
4.4. Cell Migration Assay Using Transwell
ASCs (1
×
10
4
/well) were suspended in serum-free medium and seeded on the upper side of
transwell membrane plates (BD falcon, BD Biosciences, San Jose, CA, USA). After 2 h, minoxidil
in serum-free medium were introduced into the lower chambers. Cultures were incubated for
two days to allow transwell migration. Inserts were then removed and the upper surface was cleaned
Int. J. Mol. Sci. 2018,19, 691 12 of 15
of non-migrating cells using cotton swabs and washed with PBS. The inserts were stained with
0.1% formalin/10% crystal-violet solution (Sigma-Aldrich) and cell number analyzed under a ZEISS
Observer D1 microscope. Multiple images (15–20) per insert were acquired, and average cell counts
were calculated.
4.5. Tube Formation Assay Using Matrigel
Twelve-well plates were coated with matrigel (BD matrigel matrix, BD Biosciences) and were dried
for 2 h at 37
C. ASCs in endothelial cell basal medium-2 (EBM-2, LONZA, Walkersville, MD, USA)
plus minoxidil were plated on matrigel-coated wells and incubated for 12–16 h at 37
C. The number of
tubes was analyzed under a ZEISS Observer D1 microscope [
3
], and the expression level of endothelial
cell markers was analyzed by qPCR.
4.6. RNA Extraction and Quantitative RT-PCR
Total RNA was extracted from ASCs using Trizol reagent (Invitrogen, Carlsbad, NY, USA) and
subjected to cDNA synthesis using oligodT and the HelixCript
Thermo Reverse Transcription System
(Nanohelix, Madison, WI, USA), according to the manufacturer’s instructions. BrightGreen qPCR
master mix-ROX (ABM, New York, NY, USA) was used for PCR reactions.
4.7. Western Blot
For western blot of phospho-ERK, minoxidil and PD98059 (Sigma-Aldrich) was treated for
30 min–1h. Cells were lysed with protein extraction solution (PRO-PREP
TM
; iNtRON, Seoul, Korea)
containing phosphatase inhibitor (Na
3
VO
4
; Roche, Pleasanton, CA, USA). Western blot analysis was
performed as described previously [
8
] using the following primary antibodies: rabbit anti-p42/44
(1:1500; Cell Signaling Technology, Danvers, MA, USA), mouse anti-phospho-p42/44 (1:1500; Cell
Signaling Technology), and mouse anti-
α
-tubulin (1:2000; Santa Cruz Biotechnology,
Dallas, TX, USA
).
Western blot images were obtained using ImageQuant LAS 4000 (GE Healthcare Life Science,
Pittsburgh, PA, USA).
4.8. Animal Experiment
Mice were maintained and anesthetized according to a protocol that was approved by the
US Pharmacopoeia and the Institutional Animal Care and Use Committee of Yonsei University
(IACUC120002, 17 July 2015). The dorsal area of 6.5-week-old C3H/HeN mice in the telogen stage of
the hair cycle was shaved with a clipper and electric shaver, with special care taken to avoid damaging
the bare skin. Naïve ASCs or minoxidil-treated ASCs were only once injected into the dorsal skin
of shaved mice. The growth factors (100 ng/mL per one day) were injected into the dorsal skin of
shaved mice every day for 12 days. CXCL1 (R and D Systems, Minneapolis, MN, USA), PD-ECGF
(R and D Systems) and PDGF-C (PeproTech, Rocky Hill, NJ, USA) proteins were purchased from
each companies. For application with 2% minoxidil, CXCL1, PD-ECGF or PDGF-C (1
µ
g/mL) with
2% minoxidil (Rogaine; Johnson and Johnson Healthcare Products, Skillman, NJ, USA) were applied
on the dorsal skin of shaved mice. Any darkening of the skin (indicative of hair cycle induction)
was carefully monitored by photography. After 14 days, the dorsal hair was shaved and weighed to
estimate growth rate [3,5].
4.9. Vibrissae Follicle Organ Culture
For organ culture of vibrissae hair follicle, the vibrissae hair follicle was cut from C3H/HeN
mice, washed with PBS and cultured in defined medium (Williams E medium supplemented with
2 mM L-glutamine, 10
µ
g/mL insulin, 10 ng/mL hydrocortisone, 100 U/mL penicillin, and 100
µ
g/mL
streptomycin, without serum) including CXCL1 (5 ng), PD-ECGF (20 ng) and PDGF-C (20 ng) for
three days.
Int. J. Mol. Sci. 2018,19, 691 13 of 15
4.10. BrdU Labeling
For BrdU labeling assay, 4
×
10
4
cells were seeded in six well plates and incubated for 3 days
after adding three proteins. BrdU (Sigma-Aldrich) was added in cell culture media to a final
concentration of 200 mM and incubated for 4 h at 37
C with 5% CO
2
. The cells were fixed
with 4% paraformaldehyde, incubated with mouse anti-BrdU (1:500) (Abcam, Iowa City, IA, USA)
overnight at 4
C, and then incubated with secondary antibodies, Alexa Fluor 488 goat anti-mouse IgG
(Invitrogen), for 1 h at room temperature with 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich).
Immunofluorescence staining was imaged using ZEISS LSM700 confocal microscope (Carl ZEISS,
Oberkochen, Germany). For calculation of the percentage of BrdU
+
cells, we counted the number of
BrdU
+
cells and DAPI
+
cells (all cells) in every same size pictures using Adobe photoshop CS6 extended
program (Yonsei University, Seoul, Korea), and calculated the percentage of BrdU+cells.
4.11. Hematoxylin/Eosin and Immunofluorescence Staining
For Hematoxylin and eosin staining, paraffin sections were dewaxed using xylene for 30 min,
hydrated in 100%-, 90%-, 80%- and 70% EtOH and was dipped into Mayer’s hematoxylin
(Sigma-Aldrich) for 8 min, and then rinsed in water for 1 min. Slide was dipped again into eosin Y
(Sigma-Aldrich) for 80 s, dehydrated with 70%-, 80%-, 90%- and 100% EtOH, washed with fresh xylene
for 30, dried, and mounted with mounting medium. Immunofluorescence staining was performed
using standard protocols. Briefly, paraffin sections were dewaxed using xylene for 30 min, hydrated in
100%-, 90%-, 80%- and 70% EtOH and antigen retrieval was performed by boiling using microwave
in antigen retrieval buffer (Dako, Carpinteria, CA, USA) for 2 min. The sections were treated with
rabbit Ki67 antibody (1:300) (abcam) overnight at 4
C, and were then incubated with secondary
antibodies, Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen), for 1 h at room temperature with
4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). Immunofluorescence staining was imaged
using ZEISS LSM700 confocal microscope.
4.12. Statistical Analysis
All of the experiments were performed more than three times with independent cultures. Data are
presented as mean
±
standard error (SEM). Means were compared by Student’s t-test. For all statistical
tests, a 0.05 level of confidence was accepted as statistically significant.
Supplementary Materials: Supplementary materials can be found at www.mdpi.com/xxx/s1.
Acknowledgments:
This study was supported by a grant from the National Research Foundation
(NRF-2016R1D1A1B03932050), and a grant from Small and Medium Business Administration (S2371105)
by the Korean government. Nahyun Choi was also supported by the National Research Foundation
(NRF-2017R1A6A3A11035599) funded by the Korean government.
Author Contributions:
Nahyun Choi and Jong-Hyuk Sung designed the experiments; Nahyun Choi developed
the methodology and performed the experiments; Nahyun Choi, Jong-Hyuk Sung, Soyoung Shin and
Sun U. Song
analyzed the data; Nahyun Choi and Jong-Hyuk Sung wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
ASC Adipose-derived stem cell
CXCL1 chemokine (C-X-C motif) ligand 1
PD-ECGF platelet-derived endothelial cell growth factor
PDGF-C platelet-derived growth factor-C
Int. J. Mol. Sci. 2018,19, 691 14 of 15
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Sung, J.H.; An, H.S.; Jeong, J.H.; Shin, S.; Song, S.Y. Megestrol Acetate Increases the Proliferation, Migration,
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... Specifically, the expression of genes related to the Wingless-related integration site (WNT) signaling pathway, such as WNT proteins and their receptors, plays a vital role in regulating hair growth [18]. These genes influence the expression of various growth factors like insulin-like growth factor 1 (IGF-1), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF), which are essential for initiating and maintaining the hair cycle's anagen (growth) phase [19,20]. Conversely, genes associated with the catagen (regression) phase of the hair cycle, such as transforming growth factor beta (TGF-β), IL-1, interleukin-2 (IL-2), interleukin-10 (IL-10), prostaglandin E2 (PEG-2) and TNF-α, are upregulated, leading to hair follicle apoptosis and regression [8,21]. ...
... Contemporary research in hair growth and dandruff control employs diverse materials and methodologies. Regenerative approaches involve stem cell therapy [30] and the use of topicals enriched with growth factors and peptides, such as minoxidil [20], targeting hair follicle regeneration [30]. Genetic and molecular analyses aim to identify precise therapeutic targets, while convenient treatments like laser therapy [31] and microneedling [32] offer promising options. ...
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Background Probiotics are intellectually rewarding for the discovery of their potential as a source of functional food. Investigating the economic and beauty sector dynamics, this study conducted a comprehensive review of scholarly articles to evaluate the capacity of probiotics to promote hair growth and manage dandruff. Methods We used the PRISMA 2020 with Embase, Pubmed, ClinicalTrials.gov, Scopus, and ICTRP databases to investigate studies till May 2023. Meta-analyses utilizing the random effects model were used with odds ratios (OR) and standardized mean differences (SMD). Result Meta-analysis comprised eight randomized clinical trials and preclinical studies. Hair growth analysis found a non-significant improvement in hair count (SMD = 0.32, 95 % CI -0.10 to 0.75) and a significant effect on thickness (SMD = 0.92, 95 % CI 0.47 to 1.36). In preclinical studies, probiotics significantly induced hair follicle count (SMD = 3.24, 95 % CI 0.65 to 5.82) and skin thickness (SMD = 2.32, 95 % CI 0.47 to 4.17). VEGF levels increased significantly (SMD = 2.97, 95 % CI 0.80 to 5.13), while IGF-1 showed a non-significant inducement (SMD = 0.53, 95 % CI -4.40 to 5.45). For dandruff control, two studies demonstrated non-significant improvement in adherent dandruff (OR = 1.31, 95 % CI 0.13–13.65) and a significant increase in free dandruff (OR = 5.39, 95 % CI 1.50–19.43). Hair follicle count, VEGF, IGF-1, and adherent dandruff parameters were recorded with high heterogeneity. For the systematic review, probiotics have shown potential in improving hair growth and controlling dandruff through modulation of the immune pathway and gut-hair axis. The Wnt/β-catenin pathway, IGF-1 pathway, and VEGF are key molecular pathways in regulating hair follicle growth and maintenance. Conclusions This review found significant aspects exemplified by the properties of probiotics related to promoting hair growth and anti-dandruff effect, which serve as a roadmap for further in-depth studies to make it into pilot scales.
... This unexpected finding paved the way for the development of topical formulations, heralding a new era in treating androgenetic alopecia (AGA). 2 The formulation of minoxidil, primarily as a topical solution or foam, revolutionized its application in dermatology. Understanding its mechanisms of action, including its vasodilatory properties, modulation of potassium channels and effects on hair follicle cycling has been instrumental in elucidating its diverse therapeutic roles beyond its initial indication. ...
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Minoxidil, originally developed as an antihypertensive medication, has evolved into a versatile therapeutic agent within dermatology, notably for its effectiveness in promoting hair growth. Despite its widespread use, understanding its precise mechanism of action remains a challenge. This paper addresses this gap by providing a comprehensive review of pharmacological properties, action mechanisms, clinical effectiveness and side effects associated with topical minoxidil. Furthermore, it highlights emerging trends and applications, including its role in treating androgenetic alopecia, its potential for alopecia areata management and its utilization in combination therapies. Additionally, this paper explores novel applications in scar treatment and wound healing. By synthesizing current knowledge and insights, this review adds clarity to the diverse applications of minoxidil, providing valuable guidance for clinicians and researchers aiming to optimize its therapeutic use in dermatology.
... Previous studies have reported that minoxidil promotes the proliferation of HFDPCs by activating the extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) signaling pathways. Furthermore, minoxidil prevents apoptosis in HFDPCs by increasing the ratio of B-cell lymphoma 2 (BCL-2) to BCL-2-associated X protein (Bax), both of which are key regulators of pro-and anti-apoptotic activities [25]. HFDPC cells, located at the base of the hair follicle, are crucial for hair follicle formation and the initiation of hair growth. ...
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The bioactive compounds in herbal extracts may provide effective hair loss treatments with fewer side effects compared to synthetic medicines. This study evaluated the effects of Buebang 3 CMU and Sanpatong rice bran extracts, macerated with dichloromethane or 95% ethanol, on hair growth promotion and hair loss prevention. Overall, Buebang 3 CMU extracts contained significantly higher levels of bioactive compounds, including γ-oryzanol, tocopherols, and various polyphenols such as phytic acid, ferulic acid, and chlorogenic acid, compared to Sanpatong extracts. Additionally, ethanolic extracts demonstrated greater bioactive content and antioxidant activities than those extracted with dichloromethane. These compounds enhanced the proliferation of human hair follicle dermal papilla cells (HFDPCs) by 124.28 ± 1.08% (p < 0.05) and modulated anti-inflammatory pathways by reducing nitrite production to 3.20 ± 0.36 µM (p < 0.05). Key hair growth signaling pathways, including Wnt/β-catenin (CTNNB1), Sonic Hedgehog (SHH, SMO, GLI1), and vascular endothelial growth factor (VEGF), were activated by approximately 1.5-fold to 2.5-fold compared to minoxidil. Also, in both human prostate cancer (DU-145) and HFDPC cells, the ethanolic Buebang 3 CMU extract (Et-BB3-CMU) suppressed SRD5A1, SRD5A2, and SRD5A3 expression—key pathways in hair loss—by 2-fold and 1.5-fold more than minoxidil and finasteride, respectively. These findings suggest that Et-BB3-CMU holds promise for promoting hair growth and preventing hair loss.
... To determine whether NIR LED can enhance hair growth in vivo, we irradiated NIR LED and White on the shaved back of the telogen phase in C57BL/6NCrlOri mice. Minoxidil (3%) was topically applied as a positive control [33]. We observed a color change from pink to gray skin, or the hair coat was larger on day 21 for the nNIR irradiated group compared with that of the control group. ...
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The study aimed to explore the impact of a novel near-infrared LED (nNIR) with an extended spectrum on skin enhancement and hair growth. Various LED sources, including White and nNIRs, were compared across multiple parameters: cytotoxicity, adenosine triphosphate (ATP) synthesis, reactive oxygen species (ROS) reduction, skin thickness, collagen synthesis, collagenase expression, and hair follicle growth. Experiments were conducted on human skin cells and animal models. Cytotoxicity, ATP synthesis, and ROS reduction were evaluated in human skin cells exposed to nNIRs and Whites. LED irradiation effects were also studied on a UV-induced photoaging mouse model, analyzing skin thickness, collagen synthesis, and collagenase expression. Hair growth promotion was examined as well. Results revealed both White and nNIR were non-cytotoxic to human skin cells. nNIR enhanced ATP and collagen synthesis while reducing ROS levels, outperforming the commonly used 2chip LEDs. In the UV-induced photoaging mouse model, nNIR irradiation led to reduced skin thickness, increased collagen synthesis, and lowered collagenase expression. Additionally, nNIR irradiation stimulated hair growth, augmented skin thickness, and increased hair follicle count. In conclusion, the study highlighted positive effects of White and nNIR irradiation on skin and hair growth. However, nNIR exhibited superior outcomes compared to White. Its advancements in ATP content, collagen synthesis, collagenase inhibition, and hair growth promotion imply increased ATP synthesis activity. These findings underscore nNIR therapy’s potential as an innovative and effective approach for enhancing skin and promoting hair growth.
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Introduction: Telogen effluvium (TE) is a common cause of diffuse, non-scarring hair shedding, particularly affecting women. Chronic telogen effluvium (CTE), although less common than the acute form, presents as gradual hair loss that persists for more than six months and can last for years. Current treatment options are limited, and while topical minoxidil is often used, there is insufficient evidence to fully support its effectiveness in CTE. This has led to the exploration of innovative treatment methods, such as Microinfusion of drugs into the skin (MMP®), a form of microneedling. Case Presentation: A 28-year-old female patient with a three-year history of hair loss underwent MMP® combined with 0.5% minoxidil on the right side of the scalp, targeting the frontal, parietal, and vertex areas. The treatment was administered over 3 monthly sessions, followed by the daily application of 5% minoxidil at home on both sides of the scalp. Six weeks after the final session, a trichoscopic reassessment using TrichoLAB® revealed the following results: In the frontal region, hair density increased slightly by 1 hair (from 206 to 207), while hair shaft thickness decreased by 2 µm (from 61 to 59 µm). In the parieto-vertex region, there was a more notable increase of 47 hairs (from 223 to 270), with a slight decrease in hair shaft thickness by 3 µm (from 67 to 64 µm). Conclusions: The patient reported a modest improvement in hair volume, supported by trichoscopic data, particularly in the parieto-vertex region. The combination of MMP® and minoxidil shows potential as a treatment for CTE, emphasizing the need for further research in larger, controlled trials to confirm its efficacy.
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Bicalutamide is a non-steroidal androgen receptor antagonist that exerts anti-androgenic effects on peripheral tissues. It has been recently of interest in female pattern hair loss and has led to the off-label use of this drug for the improvement of Sinclair grading in such patients. This review aims to discuss the pharmacological properties along with indications, contraindications, and safety profile of bicalutamide for its use in dermatological research.
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Objective This study was designed to evaluate the safety and efficacy of injections of concentrated growth factors (CGF) for hair growth promotion in androgenetic alopecia (AGA) patients. Methods A retrospective review of AGA patients treated with injections of CGF at our center from October 2021 to April 2023 was performed. A total of 3 injections were administered every 3–4 weeks apart, and evaluation were performed before the first injection and at 3 months, 6 months later. The outcomes were assessed by trichoscopy photomicrographs and the Global Aesthetic Improvement Scale (GAIS). Results At 3 months after the first injection, the hair density and hair growth ratio were significantly improved. Significant improvements were found in GAIS score by both patients and independent doctors and the hair growth promotion was sustained for 6 months after first treatment. Conclusions According to this tiny single‐arm trial, the use of CGF injection may help AGA by increasing terminal hair density and hair density. No severe topical or systemic adverse events occurred during the treatment.
Article
Autophagy is essential for regulating hair growth. Accordingly, we developed autophagy activator ICP5249 (pentasodium tetracarboxymethyl palmitoyl dipeptide) and investigated its potential role in hair growth. We evaluated its effect on hair growth using in vitro human dermal papilla cells (hDPCs) culture model, human hair follicles (hHFs) organ culture model, and telogenic mouse model. ICP5249 increased hDPCs proliferation and alkaline phosphatase (ALP) expression. It also increased microtubule-associated protein (MAP) light chain 3- II (LC3-II) expression and AMP-activated protein kinase α (AMPKα) and unc-51-like kinase 1 (ULK1) phosphorylation in hDPCs. ICP5249 extended the length of hHFs and increased LC3 II expression. Consistently, ICP5249 also significantly increased hair growth area, dermis thickness, and anagen and telogen ratio in telogenic mice. Furthermore, it upregulated Ki-67 and LC3-II expression and AMPKα phosphorylation on the mice's dorsal skin. To investigate whether AMPK regulates ICP5249-induced hair growth, following treatment with the compound C, AMPK inhibitor, the activity of ICP5249 was evaluated. The effects of ICP5249 on hair growth were assessed following pretreatment with the AMPK inhibitor compound C. The results showed that compound C suppressed ICP5249-mediated proliferation and hair inductivity in hDPCs. Additionally, compound C inhibited ICP5249-mediated hair growth area, dermis thickness, anagen and telogen ration, and LC3-II expression in mice, suggesting that ICP5249 promotes hair growth by modulating autophagy, with AMPKα playing a regulatory role in this process. Taken together, we demonstrate that ICP5249 has the potential as an ingredient for improving hair growth.
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Background and purpose Hair loss is a prevalent problem affecting millions of people worldwide, necessitating innovative and efficient regrowth approaches. Nanostructured lipid carriers (NLCs) have become a hopeful option for transporting bioactive substances to hair follicles because of their compatibility with the body and capability to improve drug absorption. Review approach Recently, surface modification techniques have been used to enhance hair regeneration by improving the customization of NLCs. These techniques involve applying polymers, incorporating targeting molecules, and modifying the surface charge. Key results The conversation focuses on how these techniques enhance stability, compatibility with the body, and precise delivery to hair follicles within NLCs. Moreover, it explains how surface-modified NLCs can improve the bioavailability of hair growth-promoting agents like minoxidil and finasteride. Furthermore, information on how surface-modified NLCs interact with hair follicles is given, uncovering their possible uses in treating hair loss conditions. Conclusion This review discusses the potential of altering the surface of NLCs to customize them for enhanced hair growth. It offers important information for upcoming studies on hair growth.
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Great interest remains in finding new and emerging therapies for the treatment of male and female pattern hair loss. The autologous fat grafting technique is >100 years old, with a recent and dramatic increase in clinical experience over the past 10–15 years. Recently, in 2001, Zuk et al published the presence of adipose-derived stem cells, and abundant research has shown that adipose is a complex, biological active, and important tissue. Festa et al, in 2011, reported that adipocyte lineage cells support the stem cell niche and help drive the complex hair growth cycle. Adipose-derived regenerative cells (also known as stromal vascular fraction [SVF]) is a heterogeneous group of noncultured cells that can be reliably extracted from adipose by using automated systems, and these cells work largely by paracrine mechanisms to support adipocyte viability. While, today, autologous fat is transplanted primarily for esthetic and reconstructive volume, surgeons have previously reported positive skin and hair changes posttransplantation. This follicular regenerative approach is intriguing and raises the possibility that one can drive or restore the hair cycle in male and female pattern baldness by stimulating the niche with autologous fat enriched with SVF. In this first of a kind patient series, the authors report on the safety, tolerability, and quantitative, as well as photographic changes, in a group of patients with early genetic alopecia treated with subcutaneous scalp injection of enriched adipose tissue. The findings suggest that scalp stem cell-enriched fat grafting may represent a promising alternative approach to treating baldness in men and women.
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BackgroundLL-37 is a naturally occurring antimicrobial peptide found in the wound bed and assists wound repair. No published study has characterized the role of LL-37 in the function(s) of human mesenchymal stem cells (MSCs). This study investigated the functions of adipose-derived stromal/stem cells (ASCs) activated by LL-37 by performing both in vitro assays with cultured cells and in vivo assays with C57BL/6 mice with hair loss. Methods Human ASCs were isolated from healthy donors with written informed consent. To examine the effects of LL-37 on ASC function, cell proliferation and migration were measured by a cell counting kit (CCK-8) and a Transwell migration assay. Early growth response 1 (EGR1) mRNA expression was determined by microarray and real-time PCR analyses. The protein levels of EGR1 and regenerative factors were analyzed by specific enzyme-linked immunosorbent assays and western blotting. ResultsLL-37 treatment enhanced the proliferation and migration of human ASCs expressing formyl peptide receptor like-1. Microarray and real-time PCR data showed that EGR1 expression was rapidly and significantly increased by LL-37 treatment. LL-37 treatment also enhanced the production of EGR1. Moreover, small interfering RNA-mediated knockdown of EGR1 inhibited LL-37-enhanced ASC proliferation and migration. Activation of mitogen-activated protein kinases (MAPKs) was essential not only for LL-37-enhanced ASC proliferation and migration but also EGR1 expression; treatment with a specific inhibitor of extracellular signal-regulated kinase, p38, or c-Jun N-terminal kinase blocked the stimulatory effect of LL-37. EGR1 has a strong paracrine capability and can influence angiogenic factors in ASCs; therefore, we evaluated the secretion levels of vascular endothelial growth factor, thymosin beta-4, monocyte chemoattractant protein-1, and stromal cell-derived factor-1. LL-37 treatment increased the secretion of these regenerative factors. Moreover, treatment with the conditioned medium of ASCs pre-activated with LL-37 strongly promoted hair growth in vivo. Conclusions These findings show that LL-37 increases EGR1 expression and MAPK activation, and that preconditioning of ASCs with LL-37 has a strong potential to promote hair growth in vivo. This study correlates LL-37 with MSC functions (specifically those of ASCs), including cell expansion, cell migration, and paracrine actions, which may be useful in terms of implantation for tissue regeneration.
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Although minoxidil has been used for more than two decades to treat androgenetic alopecia (AGA), an androgen-androgen receptor (AR) pathway-dominant disease, its precise mechanism of action remains elusive. We hypothesized that minoxidil may influence the AR or its downstream signaling. These tests revealed that minoxidil suppressed AR-related functions, decreasing AR transcriptional activity in reporter assays, reducing expression of AR targets at the protein level, and suppressing AR-positive LNCaP cell growth. Dissecting the underlying mechanisms, we found that minoxidil interfered with AR-peptide, AR-coregulator, and AR N/C-terminal interactions, as well as AR protein stability. Furthermore, a crystallographic analysis using the AR ligand-binding domain (LBD) revealed direct binding of minoxidil to the AR in a minoxidil-AR-LBD co-crystal model, and surface plasmon resonance assays demonstrated that minoxidil directly bound the AR with a Kd value of 2.6 µM. Minoxidil also suppressed AR-responsive reporter activity and decreased AR protein stability in human hair dermal papilla cells. The current findings provide evidence that minoxidil could be used to treat both cancer and age-related disease, and open a new avenue for applications of minoxidil in treating androgen-AR pathway-related diseases.
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Generation of reactive oxygen species (ROS) by NADPH oxidase 4 (Nox4) induces the proliferation and migration of adipose-derived stem cells (ASCs). However, the functional role of mitochondrial ROS (mtROS) generation in ASCs is unknown. Therefore, we have investigated whether hypoxia induces the differentiation of ASCs via ROS generation. We also have tried to identify the cellular mechanisms of ROS generation underlying adipocyte differentiation. Hypoxia (2%) and ROS generators, such as antimycin and rotenone, induced adipocyte differentiation, which was attenuated by an ROS scavenger. Although Nox4 generates ROS and regulates proliferation of ASCs, Nox4 inhibition or Nox4 silencing did not inhibit adipocyte differentiation; indeed fluorescence intensity of mito-SOX increased in hypoxia, and treatment with mito-CP, a mtROS scavenger, significantly reduced hypoxia-induced adipocyte differentiation. Phosphorylation of Akt and mTOR was induced by hypoxia, while inhibition of these molecules prevented adipocyte differentiation. Thus hypoxia induces adipocyte differentiation by mtROS generation, and the PI3K/Akt/mTOR pathway is involved.
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Adipose-derived stem cells (ASCs) have been used in tissue repair and regeneration. Recently, it was reported that ASC transplantation promotes hair growth in animal experiments, and a conditioned medium of ASCs (ASC-CM) induced the proliferation of hair-compositing cells in vitro. However, ASCs and their conditioned medium have shown limited effectiveness in clinical settings. ASC preconditioning is one strategy that can be used to enhance the efficacy of ASCs and ASC-CM. Therefore, we highlighted the functional role of ASCs in hair cycle progression and also the advantages and disadvantages of their application in hair regeneration. In addition, we introduced novel ASC preconditioning methods to enhance hair regeneration using ASC stimulators, such as vitamin C, platelet-derived growth factor, hypoxia, and ultraviolet B.
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Because adipose-derived stem cells (ASCs) are usually expanded to acquire large numbers of cells for therapeutic applications, it is important to increase the production yield and regenerative potential during expansion. Therefore, a tremendous need exists for alternative ASC stimuli during cultivation to increase the proliferation and adipogenic differentiation of ASCs. The present study primarily investigated the involvement of megestrol acetate (MA), a progesterone analog, in the stimulation of ASCs, and identifies the target receptors underlying stimulation. Mitogenic and adipogenic effects of MA were investigated in vitro, and pharmacological inhibition and small interfering (si) RNA techniques were used to identify the molecular mechanisms involved in the MA-induced stimulation of ASCs. MA significantly increased the proliferation, migration, and adipogenic differentiation of ASCs in a dose-dependent manner. Glucocorticoid receptor (GR) is highly expressed compared with other nuclear receptors in ASCs, and this receptor is phosphorylated after MA treatment. MA also upregulated genes downstream of GR in ASCs, including ANGPTL4, DUSP1, ERRF11, FKBP5, GLUL, and TSC22D3. RU486, a pharmacological inhibitor of GR, and transfection of siGR significantly attenuated MA-induced proliferation, migration, and adipogenic differentiation of ASCs. Although the adipogenic differentiation potential of MA was inferior to that of dexamethasone, MA had mitogenic effects in ASCs. Collectively, these results indicate that MA increases the proliferation, migration, and adipogenic differentiation of ASCs via GR phosphorylation. ©AlphaMed Press.
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Embryonic hair follicle (HF) induction and formation is dependent on signaling crosstalk between the dermis and specialized dermal condensates on the mesenchymal side and epidermal cells and incipient placodes on the epithelial side, but the precise nature and succession of signals remain unclear. Platelet Derived Growth Factor (PDGF) signaling is involved in the development of several organs and the maintenance of adult tissues, including HF regeneration in the hair cycle. As both PDGF receptors, PDGFRα and PDGFRβ, are expressed in embryonic dermis and dermal condensates, we explored in this study the role of PDGF signaling in HF induction and formation in the developing skin mesenchyme. We conditionally ablated both PDGF receptors with Tbx18(Cre) in early dermal condensates before follicle formation, and with Prx1-Cre broadly in the ventral dermis prior to HF induction. In both PDGFR double mutants, HF induction and formation ensued normally, and the pattern of HF formation and HF numbers were unaffected. These data demonstrate that mesenchymal PDGF signaling, either in the specialized niche or broadly in the dermis, is dispensable for HF induction and formation. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
Article
Platelet-derived growth factor-D (PDGF-D) was recently identified, and acts as potent mitogen for mesenchymal cells. PDGF-D also induces cellular transformation and promotes tumor growth. However, the functional role of PDGF-D in adipose-derived stem cells (ASCs) has not been identified. Therefore, we primarily investigated the autocrine and paracrine roles of PDGF-D in the present study. Furthermore, we identified the signaling pathways and the molecular mechanisms involved in PDGF-D-induced stimulation of ASCs. It is of interest that PDGF-B is not expressed, but PDGF-D and PDGF receptor-β are expressed in ASCs. PDGF-D showed the strongest mitogenic effect on ASCs, and PDGF-D regulates the proliferation and migration of ASCs through the PI3K/Akt pathways. PDGF-D also increases the proliferation and migration of ASCs through generation of mitochondrial reactive oxygen species (mtROS) and mitochondrial fission. mtROS generation and fission were mediated by p66Shc phosphorylation, and BCL2A1 and SERPINE1 mediated the proliferation and migration of ASCs. In addition, PDGF-D up-regulated the mRNA expression of diverse growth factors such as VEGFA, FGF1, FGF5, LIF, INHBA, IL11 and HBEGF. Therefore, the preconditioning of PDGF-D enhanced the hair-regenerative potential of ASCs. PDGF-D-induced growth factor expression was attenuated by a pharmacological inhibitor of mitogen-activated protein kinase pathway. In summary, PDGF-D is highly expressed by ASCs, where it acts as a potent mitogenic factor. PDGF-D also up-regulates growth factor expression in ASCs. Therefore, PDGF-D can be considered a novel ASC stimulator, and used as a preconditioning agent before ASC transplantation. Stem Cells 2014
Article
Objective: Adipose-derived stem cells (ASCs) isolated from subcutaneous adipose tissue have been tested in clinical trials. However, ASCs isolated by enzyme digestion and centrifugation are heterogeneous and exhibit wide variation in regenerative potential and clinical outcomes. Therefore, we developed a new method for isolating clonal ASCs (cASCs) that does not use enzyme digestion or centrifugation steps. Research design and methods: In addition to cell surface markers and differentiation potential, we compared the mitogenic, paracrine and hair growth-promoting effects of ASCs isolated by the gradient centrifugation method (GCM) or by the new subfractionation culturing method (SCM). Results: We selected three cASCs isolated by SCM that showed high rates of proliferation. The cell surface markers expressed by ASCs isolated by GCM or SCM were very similar, and SCM-isolated ASCs could potentially differentiate into different cell lineages. However, cASC lines exhibited better mitogenic and paracrine effects than ASCs isolated by GCM. The expression of Diras3, Myb, Cdca7, Mki67, Rrm2, Cdk1 and Ccna2, which may play a key role in cASC proliferation, was upregulated in cASCs. In addition, cASCs exhibited enhanced hair growth-promoting effects in dermal papilla cells and animal experiments. Conclusions: SCM generates a highly homogeneous population of ASCs via a simple and effective procedure that can be used in therapeutic settings.
Article
Although adipose-derived stem cells (ASCs) show promise for cell therapy, there is a tremendous need for developing ASC activators. In the present study, we investigated whether or not vitamin C increases the survival, proliferation, and hair-regenerative potential of ASCs. In addition, we tried to find the molecular mechanisms underlying the vitamin C-mediated stimulation of ASCs. Sodium-dependent vitamin C transporter 2 (SVCT2) is expressed in ASCs, and mediates uptake of vitamin C into ASCs. Vitamin C increased the survival and proliferation of ASCs in a dose-dependent manner. Vitamin C increased ERK1/2 phosphorylation, and inhibition of the mitogen-activated protein kinase (MAPK) pathway attenuated the proliferation of ASCs. Microarray and quantitative polymerase chain reaction showed that vitamin C primarily up-regulated expression of proliferation-related genes including Fos, E2F2, Ier2, Mybl1, Cdc45, JunB, FosB, and Cdca5, whereas Fos knock-down using siRNA significantly decreased vitamin C-mediated ASC proliferation. In addition, vitamin C-treated ASCs accelerated the telogen-to-anagen transition in C3H/HeN mice, and conditioned medium from vitamin C-treated ASCs increased the hair length and the Ki67-positive matrix keratinocytes in hair organ culture. Vitamin C increased the mRNA expression of HGF, IGFBP6, VEGF, bFGF, and KGF, which may mediate hair growth promotion. In summary, vitamin C is transported via SVCT2, and increased ASC proliferation is mediated by the MAPK pathway. In addition, vitamin C preconditioning enhanced the hair-growth promoting effect of ASCs. Because vitamin C is safe and effective, it could be used to increase the yield and regenerative potential of ASCs.