Activin B-activated Cdc42 signaling plays a key role in regulating adipose-derived mesenchymal stem cells-mediated skin wound healing

Abstract

Background
In our previous study, activin B in combination with ADSCs enhances skin wound healing. However, the underlying molecular mechanisms are not well studied. Cdc42 is recognized to play a critical role in the regulation of stem cells.

Methods
Pull-down assay was performed to investigate the activity of Cdc42. The dominant-negative mutant of Cdc42 (Cdc42N17) was used to explore the role of Cdc42 in activin B-induced ADSCs migration, proliferation, and secretion in vitro. Cdc42N17-transfected ADSCs were injected into a full-thickness excisional wound model to explore their efficiency in wound healing in vivo. The wound healing efficacy was evaluated by the wound closure rates and histological examination. The neovascularization and wound contraction were detected by immunohistochemistry staining of CD31 and α-SMA. Finally, the underlying mechanisms were explored by RNA sequencing.

Results
Cdc42N17 inhibited ADSCs migration, proliferation, and secretion induced by activin B. Furthermore, Cdc42N17-transfected ADSCs inhibited the wound closure rate and suppressed the expression of CD31 and α-SMA induced by activin B in vivo. The RNA sequencing showed that the differentially expressed genes in Cdc42N17-transfected ADSCs versus ADSCs were associated with cell migration, proliferation, and adhesion. Further study revealed that the Cdc42-Erk-Srf pathway was required for activin B-induced proliferation in ADSCs.

Conclusions
Our study indicates that Cdc42 plays a crucial role in ADSCs-mediated skin wound healing induced by activin B.

Graphical Abstract

Full text

Huangetal. Stem Cell Research & Therapy (2022) 13:248
https://doi.org/10.1186/s13287-022-02918-9
RESEARCH
Activin B-activated Cdc42 signaling plays
akey role inregulating adipose-derived
mesenchymal stem cells-mediated skin wound
healing
Simin Huang1†, Xueer Wang1†, Min Zhang1, Mianbo Huang1, Yuan Yan1, Yinghua Chen1, Yijia Zhang1, Jinfu Xu1,
Lingwei Bu1, Ruyi Fan1, Huiyi Tang1, Canjun Zeng3, Lu Zhang2* and Lin Zhang1*
Abstract
Background: In our previous study, activin B in combination with ADSCs enhances skin wound healing. However,
the underlying molecular mechanisms are not well studied. Cdc42 is recognized to play a critical role in the regulation
of stem cells.
Methods: Pull-down assay was performed to investigate the activity of Cdc42. The dominant-negative mutant of
Cdc42 (Cdc42N17) was used to explore the role of Cdc42 in activin B-induced ADSCs migration, proliferation, and
secretion in vitro. Cdc42N17-transfected ADSCs were injected into a full-thickness excisional wound model to explore
their efficiency in wound healing in vivo. The wound healing efficacy was evaluated by the wound closure rates and
histological examination. The neovascularization and wound contraction were detected by immunohistochemistry
staining of CD31 and α-SMA. Finally, the underlying mechanisms were explored by RNA sequencing.
Results: Cdc42N17 inhibited ADSCs migration, proliferation, and secretion induced by activin B. Furthermore,
Cdc42N17-transfected ADSCs inhibited the wound closure rate and suppressed the expression of CD31 and α-SMA
induced by activin B in vivo. The RNA sequencing showed that the differentially expressed genes in Cdc42N17-trans-
fected ADSCs versus ADSCs were associated with cell migration, proliferation, and adhesion. Further study revealed
that the Cdc42-Erk-Srf pathway was required for activin B-induced proliferation in ADSCs.
Conclusions: Our study indicates that Cdc42 plays a crucial role in ADSCs-mediated skin wound healing induced by
activin B.
Keywords: ADSCs, Wound healing, Cdc42, RNA-seq
© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
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Open Access
†Simin Huang and Xueer Wang have contributed equally to this work
*Correspondence: zlulu70@126.com; zlilyzh@126.com
1 Department of Histology and Embryology, NMPA Key Laboratory for Safety
Evaluation of Cosmetics, Key Laboratory of Construction and Detection
in Tissue Engineering of Guangdong Province, School of Basic Medical
Sciences, Southern Medical University, Guangzhou 510515, People’s Republic
of China
2 Key Laboratory of Functional Proteomics of Guangdong Province, Key
Laboratory of Mental Health of the Ministry of Education, School of Basic
Medical Sciences, Southern Medical University, Guangzhou 510515, China
Full list of author information is available at the end of the article
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Introduction
Stem-cell-based therapeutic strategies have shown con-
siderable potential to improve the rate and quality of skin
wound healing [1, 2]. Among a wide variety of stem cells
that show efficacies in wound healing, adipose-derived
mesenchymal stem cells (ADSCs) seem to possess the
least limitations for clinical applications [3, 4]. Trans-
plantation of ADSCs into full-thickness wounds is able
to improve wound healing by promoting angiogenesis,
modulating immune response, and inducing epitheliali-
zation in the wound [5, 6].
Many growth factors and cytokines were shown to play
a key role in the process of cutaneous wound healing
[7–10]. For example, activin B, a member of the TGF-β
superfamily, is involved in the process of skin wound
repair [11, 12]. In a previous study, we reported that
activin B was able to induce actin stress fiber formation
and migration of ADSCs to promote skin wound heal-
ing [13]. However, the mechanism in this process is still
poorly understood.
Cell division cycle 42 (Cdc42), a member of the Rho
GTPases family, contributes to cell migration by con-
trolling protrusions, adhesion, and contraction [14–17].
Cdc42 is crucial for the regulation of stem cells’ behav-
iors. Loss of Cdc42 reduces α-SMA expression in mesen-
chymal stem cells [18]. e elevated activity of the Cdc42
in aged hematopoietic stem cells (HSCs) correlates with a
loss of polarity [19]. In a recent study, we have found that
Cdc42 influences the morphology and skeleton in bone
marrow-derived mesenchymal stem cells (BMSCs) [20].
However, the role of Cdc42 in ADSCs-mediated wound
healing remains unclear.
In this study, we used the dominant-negative mutant of
Cdc42 (Cdc42N17) to explore the role of Cdc42 in activin
B-induced ADSCs migration, proliferation, and secretion
in vitro. Cdc42N17-transfected ADSCs were injected
into a full-thickness excisional wound model to explore
their efficiency in wound healing invivo. Moreover, RNA
sequencing was used to explore the downstream mecha-
nism of Cdc42 in the regulation of wound healing pro-
cesses. Our study reveals an essential role of Cdc42 in
ADSCs-mediated wound healing.
Material andmethods
Animals
One hundred and eight specific pathogen-free (SPF)
class C57BL/6 male mice (26–28g) of 3months old were
purchased from Laboratory Animal Center of Southern
Medical University (Guangzhou, China; SCXK 2021-
0041). e mice were housed in a temperature-controlled
room (23 ± 2°C) under a 12-h light/dark cycle with avail-
able food and water adlibitum. e mice were allowed
1 week to adapt to the environment before experiments.
All the procedures related to the care and use of animals
in this study were in accordance with the guidelines of
National Institutes of Health (NIH) and was approved by
the Bioethics Committee of Southern Medical University
(Approval number: L2019084).
Isolation, identication, andlabeling ofADSCs
ADSCs’ isolation was performed as previously reported
[21, 22]. Mice were killed by cervical dislocation and
asepticized by soaking in 75% ethanol for 5 min. e
inguinal adipose tissues were withdrawn and washed
with PBS at 4 °C for three times. After removing the
blood vessels and lymph nodes carefully, the adipose
was cut into pieces and digested for 90 min at 37 °C
using 10% fetal bovine serum (FBS) and 125 U/mL col-
lagenase (Cat# 17018029, Collagenase Type I, Gibco)
[23]. By filtrating through 100-μm nylon filter mesh (Cat#
Graphical Abstract
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Huangetal. Stem Cell Research & Therapy (2022) 13:248
352360, BD Falcon) and concentrated by centrifugation
at 300g for 5min, the stromal vascular fraction (SVF) was
attained [24]. en, ADSCs were resuspended in com-
plete medium (high-glucose Dulbecco’s modified Eagle’s
medium (H-DMEM) with 10% FBS and 1% penicillin-
streptomycin solution (Cat# C0222, Beyotime, China))
and cultured in a humid atmosphere with 5% CO2 at
37°C. e culture medium was updated every 48h. e
cells were monitored daily under an inverted phase-con-
trast microscope (Leica DMI4000 B) and passaged when
they reached 80–90% confluence.
Cells at passage three were used for 5,6-carboxyfluo-
rescein diacetate succinimidyl ester (CFSE) (Cat# C1157,
Invitrogen) labeling and identification according to the
standard listed in Additional file1.
Lentivirus infection
e dominant negative mutant Cdc42N17, the constitu-
tively active mutant Cdc42L61, and the EGFP lentiviral
vector were generated as described in our previous study
[20, 22]. e titers of Cdc42N17, Cdc42L61, and EGFP
vector were 1.63 × 109 TU/ml, 6.93 × 108 TU/ml, and
4.53 × 108TU/ml, respectively. e lentivirus was serum-
free incubated with ADSCs for 8 h. On the day 3 after
lentivirus infection, the infection efficiency was deter-
mined by examining green fluorescence in the cells using
fluorescent microscopy and pull-down assay.
The inhibitor treatment
Cells were serum-starved for 12h and then treated with
serum-free H-DMEM containing Srf inhibitor CCG-
100602 (15μM, Cat# HY-120855, Mce) for 24h [25, 26]
or Erk1/2 inhibitor SCH772984 (5μM, Cat# S7101, Sell-
eck) for 2h [27].
GST pull‑down assay
GTPase pull-down assay was performed according to
the manufacturer’s protocol (1:100, Cat # 14-325, Rac/
cdc42 Assay Reagent, Millipore) as described previously
[20, 28]. In short, after lysed with MLB buffer and deter-
mined the total protein concentration, the cell lysates
were divided into two equal parts: One was blotted for
total Cdc42, and the other was subjected to a PAK1 PBD
binding assay. e beads with Cdc42 GTP-bound were
captured, and the activation levels of Cdc42 were ana-
lyzed by western blot using an anti-Cdc42 antibody. e
detailed protocol for pull-down assay is described in
Additional file1.
Western blot analysis
Cells were lysed using radio-immunoprecipitation assay
(RIPA) buffer (Cat# P0013B, Beyotime, China), and
total protein was extracted and normalized according to
the concentrations determined by Enhanced BCA Pro-
tein Assay Kit (Cat# P0010, Beyotime, China). e total
protein was then re-suspended with 5 × loading buffer
(Ca# P0286, Beyotime, China) and boiled for 5 min.
Twenty-five mg protein from each sample was separated
by electrophoresis on 10% polyacrylamide gels (Cat#
PG112, Epizyme, China) and transferred onto polyvi-
nylidene difluoride membranes. e blots were blocked
with 5% skim milk (Cat# 232100, Genebase, China) and
incubated with a primary antibody Cdc42 (1:2000, Cat#
ab187643, Abcam), Srf (1:1000, Cat# 16821-1-AP, Pro-
teintech, China), or phosphorylation Erk (p-Erk) (1:1000,
Cat# 4370S, Cell Signaling) at 4°C overnight. After fur-
ther incubated with HRP-conjugated anti-rabbit IgG
(1:2000, Cat# ab6721, Abcam) secondary antibody for
1h at room temperature, the bands’ intensity was visual-
ized using the enhanced chemiluminescence (ECL) lumi-
nescence reagent (Cat# MA0186-1, Meilunbio, China)
and Gel Image System (Tanon-5200CE). e blots were
then washed with a commercial stripping buffer (Cat#
P0025N, Beyotime, China) for 10min at room temper-
ature and re-probed with antibodies against total Erk
(1:1000, Cat# 4695S, Cell Signaling). en, the intensity
of the band was quantified using ECL and Gel Image Sys-
tem (Tanon-5200CE).
Migration assay
ADSCs and cells transduced with Cdc42N17, Cdc42L61,
or EGFP vector were seeded into 24-well plastic plates
at the density of 1 × 105 cells per well and cultured until
they reached a confluence of 90%. After serum-starved
for 12h, the cells were divided into seven groups. Among
them, four groups (ADSCs, Cdc42N17, Cdc42L61, and
EGFP vector) were treated with serum-free H-DMEM,
while the other three groups (activin B+ADSCs, activin
B+Cdc42N17, and activin B+Cdc42L61) were treated
with H-DMEM containing 10ng/ml activin B. en, the
migration properties of cells were analyzed by the follow-
ing two methods.
For the scratch wound healing assay, the uniform
scratch wounds were scraped and photographed at 0, 24,
and 48h after being washed with PBS. e Image-Pro
Plus 6.0 software was used to calculate the scratching
area.
For the transwell assay, 500 µl preheated serum-free
H-DMEM was added to the upper and lower chamber,
respectively, and incubated for 1h at 37 °C. After dis-
carding the medium, a total of 200μl serum-free media
including 5 × 104 cells has been added into the upper
chamber, while the lower chamber was filled with 300µl
different culture media (H-DMEM or H-DMEM contain-
ing 10ng/ml activin B) as designed previously. After 24-h
incubation, the migrated cells under the chamber were
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Huangetal. Stem Cell Research & Therapy (2022) 13:248
fixed with 4% paraformaldehyde solution (PFA) (Cat#
G0528, GBCBIO, China) for 15min, stained with crystal
violet staining solution (Cat# C0121, Beyotime, China)
and imagined by microscope.
Proliferation assay
For the cell proliferation experiment, ADSCs and cells
transduced with Cdc42N17, Cdc42L61, or the mock
vector were seeded on cover glass into 24-well plas-
tic plates at the density of 1 × 105cells per well. After
serum-starved for 12h, the cells were divided into eleven
groups. Among them, six groups (ADSCs, Cdc42N17,
Cdc42L61, CCG-100602, SCH772984, and EGFP vector)
were treated with serum-free H-DMEM, while the other
five groups (activin B+ADSCs, activin B+Cdc42N17,
activin B+Cdc42L61, activin B+CCG-100602, and
activin B+SCH772984) were treated with H-DMEM con-
taining 10ng/ml activin B. en, an addition of 12.5μM
EdU was added in each group. After incubating for 6h
at 37°C, cells were fixing, Apollo- and nuclear-stained
as manufacturer’s instructions (Cat# C10310-1, RiboBio,
China). e EdU-positive cells were observed under fluo-
rescence microscope.
Cytokine analysis
e cells of ADSCs and cells transduced with Cdc42N17
or Cdc42L61 have been selected for a 12-h serum-
free starve. After incubating for 24 h with H-DMEM
(for ADSCs, Cdc42N17, and Cdc42L61) or H-DMEM
with 10ng/ml activin B (for activin B+ADSCs, activin
B+Cdc42N17, and activin B+Cdc42L61), the cell cul-
ture supernatant was collected by centrifuging at 3000g
for 15min and filtering with a 0.22-μm filter to remove
apoptotic cells and debris. en, the secretion levels of
Col 1 (Cat# MU30364, Bisowamp, China) and VEGF
(Cat# MU30236, Bisowamp, China) were detected using
a commercially available enzyme-linked immunosorbent
assay (ELISA) system following the kit’s instructions.
Wound healing model
e wound healing model was established as we
described previously [13]. Mice were anesthetized by
injecting 2% pentobarbital sodium (1.5mL/kg) into their
intraperitoneal. After the dorsal hair was depilated using
a honey and wax mixture and cleaned with 70% ethanol,
skin punch was used to delineate an 8-mm round-shaped
image on the skin. en, sterile scissors were applied to
create full-thickness skin wounds on both the right and
left shaved dorsal skin after povidone iodine disinfection.
Each mouse had retained the capability to free ingest
food and water after the operation.
After wounding, mice were randomly divided into six
groups (n = 6 for each group) according to the treatment
on the site surrounding the wound (Table1) daily for 3
days. e cells and/or activin B were resuspended in PBS
and administered to the wounds by subcutaneous injec-
tion. Wound areas were measured photographically at
days 0, 3, 7, and 14 after surgery, and the rate of wound
closure was calculated with the following equation:
wound closure rate (%) = [(original wound area − open
area on final day)/original wound area] × 100%.
Frozen sections
For analysis of the applied ADSCs within the wound site,
the wound and surrounding tissue were collected on day
3 and frozen with optimal cutting temperature (OCT)
compound. Serial sections(15μm) were taken from the
wound center to the edge by a freezing microtome at
− 20°C and adhered to the slides. After washing with
PBS and blocking in PBS containing 5% bovine serum
albumin (BSA) (Cat# ST023, Beyotime, China), the sec-
tions were stained with Hoechst 33258 (1:500, Cat#
H21491, Invitrogen) for 10min and examined by fluores-
cence microscopy.
Hematoxylin andeosin (H&E) staining andhistological
evaluation
Mice were killed at postoperative 3rd, 7th, and 14th day,
respectively. en, complete wound with 0.5-cm mar-
gin was carefully cut down and rinsed with PBS before
and after fixing by 4% paraformaldehyde solution (PFA)
(Cat# G0528, GBCBIO, China) for 3 days. en, the sam-
ples were dehydrated and embedded in paraffin. Serial
Table 1 Group on wound healing model
Group Name Treatment
1 Control 0.4 ml PBS
2Act B 0.4 ml 10 ng/ml activin B
3 ADSCs 0.4 ml 6 × 106/ml ADSCs
4Act B+ADSCs 0.4 ml 6 × 106/ml ADSCs induced by 10 ng/ml activin B for 12 h
5 ADSCs (Cdc42N17) 0.4 ml 6 × 106/ml ADSCs transduced with Cdc42N17
6Act B+ADSCs (Cdc42N17) 0.4 ml 6 × 106/ml ADSCs transduced with Cdc42N17 and
induced by 10 ng/ml activin B for 12 h
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Huangetal. Stem Cell Research & Therapy (2022) 13:248
sections (5µm) were collected and H&E-stained accord-
ing to the detailed method described in Additional file1.
For histological evaluation, re-epithelialization and
granulation tissue formation was evaluating by using
H&E staining. e length of epithelial tongues of wound
skin sections on each group sample (n = 6) was meas-
ured between the wound edge and the leading edge of
migrating epithelial tongue with ImageJ 1.52a software
(National Institutes of Health, Bethesda, MD, USA).
For the granulation tissue formation, each group sam-
ple (n = 6) was given a granulation tissue score rang-
ing from 1 to 12. e criteria used for granulation
tissue scores of wound healing were referred to previ-
ous researches [29–32] and our previous study [33] and
summarized in Additional file1: TableS1.
Masson’s trichrome staining
e slices adjacent to the wound center were used for
Masson’s trichrome staining using Masson’s trichrome
staining kit (Cat# MST-8004, Maixin-Bio, China) accord-
ing to the manufacturer’s instructions.
Immunohistochemical assay
e tissue slices were deparaffinized and rehydrated
(detailed procedure as previously described in H&E
staining). In order to retrieve antigen, samples were first
immersed in 0.1M citrate buffer at 96°C for 10min and
later incubated in 5% BSA for 2h. e slices were incu-
bated with antibody against CD31 (1:100, Cat# ab281583,
Abcam) and α-SMA (1:200, Cat# ab32575, Abcam) at
4 °C overnight. After rinsing with PBS and incubated
with biotinylated goat anti-rabbit secondary antibody
(Cat# PV-6001, ZSGB-BIO, China) for 2 h, the tissue
slices were colored with 3,3-diaminobenzidine (DAB)
(Cat# AR1022, Boster, China), stained with hematoxylin,
dehydrated with a gradient ethanol series, soaked with
xylene, and then sealed with resin. Finally, five random
locations of each tissue slice were selected to count the
new capillaries with endothelial cells positive for CD31
via microscope (400 ×) or to analyze the average opti-
cal density values for α-SMA expression at the indicated
time points using Image-Pro Plus 6.0 Software.
Total RNA preparation
ADSCs transduced with or without Cdc42N17
were seeded in 60-mm plastic culture plates. After
12-h serum-free starve, they were divided into four
groups according to different processing methods
with H-DMEM (ADSCs, Cdc42N17) or H-DMEM
with 10 ng/ml activin B (Act B+ADSCs and Act
B+Cdc42N17) and incubated for another 24h. Total
RNA of those cells was extracted, and genomic DNA
was simultaneously eliminated using EZ-10 DNAaway
RNA Mini-Preps Kit (Cat# B618133, Sangon Biotech,
China) according to the manufacturer’s instructions.
e quality and purity of the resulting RNA were
checked using the NanoDrop™ spectrophotometer;
only high-quality RNA sample (OD260/280 = 1.8–2.2,
OD260/230 ≥ 2.0) could be used for sequencing and
qPCR analysis.
RNA sequencing
For the transcriptomic analysis, 1 μg total RNA was
used to prepare RNA-seq transcriptome library using
TruSeq™ RNA sample preparation kit from Illu-
mina (San Diego, CA). e raw paired-end reads were
trimmed, and the quality was controlled by SeqPrep
and Sickle with default parameters. e high-quality
clean data were aligned to the mouse genome using
HISAT2 software [34], and mapped data (reads) of each
sample were assembled by StringTie in a reference-
based approach [35].
To identify differential expression genes (DEGs)
between these samples, the expression levels of each
transcript and gene abundance were calculated according
to the transcripts per million reads (TPM) method and
RSEM, respectively [36]. Essentially, differential expres-
sion analysis was performed using the DESeq2 [37]/
EdgeR [38] with Q value ≤ 0.05; DEGs with |log2FC|> 1
and Q value ≤ 0.05 (DESeq2 or EdgeR)/Q value ≤ 0.001
(DEGseq) were considered to be significantly differen-
tially expressed genes. In addition, functional enrichment
analysis including GO and KEGG was performed to
identify which DEGs were significantly enriched in GO
terms and metabolic pathways at Bonferroni-corrected
P value ≤ 0.05 compared with the whole-transcriptome
(See figure on next page.)
Fig. 1 Cdc42 regulates activin B-mediated ADSCs migration in vitro. A Pull-down assays were performed to detect the amounts of GTP-bound
Cdc42 in ADSCs, ADSCs treated with activin B, respectively. B The ratios of GTP-Cdc42 versus total Cdc42 levels were analyzed. C Scratch wound
healing assays were performed to detect the migration of cells treated by seven methods, and photographs were taken at 0, 24, and 48 h after
scratch injury. Scale bar = 500 μm. D The healing rates were quantified by measuring the area of the injured region. E Transwell assays were
performed to detect the invasion of these cells, and photographs were taken at 24 h after seeds. Scale bar = 200 μm. F The migrated cells were
counted. All values are expressed as mean ± SD from three independent repeats. *P < 0.05
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Fig. 1 (See legend on previous page.)
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Huangetal. Stem Cell Research & Therapy (2022) 13:248
background. GO functional enrichment and KEGG path-
way analysis were carried out by Goatools and KOBAS
[39].
qPCR analysis
To further verify RNA-seq data, the genes described in
Table S2 with significant expression differences were
analyzed by qPCR using the Maxima SYBR Green/ROX
qPCR Master Mix kit (Cat# K0221, ermo Scientific)
according to the manufacturer’s instructions. To be spe-
cific, 1.0 μg total RNA was converted to cDNA using
MonScript™ RTIII All-in-One Mix with dsDNase kit
(Cat# MR05101S, Monad Biotech, China), and the qPCR
reactions were performed on the ABI 7900 system using
the following program: 95°C for 10min, and 40 cycles of
95°C for 10s, 60°C for 10s, and 72°C for 30s, followed
by dissociation steps. Reactions were run in triplicates.
e GAPDH housekeeping gene was used as an internal
control for data normalization across samples. e nor-
malization of Ct values for each gene and the determina-
tion of fold changes in gene expression (normalized to
control group) were calculated using the 2−ΔΔCt method.
Statistical analysis
Numerical data were expressed as the means ± stand-
ard deviation (SD). All experiments were performed
at least three times. Statistical differences between the
groups were assessed using one-way analysis of vari-
ance (ANOVA) in SPSS20.0 software (IBM, Armonk, NY,
USA). A p value of less than 0.05 (*P < 0.05) was consid-
ered to be statistically significant.
Fig. 2 Cdc42 regulates activin B-mediated ADSCs’ proliferation and secretion in vitro. A Representative fluorescence imaging of EdU staining of
seven groups cells at 6 h. Scale bar = 50 μm. B The proliferation rates were quantified by calculating the percentage of EdU-positive cells. C Average
concentration of Col 1 and VEGF in the conditioned cell medium of six groups. All values are expressed as mean ± SD from three independent
repeats. *P < 0.05
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Results
Characterization ofisolated ADSCs
To validate ADSCs isolated from mice, we assessed cell
surface markers using immunocytochemistry. In support
of previous reports [13, 40], ADSCs were positive for
CD44 and CD90, while nearly negative for CD31 and
CD80 (Additional file1: Fig. S1A). e flow cytometry
was also assessed to characterize isolated ADSCs cell
Fig. 3 Cdc42-regulated activin B-induced ADSCs-mediated skin wound healing in vivo. A Representative macroscopic images of wounds treated
with PBS, activin B, ADSCs, activin B+ADSCs, ADSCs (Cdc42N17), or activin B+ADSCs (Cdc42N17) on days 0, 3, 7, and 14. B Quantitative analysis of
wound closure rate of six mice per group. All values are expressed as mean ± SD from six independent repeats. *P < 0.05
Fig. 4 Cdc42-regulated activin B-induced ADSCs-mediated re-epithelialization and granulation tissue formation in skin wound healing. A H&E
staining showed the re-epithelialization and granulation tissue formation of the six groups after wounding for 3, 7, and 14 days. Black and green
arrows represent the dermal border and epidermal margin (scale bar = 500 μm), whereas insets of main figures (1) and (2) represent wound
re-epithelialization and granulation tissue (scale bar = 100 μm), respectively. B The percentage of re-epithelialization ratio of six experiments. C
Histological scores of granulation tissue thickness. All values are expressed as mean ± SD from three independent repeats. *P < 0.05
(See figure on next page.)
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Fig. 4 (See legend on previous page.)
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Huangetal. Stem Cell Research & Therapy (2022) 13:248
surface markers. ADSCs were positive for CD29, CD44,
CD90.2 and nearly negative for CD31, CD45, and CD117
(Additional file1: Fig. S1B). ADSCs were further char-
acterized by confirming their ability to undergo specific
osteogenic, adipogenic, and chondrogenic differentiation.
ese cells were positive for Alizarin Red staining (Addi-
tional file1: Fig. S1C), oil red O staining (Additional file1:
Fig. S1D), and Alcian Blue staining (Additional file1: Fig.
S1E), indicating osteogenic, adipogenic, and chondro-
genic respective cell-type differentiation. Only cells that
met these criteria were used in subsequent experiments.
Cdc42 regulates activin B‑induced ADSCs migration
We then investigated whether activin B regulated the
activity of Cdc42 in ADSCs by GST pull-down assay. We
found that the GTP-bound Cdc42 was increased from 30
to 120min after activin B treatment (Fig.1A, B).
To study the role of Cdc42 in activin B-induced
ADSCs migration and other biological functions, the
mock vector or the dominant-negative mutant of Cdc42
(Cdc42N17) and the constitutively active mutant of
Cdc42 (Cdc42L61) were transduced into ADSCs, respec-
tively. e efficient transduction of Cdc42N17 and
Cdc42L61 was confirmed by GFP imaging and pull-down
assay (Additional file1: Fig. S2).
We next examined whether Cdc42 was involved
in activin B-induced ADSCs migration. In a scratch-
ing assay, as reported previously [13], 10ng/ml activin
B effectively induced ADSCs migration (Fig. 1C, D).
However, Cdc42N17 inhibited ADSCs migration with
or without activin B after 24 and 48h (Fig.1C, D). e
wound healing rate of activin B-stimulated Cdc42N17-
transduced ADSCs was comparable to that of Cdc42N17-
transduced ADSCs without activin B stimulation
(Fig.1C, D). ese findings suggest that Cdc42N17 abol-
ished activin B-induced ADSCs migration. Consistent
with this finding, Cdc42N17 inhibited activin B-induced
ADSCs migration to the lower chamber in a transwell
assay (Fig.1E, F). However, Cdc42L61 did not promote
ADSCs migration with or without activin B (Fig.1C–F).
In summary, these results suggest that Cdc42 is nec-
essary but not sufficient for activin B-induced ADSCs
migration.
Cdc42 regulates activin B‑induced ADSCs proliferation
andsecretion
Next, we investigated the role of Cdc42 in the prolif-
eration and secretion of ADSCs treated with or without
activin B.
EdU-stained cell number was markedly increased in
the activin B group compared with the control group.
Cdc42N17 significantly decreased EdU-stained cell num-
ber in the ADSCs treated with or without activin B. Of
note, EdU-stained cell number in activin B-stimulated
Cdc42N17-transduced ADSCs was comparable to that in
Cdc42N17-transduced ADSCs without activin B stimula-
tion (Fig.2A, B). ese findings reveal that Cdc42 may
regulate ADSCs’ proliferation induced by activin B.
Moreover, the concentrations of Col 1 and VEGF were
markedly increased in the activin B group compared with
that in the control group, whereas Cdc42N17 signifi-
cantly inhibited the secretion of Col 1 and VEGF in the
ADSCs with or without activin B. us, Cdc42 contrib-
utes to ADSCs’ secretion induced by activin B (Fig.2C).
However, Cdc42L61 did not promote ADSCs’ prolifera-
tion and secretion with/without activin B (Fig. 2A-C).
Taken together, these findings suggest that Cdc42 is nec-
essary but not sufficient for ADSCs’ proliferation and
secretion induced by activin B.
Cdc42 promotes activin B‑induced ADSCs‑mediated skin
wound healing invivo
We then examined the role of Cdc42 on activin
B-induced ADSCs-mediated cutaneous wound healing
invivo.
To assess ADSCs administered, 5, 6-carboxyfluores-
cein diacetate succinimidyl ester (CFSE) was used to
label ADSCs before transplantation (Additional file 1:
Fig. S3A) and then we collected the frozen sections on
day 3 after ADSCs transplantation. Green fluorescence
(EGFP+) cells were detected in the ADSCs group, activin
B+ADSCs group, ADSCs (Cdc42N17) group, and activin
B+ADSCs (Cdc42N17) group but not in the activin B
group or control group (Additional file1: Fig. S3B). ese
data suggest that ADSCs were incorporated into wound
sites on day 3 after transplantation.
Wound closure rates were then evaluated. Consistently
with the reported study [13], the wound closure rates
(See figure on next page.)
Fig. 5 Cdc42 regulates activin B-induced ADSCs-mediated neovascularization and wound contraction in vivo. A Representative photomicrographs
of CD 31 immunohistochemical staining treated with PBS, activin B, ADSCs, activin B+ADSCs, ADSCs (Cdc42N17), and activin B+ADSCs (Cdc42N17),
respectively, at the specified time. The black arrow indicates positive staining of CD31. Scale bars = 200 μm. B Representative photomicrographs of
α-SMA immunohistochemical staining of wounds per group on days 3 and 7 after wounding. Scale bars = 200 μm. The areas stained with α-SMA
were determined by planimetric image analysis using Image-Pro Plus 6.0 software. All values are expressed as mean ± SD from three independent
repeats, *P < 0.05
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Fig. 5 (See legend on previous page.)
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Huangetal. Stem Cell Research & Therapy (2022) 13:248
in the activin B+ADSCs group were significantly accel-
erated compared with that of the control, activin B, or
ADSCs group (Fig.3A, B). However, the wound closure
rate of ADSCs (Cdc42N17) was significantly decreased
compared with that of the ADSCs group on days 3 and
7 after treatment (Fig. 3A, B). In addition, the wound
healing rate in the activin B+ADSCs (Cdc42N17) group
was also decreased compared with that of the activin
B+ADSCs group (Fig. 3A, B). As there was no differ-
ence in wound healing rate between ADSCs (Cdc42N17)
group and activin B+ADSCs (Cdc42N17) group, these
data suggest that Cdc42N17 inhibited wound healing rate
induced by activin B and ADSCs invivo.
Cdc42 promotes activin B‑induced re‑epithelialization,
granulation tissue formation, andcollagen deposition
inskin wound healing
e results of H&E staining revealed that re-epithe-
lialization in ADSCs (Cdc42N17) group was impaired
compared with that of ADSCs groups on day 3 and 7
with or without activin B (Fig.4A, B). Moreover, granu-
lation tissue scores of ADSCs (Cdc42N17) and activin
B+ADSCs (Cdc42N17) groups were much lower than
those of the ADSCs and activin B+ADSCs groups,
respectively. Re-epithelialization and granulation tissue
scores in ADSCs (Cdc42N17) group were similar to that
in activin B+ADSCs (Cdc42N17) group(Fig.4A–C).
e Masson trichrome staining experiment was used to
investigate the collagen deposition on day 7 and day 14
after treatment. After 7days of treatment, there was lit-
tle collagen deposition in the control group. In contrast,
more newly formed collagen appeared in the ADSCs and
activin B+ADSCs groups. However, there was still a large
area of tissue without collagen deposition in the ADSCs
(Cdc42N17) and activin B+ADSCs (Cdc42N17) groups.
After 14days of treatment, the collagen deposition pat-
tern in the activin B+ADSCs group was similar to that
in normal skin at the wound gap. However, the collagen
deposition pattern in the ADSCs (Cdc42N17) group was
similar to that in activin B+ADSCs (Cdc42N17) group,
which was significantly less mature than that in the
ADSCs group (Additional file1: Fig. S4).
ese data reveal that Cdc42 regulates activin
B-induced re-epithelialization, granulation tissue
formation, and collagen deposition or maturation after
wounding.
Cdc42 regulates theneovascularization andwound
contraction inactivin B‑induced ADSCs‑mediated skin
wound healing
Immunohistochemistry staining of CD31 and α-SMA
was used to evaluate the neovascularization and wound
contraction. We found that the expression of CD31 and
α-SMA in the wounds treated with ADSCs (Cdc42N17)
was decreased compared with that of ADSCs group on
days 7 and 14 (Fig.5A, B). Similarly, less expression of
CD31 and α-SMA in activin B+ADSCs (Cdc42N17)
group was observed compared with that of activin
B+ADSCs group (Fig.5A, B). Cdc42N17 inhibited CD31
and α-SMA expression in activin B+ADSCs group to the
levels comparable to that in ADSCs (Cdc42N17) group
(Fig. 5A, B). ese data suggest that Cdc42 promotes
the neovascularization and wound contraction in activin
B-induced ADSCs-mediated skin wound healing.
RNA sequencing identies possible mechanism ofCdc42
intheregulation ofADSCs biological function
RNA sequencing was further conducted in ADSCs group
and ADSCs (Cdc42N17) group to explore global gene
expression changes induced by Cdc42N17. By principal
component analysis (PCA), we found that there was a
strong correlation between biological duplicate samples
(Additional file1: Fig. S5). Compared with ADSCs group,
a total of 216 upregulated and 266 downregulated DEGs
were detected in ADSCs (Cdc42N17) (Fig.6A).
e KEGG pathway enrichment analysis found that
compared with ADSCs group, the upregulated DEGs
in ADSCs (Cdc42N17) group were enriched in micro-
RNAs in cancer, pathways in cancer, wnt, cytokine–
cytokine receptor interaction, cellular senescence, and
foxo (Fig. 6B). e downregulated DEGs in ADSCs
(Cdc42N17) were enriched in the pathways of cell cycle,
p53, regulating pluripotency of stem cells, focal adhesion,
TGF-β, and gap junction pathways (Fig.6C).
e GO enrichment analysis revealed that compared
with ADSCs group, the upregulated DEGs in ADSCs
(Cdc42N17) group were significantly associated with cell
differentiation, negative regulation of cell proliferation,
Fig. 6 Cdc42 deletion in ADSCs exhibits a distinct transcriptional signature. A Heatmap showing differentially expressed genes (DEGs) between
ADSCs (Con) and ADSCs transduced with Cdc42N17. There were 216 upregulated and 266 downregulated DEGs in ADSCs (Cdc42N17), respectively,
and were subjected to Kyoto Encyclopedia of Genes and Genomes pathways enriched (KEGG) and gene ontology (GO) analysis. B, C showing
the top 15 enrich pathways in upregulated and downregulated DEGs, while D showing the top 15 biological processes in upregulated and
downregulated DEGs. E Selected gene expression in some of the GO biological processes. F qPCR analysis of relative expression level of Cdkn2b,
Wnt11, Pdgfb, Iqgap3, Racgap1 and Cd248 in ADSCs (Cdc42N17). All values are expressed as mean ± SD from three independent repeats. *P < 0.05
(See figure on next page.)
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Fig. 6 (See legend on previous page.)
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Huangetal. Stem Cell Research & Therapy (2022) 13:248
transcription and cell migration, positive regulation of
apoptotic process, and intracellular signal transduction
(Fig.6D). On the other hand, the downregulated DEGs
were associated with cell cycle, cell division, mitotic
nuclear division, cell adhesion, and cell proliferation
(Fig.6D).
We listed the upregulated and downregulated genes in
some of the GO enrichment terms (Fig.6E). e expres-
sion levels of Cdkn2b, Wnt11, Pdgfb, Iqgap3, Racgap1,
and Cd248 were verified by qPCR, and the results were
consistent with the RNA-seq (Fig.6F).
Taken together, these data suggest that Cdc42 plays a
role in cell proliferation, migration, adhesion, and cell
cycle of ADSCs. Furthermore, the signaling in wnt, foxo,
cell cycle, p53, and TGF-β pathway may contribute to
Cdc42-regulated biological function of ADSCs.
RNA sequencing identies thepossible mechanism
ofCdc42 intheregulation ofactivin B‑induced ADSCs
biological function
To further identify the mechanism of Cdc42 in the regu-
lation of activin B-induced ADSCs biological function,
RNA sequencing was performed in activin B+ADSCs
group and activin B+ADSCs (Cdc42N17) group. ere
was obvious difference between the two groups (Addi-
tional file 1: Fig. S6). Activin B+ADSCs (Cdc42N17)
group showed 355 upregulated DEGs and 615 downregu-
lated DEGs (Fig.7A).
KEGG pathway enrichment analysis revealed that
the upregulated DEGs were enriched in the pathway of
metabolism of xenobiotics by cytochrome P450, drug
metabolism—cytochrome P450, glutathione metabo-
lism, cytokine–cytokine receptor interaction, and pro-
teoglycans in cancer (Fig.7B). e downregulated DEGs
were enriched in the pathway of ECM–receptor interac-
tion, focal adhesion, Rap1, MAPK, TGF-β, and PI3K-Akt
(Fig.7C).
In the GO annotations analysis, the upregulated DEGs
were enriched in the biological processes of oxidation–
reduction process, transport, negative regulation of cell
proliferation, and aging (Fig. 7D). e downregulated
DEGs were enriched in the biological processes of cell
adhesion, signal transduction, positive regulation of cell
proliferation and migration, cell cycle, and angiogenesis
(Fig.7D). e detailed selected gene expression changes
in the biological processes of apoptosis, negative regula-
tion of proliferation, aging, migration, proliferation, and
differentiation are shown in Fig.7E.
Moreover, the changes in selected gene expression such
as Bmp6, Nkd1, Cd248, Id4, and Sox11 were confirmed
by qPCR (Fig.7F).
Taking together, these data imply that Cdc42 plays a
role in activin B-induced signal transduction, cell adhe-
sion, cell migration, and cell proliferation of ADSCs. Fur-
thermore, the signaling pathway of PI3K-Akt, pathway
in cancer, focal adhesion, Rap1, MAPK, and TGF-β may
contribute to Cdc42’s regulation of activin B-induced
biological function of ADSCs.
The Erk‑Srf pathway isinvolved inCdc42‑mediated activin
B‑induced ADSCs proliferation
Among the differentially expressed genes in the activin
B+ADSCs and activin B+ADSCs (Cdc42N17) group,
several members of the MAPK signaling pathways were
downregulated in activin B+ADSCs (Cdc42N17) group
(Fig. 8A). We confirmed the downregulation of Srf in
activin B+ADSCs (Cdc42N17) group by qPCR and west-
ern blot (Fig.8B, C). For Srf is a major downstream cyto-
solic transcription factor of Erk/MAPK signaling [41],
the effect of Cdc42 on Erk/MAPK activity and Srf expres-
sion was further explored. e result of qPCR and west-
ern blot confirmed that while Erk and its downstream Srf
were increased upon activin B treatment, this increase
was abolished by inhibition of Cdc42 (Fig.8B–F). Further
study of the mechanism revealed that the activation of
Erk signal in ADSCs by activin B was able to induce pro-
liferation. However, Erk inhibitor SCH772984 abolished
the growth of ADSCs with activin B (Fig. 8G, H). e
inhibition of Srf by CCG-100602 also suppressed ADSCs
proliferation induced by activin B (Fig.8G, H). ese data
suggest that Erk-Srf pathway is involved in Cdc42-medi-
ated activin B-induced ADSCs proliferation.
Discussion
We have previously found that activin B is able to induce
migration of ADSCs to promote skin wound healing
[13]. However, whether Cdc42 signaling is important in
ADSCs remained unknown. e current study found
that Cdc42 signaling is essential for ADSCs-mediated
(See figure on next page.)
Fig. 7 Cdc42 regulates activin B-induced ADSCs transcriptional signature. A Heatmap showing differentially expressed genes (DEGs) between
activin B+ADSCs and activin B+ADSCs (Cdc42N17). There were 355 upregulated (Red) and 615 (Blue) downregulated DEGs in activin B+ADSCs
(Cdc42N17), respectively, and were subjected to Kyoto Encyclopedia of Genes and Genomes pathways (KEGG) enriched and gene ontology (GO)
functional enrichment analysis. B and C showing the genes top 15 enrich pathway, while D showing the top 15 GO terms in upregulated and
downregulated DEGs. E Selected gene expression in some of the GO terms. F qPCR analysis of relative expression level of Bmp6, Nkd1, Cd248, Id4,
and Sox11 in Act B+ADSCs (Cdc42N17). All values are expressed as mean ± SD from three independent repeats. *P < 0.05
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Fig. 7 (See legend on previous page.)
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Huangetal. Stem Cell Research & Therapy (2022) 13:248
Fig. 8 Activin B-induced ADSCs proliferation by activating Cdc42-Erk-Srf pathway. A Genes expression related to MAPK signaling in Act B+ADSCs
(Cdc42N17) group. B qPCR analysis was performed to confirm the relative mRNA expression level of Srf in four groups. C, E Western blot assays
were performed to detect the level of Srf and phosphorylation Erk (p-Erk) in ADSCs, activin B+ADSCs, ADSCs (SCH772984) (Erk1/2 inhibitor) or
ADSCs (Cdc42N17) treated with or without activin B, respectively. D, F The band and the relative quantification levels of the expression of Srf
and phosphorylation Erk were analyzed. G Representative fluorescence imaging of EdU staining of ADSCs and ADSCs treated with Srf inhibitor
CCG-100602 or Erk1/2 inhibitor SCH772984 and with/without activin B at 6 h. Scale bar = 50 μm. H The proliferation rates were quantified by
calculating the percentage of EdU-positive cells. All values are expressed as mean ± SD from three independent repeats. *P < 0.05
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Huangetal. Stem Cell Research & Therapy (2022) 13:248
cutaneous wound healing as well as proliferation and
secretion. While our previous study found that Cdc42
regulates BMSCs-mediated wound healing by affecting
Golgi reorientation [20], this study found that Cdc42
regulates ADSCs-mediated cutaneous wound healing by
affecting VEGF-mediated vascularization, myofibroblast
differentiation-mediated granulation tissue formation,
and Erk-Srf signaling pathway.
Cutaneous wound healing is a dynamic process that
involves overlapping phases, including inflammation,
granulation tissue formation, and matrix remodeling [42].
ADSCs have been shown to enhance wound healing via
their paracrine function [43]. e present study suggests
that Cdc42 signaling is partially responsible for activin
B-induced secretion of VEGF in ADSCs. VEGF stimulates
wound healing through angiogenesis [44]. Consistently,
we have found that the ADSCs-mediated vascularization
in cutaneous wound healing is suppressed upon Cdc42
suppression, as the expression of CD31 is decreased in
ADSCs (Cdc42N17) group induced by activin B. ese
data suggest that Cdc42 regulates activin B-induced
ADSCs-mediated vascularization in cutaneous wound
healing via regulation of VEGF secretion.
Myofibroblast, mostly characterized by high expression
of α-smooth muscle actin (α-SMA), is the major cell type
responsible for ECM production during wound healing
[45]. A recent study has shown that activin A promotes
myofibroblast differentiation of endometrial mesenchy-
mal stem cells [46]. In the present study, we found that
activin B may also promote myofibroblast differentiation
of ADSCs, as evidenced by an up-regulation of the expres-
sion of α-SMA in the wounds of activin B+ADSCs group.
In addition, we have found that the expressions of α-SMA
in the wounds treated with ADSCs (Cdc42N17) were
decreased compared with that of ADSCs group. Further-
more, our animal studies have revealed that the wound
granulation tissue formation is impaired upon Cdc42
suppression. ese data indicate that Cdc42 regulates the
ADSCs-mediated granulation tissue formation in cutane-
ous wound healing via regulation of myofibroblast differ-
entiation. In line with this, Ge’s study shows that Cdc42 is
required for TGFβ1-induced α-SMA expression in MSCs
[18]. ey have found that TGFβ1-induced expression
of α-SMA is significantly decreased in Cdc42 knockout
BMSCs after 24-h stimulation [18]. Hence, Cdc42 is nec-
essary for myofibroblast differentiation of MSCs (e.g.,
BMSCs and ADSCs).
Comprehensive understanding of the mechanism of
ADSCs in the wound healing process is of great signifi-
cance for further improving wound healing effect. Our
RNA sequencing begins to reveal potential mechanism
of Cdc42 in the regulation of activin B-induced ADSCs
biological function. e RNA sequencing results provide
evidence that Cdc42 globally controls gene expression pat-
tern of ADSCs treated with/without activin B and indicate
possible signaling pathways downstream of Cdc42 that is
associated with the regenerative ability of ADSCs. e Erk/
MAPK pathway is an essential intracellular signal trans-
duction pathway that controls cell proliferation [47, 48].
e previous study showed that exendin-4 treatment pro-
motes ADSCs growth via ERK signaling pathways [49]. In
the present study, we found that activin B contributes to
ADSCs proliferation via Cdc42-Erk-Srf pathway. Our find-
ings may help understand the comprehensive molecular
mechanisms of ADSCs in wound healing.
Conclusions
e present study shows that Cdc42 is a key regulator
of ADSCs-mediated cutaneous wound healing induced
by activin B. Firstly, Cdc42 regulates activin B-induced
migration and proliferation in ADSCs. Secondly, Cdc42
regulates ADSCs-mediated vascularization in cutaneous
wound healing via regulation of VEGF secretion. irdly,
Cdc42 regulates ADSCs-mediated granulation tissue for-
mation in cutaneous wound healing via regulation of
myofibroblast differentiation. In addition, RNA sequenc-
ing further identifies the potential mechanism of Cdc42
in the regulation of activin B-induced ADSCs biological
function. Last but not the least, activin B might activate
the Cdc42-Erk-Srf signaling pathway to promote the pro-
liferation of ADSCs in the skin wound healing process.
is study significantly advances the understanding of
the function of Cdc42 in the ADSCs-mediated cutaneous
wound healing.
Abbreviations
ADSCs: Adipose-derived mesenchymal stem cells; Act B: Activin B; Cdc42N17:
Cdc42 dominant-negative mutant; Cdc42L61: Cdc42 constitutively active
mutant; Srf: Serum response factor; Erk: Extracellular signal-regulated kinase;
MAPK: Mitogen-activated protein kinase; H-DMEM: High-glucose Dulbecco’s
modified Eagle’s medium; PBS: Phosphate-buffered saline; DAPI: 4,6-Diamid-
ino-2-phenylindole; EdU: 5-Ethynyl-2’-deoxyuridine; IHC: Immunohistochem-
istry; SDS-PAGE: Sodium dodecyl sulfate–polyacrylamide gel electrophoresis;
RNA-seq: RNA sequencing; DEGs: Differential expression genes; GO: Gene
ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; qPCR: Quantita-
tive real-time polymerase chain reaction; H&E: Hematoxylin and eosin.
Supplementary Information
The online version contains supplementary material available at https:// doi.
org/ 10. 1186/ s13287- 022- 02918-9.
Additional le1. Experimental details (characterization of ADSCs; the
detail methods for dehydration, paraffin embedding, and H&E; GST
pull-down assay and western blot); the criteria for histological evaluation
of cutaneous wound healing; the primers used in this study; characteriza-
tion of ADSCs; ADSCs transduced with lentivirus containing Cdc42N17,
Cdc42L61, and the EGFP vector; ADSCs participated in cutaneous wound
healing; Cdc42 regulates activin B-induced collagen deposition and matu-
ration after wounding; gene expression patterns analysis between ADSCs
and ADSCs (Cdc42N17); gene expression patterns analysis between
activin B+ADSCs and activin B+ADSCs (Cdc42N17).s
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Acknowledgements
Not applicable.
Author contributions
SH and XW were responsible for the design of the study, performed the
experiments, collected and analyzed data, and wrote the manuscript. MZ,
MH, YY, YC, and CZ helped perform the main experiments and helped with
the surgical design of the animal model. YZ, JX, LB, RF, and HT participated in
cell experiments, animal surgeries, and animal care. Lu Z and Lin Z collectively
oversaw the collection of data and data interpretation and revised the manu-
script. All authors read and approved the final manuscript.
Funding
This work is supported by the National Natural Science Foundation of China
(81872514, 82073417, 81971297), Guangdong Basic and Applied Basic
Research Foundation (2020A1515011067, 2019A1515011213), Natural Science
Foundation of Guangdong Province (2018B030311062), and Program for
Changjiang Scholars and Innovative Research Team in University (IRT 16R37).
Availability of data and materials
The raw sequence data reported in this paper have been deposited in the
Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2021)
in National Genomics Data Center (Nucleic Acids Res 2022), China National
Center for Bioinformation/Beijing Institute of Genomics, and Chinese Acad-
emy of Sciences (GSA: CRA006666) that are publicly accessible at https:// ngdc.
cncb. ac. cn/ gsa. All data generated or analyzed during this study are included
in this published article and its Additional file 1.
Declarations
Ethics approval and consent to participate
All animal experiments were performed under an institutionally approved pro-
tocol for the use of animal research of Southern Medical University (Approval
number: L2019084).
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1 Depar tment of Histology and Embryology, NMPA Key Laboratory for Safety
Evaluation of Cosmetics, Key Laboratory of Construction and Detection in Tis-
sue Engineering of Guangdong Province, School of Basic Medical Sciences,
Southern Medical University, Guangzhou 510515, People’s Republic of China.
2 Key Laboratory of Functional Proteomics of Guangdong Province, Key Labo-
ratory of Mental Health of the Ministry of Education, School of Basic Medical
Sciences, Southern Medical University, Guangzhou 510515, China. 3 Depart-
ment of Orthopedics, Third Affiliated Hospital of Southern Medical Univer-
sity, Academy of Orthopedics Guangdong Province, Guangzhou 510630,
Guangdong, China.
Received: 8 October 2021 Accepted: 2 April 2022
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