Cardiac Differentiation of Adipose Tissue-Derived Stem Cells Is Driven by BMP4 and bFGF but Counteracted by 5-Azacytidine and Valproic Acid
Hasani S, Javeri A, Asadi A, Taha MF. Cardiac differentiation of adipose tissue-derived stem cells is driven by BMP4 and bFGF but counteracted by 5-azacytidine and valproic acid. Cell J. 2020; 22(3): 273-282. doi:10.22074/cellj.2020.6582.
Bone morphogenetic protein 4 (BMP4) and basic fibroblast growth factor (bFGF) play important roles in embryonic heart development. Also, two epigenetic modifying molecules, 5ˊ-azacytidine (5ˊ-Aza) and valproic acid (VPA) induce cardiomyogenesis in the infarcted heart. In this study, we first evaluated the role of BMP4 and bFGF in cardiac trans-differentiation and then the effectiveness of 5´-Aza and VPA in reprogramming and cardiac differentiation of human adipose tissue-derived stem cells (ADSCs).
Materials and Methods
In this experimental study, human ADSCs were isolated by collagenase I digestion. For cardiac differentiation, third to fifth-passaged ADSCs were treated with BMP4 alone or a combination of BMP4 and bFGF with or without 5ˊ-Aza and VPA pre-treatment. After 21 days, the expression of cardiac-specific markers was evaluated by reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR, immunocytochemistry, flow cytometry and western blot analyses.
BMP4 and more prominently a combination of BMP4 and bFGF induced cardiac differentiation of human
ADSCs. Epigenetic modification of the ADSCs by 5ˊ-Aza and VPA significantly upregulated the expression of OCT4A,
Our findings demonstrated the inductive role of BMP4 and especially BMP4 and bFGF combination in cardiac trans-differentiation of human ADSCs. Treatment with 5ˊ-Aza and VPA reprogrammed ADSCs toward a more pluripotent state and increased tendency of the ADSCs for mesodermal differentiation. Although pre-treatment with 5ˊ-Aza and VPA counteracted the cardiogenic effects of BMP4 and bFGF, it may be in favor of migration, engraftment and survival of the ADSCs after transplantation.
Cardiovascular diseases are the most common causes of deaths worldwide (1). Despite the great advances in medical and surgical therapies, functional recovery of the infarcted heart remains elusive. A novel strategy for the treatment of advanced myocardial infarction is transplantation of stem cells or stem cell-derived cardiac progenitor cells into the damaged heart with the expectation that these cells can produce or stimulate generation of new cardiomyocytes and blood vessels in the injured tissue (2).
Adipose tissue has been considered as a valuable source of autologous mesenchymal stem cells for heart tissue engineering and cardiac repair. Beneficial role of adipose tissue-derived stem cells (ADSCs) in regeneration of ischemic heart disease is emanated from several properties, including differentiation to cardiomyocytes, endothelial cells and smooth muscle cells (3, 4), secretion of several angiogenic and anti-apoptotic factors (3, 5) and recruitment of endogenous stem cells into the damaged area (6). Although accumulating evidence has shown the capability of ADSCs for differentiation into cardiomyocytes and improvement of ventricular function in animal models of myocardial infarction (7-9), a highly efficient protocol for cardiac differentiation of human ADSCs is yet to be reported. Further studies are required to develop optimal media formulations which generate a large number of functional cardiomyocytes for embryology, toxicology, pharmacology and transplantation therapy purposes. In this regard, better understanding of the role of cardiogenic growth factors and small molecules which can reprogram somatic cells toward a more undifferentiated state is of utmost importance.
Bone morphogenetic protein 4 (BMP4) and basic fibroblast growth factor (bFGF) signaling play important roles in embryonic heart development (10, 11). A combination of bFGF and BMP2/4 has been shown necessary to induce Nkx2.5 expression and contractile phenotype in non-precardiac mesoderm of chicken embryos (12). In fact, BMPs and FGFs have complementary roles in cardiac development; BMP induces the specification of non-precardiac mesoderm cells to cardiac cell lineage, while FGF supports terminal differentiation of cardiomyocytes (13, 14).
5ʹ-azacytidine (5ʹ-Aza) and valproic acid (VPA) are two small molecules which regulate chromatin remodeling through inhibition of DNA methyltransferases and histone deacetylases, respectively (14). The positive role of 5ʹ-Aza and VPA in cardiac differentiation has been demonstrated by different groups (14-16), although contradictory results have also been reported (17, 18). In an attempt by Thal and colleagues (14), treatment of the endothelial progenitor cells with 5ʹ-Aza and VPA significantly upregulated the expression of pluripotency and cardiacspecific genes and increased the cardiogenic potential of the reprogrammed cells. However, this should be kept in mind that reactivation of previously silent genes by epigenetic modifiers like 5ʹ-Aza and VPA is not limited to pluripotency-associated or cardiacspecific genes but is rather indicative of a global gene transcription. So, an appropriate culture condition is necessary to direct the fate of reprogrammed cells towards a cardiogenic lineage (14).
Despite the available evidence demonstrating the inductive role of BMP4 and bFGF growth factors (10- 12) as well as small molecules like 5ʹ-Aza and VPA (14- 16) in cardiac differentiation, there is no report regarding the impact of these factors on cardiac differentiation of hADSCs. We previously showed that BMP4 treatment induces the expression of cardiac-specific markers in mouse ADSCs (19). In the current study, we first evaluated the role of BMP4 individually, and then in combination with bFGF in cardiac trans-differentiation of human ADSCs and finally examined the impact of 5ʹ-Aza and VPA on reprogramming and cardiac differentiation of the ADSCs.
Materials and Methods
Isolation and culture of human adipose tissue-derived stem cells
In this experimental study, adipose tissue samples were harvested from five 40-45 years old women undergoing elective abdominoplasty after obtaining informed consent. The study was approved by the Ethics Committee of National Institute of Genetic Engineering and Biotechnology (7-8-93/NIGEB).
Isolation and characterization of the ADSCs was performed as described previously (20). Briefly, adipose tissue was minced and digested by 2 mg/ml collagenase I (Thermo Fisher Scientific, USA) in PBS containing 2% bovine serum albumin (BSA, Sigma Aldrich, USA). The stromal vascular fraction (SVF) was plated at 5×104 cells/ ml in tissue culture flasks. Growth medium contained Dulbecco’s Modified Eagle’s Medium (DMEM), 20% fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin (all from Gibco, Thermo Fisher Scientific, USA). Medium was changed every other day, and the cells were subcultured after reaching 80-90% confluency.
Cardiac differentiation of human adipose tissuederived stem cells
For cardiac differentiation, third to fifth-passaged ADSC were seeded into 0.1% gelatin-coated 6-well tissue culture plates with a density of 105 cell/ml (2 ml per each well). After 24 hours, the cells were induced for cardiac differentiation with 10 ng/ml bFGF (Sigma-Aldrich, USA) and 20 ng/ml BMP4 (Thermo Fisher Scientific, USA) for four days. After the induction stage, growth factors were omitted completely and differentiation of the cells was continued in 10% FBS-containing medium up to three weeks. The ADSCs that were cultured in the same medium without bFGF and BMP4 treatment were used as the control group.
To investigate the impact of DNA methyltransferase and histone deacetylase inhibitors on cardiac differentiation of ADSCs, the cells were pre-treated with 10 µM 5ʹ-Azacitidine (Sigma-Aldrich, USA) and 500 nM VPA (Sigma-Aldrich, USA) for 24 hours and then were treated with 10 ng/ml bFGF and 20 ng/ml BMP4 as described above.
Gene expression analysis
Total RNAs were extracted from three-week
differentiated ADSCs using High Pure RNA Isolation
Kit (Roche Applied Science, Germany). Briefly,
cDNA was synthesized from 1 µg of total RNA
using cDNA Synthesis Kit (Thermo Fisher Scientific,
USA). PCR amplification on the cDNA samples
was performed using PCR master mix (Ampliqon,
Denmark) and specific primers, as described in Table
S1, (See Supplementary Online Information at
RealQ PCR Master (Ampliqon, Denmark) were used
for quantitative assessment of gene expression by realtime polymerase chain reaction (qPCR) on a RotorGeneTM 6000 (Corbett Research, Australia) real-time
analyzer. β2 microglobulin (
Comparative quantification was performed using
REST 2009 (Relative Expression Software Tool,
Qiagen) based on Pair Wise Fixed Reallocation
Randomization Test® (21). At least, three biological
replicates of each group were included in the qPCR
Since cell density of three-week differentiated ADSCs was too high, the cells were dissociated using trypsin-EDTA (Gibco, USA) and cultured at half the density in gelatin-coated 4-well tissue culture plates. After 24 hours, the cells were fixed using 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. 10% goat serum was used to block non-specific binding sites. Next, the cells were incubated with the primary monoclonal antibodies against α-actinin (Sigma-Aldrich, USA) and cardiac troponin I (Santa Cruz Biotechnology, USA) and then with goat antimouse FITC-conjugated IgG (Sigma-Aldrich, USA). The stained cells were observed by a fluorescence microscope (Nikon, Japan).
Flow cytometry analysis
Three-week differentiated cells were dissociated using trypsin-EDTA and fixed in cold 70% ethanol. The cells were permeabilized with 0.2% Triton X-100. After washing, the cells were incubated with the primary antibody against cardiac troponin I (Santa Cruz Biotechnology, USA) and then with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Sigma-Aldrich, USA). Some cells were only stained with the secondary antibody and were used as the negative control. Flow cytometry was performed using an Attune® Acoustic Focusing Cytometer (Applied Biosystems, Thermo Fisher Scientific, USA). FlowJo vX.0.6 software (Tree Star Inc., Ashland, USA) was used for analysis of the results.
Western blot analysis
For protein analysis by western blot, three-week differentiated ADSCs were homogenized in ice-cold Radioimmunopercipitation assay (RIPA) lysis buffer and were centrifuged at 13000 g for 15 minutes at 4˚C. After collecting the supernatant, protein concentration was determined by Bradford assay. For each sample, 50 µg of protein was separated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDSPAGE) and transferred to polyvinylidene difluoride (PVDF, Roche) membranes. Blocking of non-specific binding sites was achieved by 5% non-fat dried milk in Tris-buffered saline containing 0.1% Tween-20 (TBST). After blocking, the membranes were incubated with the diluted primary antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Sigma-Aldrich, G8795), α-actinin (Sigma-Aldrich, USA), desmin (Sigma-Aldrich, USA) and connexion 43 (Sigma-Aldrich, USA) overnight at 4˚C. Then, the membranes were incubated with goat anti-mouse horseradish peroxidase (HRP)-conjugated secondary IgG for 1 hour at room temperature. Enhanced chemiluminescence (ECL) kit (Najm Biotech Co., Iran) was used to detect the immunoreactive bands.
Isolation, culture and differentiation of human adipose tissue-derived stem cells
Within 3-4 hours, the ADSCs attached to the
growth surfaces of tissue culture plates. The ADSCs
proliferated rapidly and were passaged 2-3 times a
week. The undifferentiated cells showed a fibroblastlike morphology (Fig .1A,). The mesenchymal stem cell
feature and multipotential differentiation capability of
the ADSCs was determined as described previously by
our team (20, 22, 23). Third-passaged ADSCs expressed
cardiac transcription factors,
After three weeks cardiac differentiation in different experimental groups, including control (no treatment, Fig .1C,), BMP4 alone (Fig .1D,) and a combination of bFGF and BMP4 with or without pre-treatment with 5-Aza and VPA (Fig .1E, F,, respectively), differentiating ADSCs showed an elongated morphology.
BMP4 induces cardiac trans-differentiation of human adipose tissue-derived stem cells
We previously showed that BMP4 induces the expression
of cardiac-specific genes in mouse ADSCs (19). In the
current study, treatment of human ADSCs with 20 ng/
ml BMP4 upregulated the expression of
A combination of BMP4 and bFGF augments cardiac trans-differentiation of human ADSCs
To investigate the synergistic effect of BMP4 and
bFGF in cardiac differentiation of human ADSCs, third
to fifth-passaged ADSCs were simultaneously treated
with 10 ng/ml bFGF and 20 ng/ml BMP4 for the first
four days of differentiation. The expression levels of
Treatment of the ADSCs with 5ʹ-Aza and VPA upregulated the expression of some pluripotency and mesodermal genes
24 hours treatment of the undifferentiated ADSCs with
5ʹ-Aza and VPA upregulated the expression of
Pre-treatment with 5ʹ-Aza and VPA affected cardiac differentiation of human ADSCs
To elucidate the influence of DNA methyltransferase
and histone deacetylase inhibitors on cardiac
differentiation of human ADSCs, the cells were pretreated with 10 µM 5ʹ-Aza and 0.5 µM VPA for 24
hours and then were induced with 10 ng/ml bFGF and
20 ng/ml BMP4 in 10% FBS-containing medium. As
revealed by qPCR analysis, pre-treatment with 5ʹ-Aza
and VPA downregulated the expression of
Protein expression analysis
BMP4 treatment group and the groups which received a combination of BMP4 and bFGF with or without 5ʹ-Aza and VPA pre-treatment were assessed for the expression of α-actinin and cardiac troponin I as two cardiac-specific proteins. As revealed by immunocytochemistry, after trypsinization and re-plating the differentiated cells, ADSC-derived cardiomyocyte-like cells tend to form aggregations which showed positive immunostaining for α-actinin (Fig .5A-F,) and cardiac troponin I (Fig .5G-L,) proteins.
Western blot analysis demonstrated the expression of α-actinin, desmin and connexin 43 proteins in the differentiated cells. α-actinin and connexin 43 showed their maximum expression in the cells pre-treated with 5-Aza and VPA and followed by BMP4 and bFGF treatment (Fig .6A,). Based on flow cytometry analysis, about 21% of the cells in the BMP4 treatment group, 39% of the cells which treated with BMP4 and bFGF combination without 5-Aza and VPA pre-treatment and 18% of the cells pre-treated with 5-Aza and VPA and induced with BMP4 and bFGF combination showed positive staining for cardiac troponin I protein. In the control group, about 1.5% of the cells expressed cardiac troponin I protein (Fig .6B,).
In the current study, we first examined the influence of
BMP4 on cardiomyocyte trans-differentiation of human
ADCSs. BMPs are members of TGFβ superfamily
with essential roles in both mesoderm induction and
embryonic heart development (11). While increasing
evidence support the inductive role of BMPs in cardiac
differentiation, some studies point to the temporally
and spatially regulated expression of BMPs and BMP
antagonists during heart development (24). BMP2 and
BMP4 inhibit cardiomyogenesis during gastrula stage of
chicken embryos (25). In mouse, noggin show a transient
but strong expression in the anterolateral plate mesoderm
and has a critical role in cardiac differentiation (26).
Similar contradictory results have been obtained during
cardiac differentiation of embryonic and adult stem
cells. As reported by Yuasa et al. (26), inhibition of BMP
signalling in a period between the undifferentiated state
and early phase of embryoid body formation increases
the incidence of beating EBs and the expression of
cardiac transcription factors. We showed previously
that BMP4 treatment inhibits cardiac differentiation of
mouse embryonic stem cells (ESCs) in serum-containing
media (27), although the complete removal of serum is
not in favour of cardiomyocyte development (28). Some
other investigators have demonstrated the inductive role
of BMP4 in cardiac differentiation of human ESCs in a
serum-based condition (29). Treatment of human bone
marrow-derived mesenchymal stem cells (BM-MSCs)
with BMP4 shifts the fate of cells toward a cardiac
phenotype rather than the skeletal-like myocytes (30).
We previously showed that BMP4 treatment of mouse
ADSCs, especially in a knockout serum replacement
(KoSR)-containing medium, induces the expression of
cardiac-specific markers (19). In the current study, we
examined the effect of BMP4 on cardiac differentiation of
human ADSCs and showed that treatment of the ADSCs
with 20 ng/ml BMP4 increases the expression of
bFGF is a paracrine FGF with significant roles in
development and pathophysiology of the heart (10).
Barron et al. (12) showed that treatment of non-precardiac
mesoderm of stage 6 chicken embryos with a combination
of bFGF and BMP2/4 is necessary to induce Nkx2.5
expression and to promote contractile phenotype. In fact,
both BMPs and FGFs act as cardiac specification factors;
BMP specifies non-precardiac mesoderm cells to cardiac
lineage (12), while FGF functions as a survival factor
and supports their terminal differentiation (13). Here we
examined the role of bFGF-BMP4 combination in cardiac
differentiation of human ADSCs and showed that except
In this study,
Previous human clinical trials demonstrate the safety and efficacy of ADSCs for regeneration of myocardial infarction (34). However, a significant portion of this reparative function is emanated from secretion of several angiogenic and anti-apoptotic factors (3, 5) and recruitment of endogenous stem cells into the injury site (6). ADSCs rarely differentiate into cardiomyocytes in vivo (35), and even when collected from aged patients, they have a diminished capability for proliferation and differentiation (36). Epigenetic modification of ADSCs by small molecules may reprogram ADSCs towards a more pluripotent state, enhance their functional properties and improve their functionality after transplantation. In this study, we examined effectiveness of two epigenetic modifying molecules, 5ʹ-Aza and VPA, for reprogramming of human ADSCs towards a more undifferentiated state. 5ʹ-Aza and VPA, which are inhibitors of DNA methyltransferases and histone deacetylases respectively, have been used in generation of induced pluripotent stem cells (iPSCs) to improve reprogramming efficiency (37).
24 hours treatment of the undifferentiated ADSCs
with a combination of 5ʹ-Aza and VPA upregulated
the expression of some pluripotency transcription
We assessed the influence of 5ʹ-Aza and VPA on
cardiac differentiation of human ADSCs. It has been
shown that both chemical factors remodel chromatin to
allow expression of transcriptionally inactivated genes
and to induce differentiation toward cardiomyocytes
(15, 16). In 2012, Thal et al. (14) showed that epigenetic
reprogramming of endothelial progenitor cells with
5ʹ-Aza and VPA improves repair of infarcted hearts by
both cardiomyogenesis and vascularization. In contrast,
we showed here that pre-treatment with a combination
of 5ʹ-Aza and VPA downregulated the expression of
Stromal cell-derived factor (SDF)-1 and its membrane
receptor, CXCR4, play pivotal roles in the migration,
homing and engraftment of multiple stem cell types. At
the injury site, SDF-1 expression increases and recruits
circulating CXCR4-expressing MSC. Strategies to
induce CXCR4 upregulation increases the migration and
engraftment of MSCs
Our findings demonstrated that cardiac differentiation
of human ADSCs can be induced by BMP4 but more
significantly by a combination of BMP4 and bFGF.
Treatment of the ADSCs with a combination of 5ʹ-Aza and
VPA, which are respectively DNA methyltransferase and
histone deacetylase inhibitors, significantly upregulated
the expression of pluripotency transcription factors which
indicates reprogramming of the ADSCs towards a more
undifferentiated state. Downregulation of GSC and NES
and upregulation of Brachyury/T and
There is no financial support and conflict of interest in this study.
S.H.; Performed the experiments and wrote the draft. A.J.; Performed gene expression and flow cytometry analyses and contributed to editing and approving the manuscript for submission. A.A.; Contributed to the project as co-supervisor and participated in drafting. M.F.T.; Designed the study, supervised the project, performed western blot analysis and edited and approved the final version of manuscript for submission. All authors read and approved the final manuscript.