Exploring the Potential Effects of Cryopreservation on the Biological Characteristics and Cardiomyogenic Differentiation of Rat Adipose-Derived Mesenchymal Stem Cells.

Cryopreservation is essential for the broad clinical application of mesenchymal stem cells (MSCs), yet its impact on their cellular characteristics and cardiomyogenic differentiation potential remains a critical concern in translational medicine. This study aimed to evaluate the effects of cryopreservation on the biological properties and cardiomyogenic capacity of rat adipose-derived MSCs (AD-MSCs). We examined their cellular morphology, surface marker expression (CD29, CD90, CD45), trilineage differentiation potential (adipogenic, osteogenic, chondrogenic), and gene expression profiles for the pluripotency marker REX1 and immunomodulatory markers TGFß1 and IL-6. After inducing cardiomyocyte differentiation, we assessed cardiac-specific gene expressions (Troponin I, MEF2c, GSK-3ß) using quantitative RT-qPCR, along with live/dead cell staining and immunofluorescence for cardiac-specific proteins (Troponin T, α-actinin, Myosin Heavy Chain). Cryopreserved AD-MSCs preserved their morphology, surface markers, and differentiation potential, but exhibited a reduced expression of REX1, TGFß1, and IL-6. Additionally, cryopreservation diminished cardiomyogenic differentiation, as indicated by the lower levels of Troponin I, MEF2c, and GSK-3ß seen compared to non-cryopreserved cells. Despite this, high cell viability (>90%) and maintained cardiac protein expression were observed post-cryopreservation. These findings highlight the necessity of optimizing cryopreservation protocols to ensure the full therapeutic potential of AD-MSCs, particularly in applications related to cardiac regenerative medicine.

Optimizing Cardiomyocyte Differentiation: Comparative Analysis of Bone Marrow and Adipose-Derived Mesenchymal Stem Cells in Rats Using 5-Azacytidine and Low-Dose FGF and IGF Treatment.

Mesenchymal stem cells (MSCs) exhibit multipotency, self-renewal, and immune-modulatory properties, making them promising in regenerative medicine, particularly in cardiovascular treatments. However, optimizing the MSC source and induction method of cardiac differentiation is challenging. This study compares the cardiomyogenic potential of bone marrow (BM)-MSCs and adipose-derived (AD)-MSCs using 5-Azacytidine (5-Aza) alone or combined with low doses of Fibroblast Growth Factor (FGF) and Insulin-like Growth Factor (IGF). BM-MSCs and AD-MSCs were differentiated using two protocols: 10 µmol 5-Aza alone and 10 µmol 5-Aza with 1 ng/mL FGF and 10 ng/mL IGF. Morphological, transcriptional, and translational analyses, along with cell viability assessments, were performed. Both the MSC types exhibited similar morphological changes; however, AD-MSCs achieved 70-80% confluence faster than BM-MSCs. Surface marker profiling confirmed CD29 and CD90 positivity and CD45 negativity. The differentiation protocols led to cell flattening and myotube formation, with earlier differentiation in AD-MSCs. The combined protocol reduced cell mortality in BM-MSCs and enhanced the expression of cardiac markers (MEF2c, Troponin I, GSK-3ß), particularly in BM-MSCs. Immunofluorescence confirmed cardiac-specific protein expression in all the treated groups. Both MSC types exhibited the expression of cardiac-specific markers indicative of cardiomyogenic differentiation, with the combined treatment showing superior efficiency for BM-MSCs.

Molecular changes in endometrium origin stromal cells during initiation of cardiomyogenic differentiation induced with Decitabine, Angiotensin II and TGF- ß1.

Stem cells' differentiation toward cardiac lineage is a complex process dependent on various alterations in molecular basis and regulation pathways. The aim of the study is to show that endometrium-derived stromal cells - menstrual, endometrial and endometriotic, could be an attractive source for examination of the mechanisms underlying cardiomyogenesis. After treatment with Decitabine, Angiotensin II and TGF-ß1, cells demonstrated morphological dedifferentiation into early cardiomyocyte-like cells and expressed CD36, CD106, CD172a typically used to sort for human pluripotent stem cell-derived cardiomyocytes. RT-qPCR revealed changed cells' genetic profiles, as majority of cardiac lineage differentiation related genes and cardiac ion channels (calcium, sodium, potassium) coding genes were upregulated after 6 and 13 days of exposure. Additionally, analysis of expression of various signaling proteins (FOXO1, PDGFB, TGFBR1, mTOR, VEGFA, WNT4, Notch1) coding genes showed differences between cell cultures as they seem to employ distinct signaling pathways through differentiation initiation. Early stages of differentiation had biggest impact on cardiomyogenesis related proteins (Nkx-2.5, EZH2, FOXO3a, H3K9Ac) levels, as we noticed after conducting Western blot and as expected, early cardiac transcription factor Nkx-2.5 was highly expressed and localized in nucleus of differentiating cells. These findings led us to assess endometrium origin stromal cells' potential to differentiate towards cardiomyogenic lineage and better understand the regulation of complex differentiation processes in ex vivo model systems.

Investigation of impacts of decellularized heart extracellular matrix and VEGF on cardiomyogenic differentiation of mesenchymal stem cell through Notch/Hedgehog signaling pathways.

OBJECTIVE: Decellularization is the process to obtain natural scaffolds with tissue integrity and extracellular matrix components, and recellularization is used to produce tissue-like constructs with specific cell types. In this study, rat bone marrow-derived mesenchymal stem cells (rBM-MSCs) were cultured on decellularized heart extracellular matrix. These cells were then induced to differentiate into cardiomyogenic cells under the stimulatory effect of vascular endothelial growth factor (VEGF) and other chemicals. This study aimed to investigate the effect of the cardiac extracellular matrix and VEGF on cardiomyogenic differentiation in the context of the Notch and Hedgehog signaling pathways. METHODS: Heart samples extracted from rats were decellularized by serial application of detergent to remove cells from the tissue, and then recellularized with rBM-MSCs. The recellularized tissue matrices were then analyzed for cardiomyogenesis. Cardiomyogenic differentiation was performed on decellularized heart extracellular matrix (ECM; three-dimensional scaffolds) and culture plates (two-dimensional cell culture system) for 28 days to understand the effects of the heart extracellular matrix. In addition, differentiation was induced with and without the stimulatory effect of VEGF to understand the effect of VEGF on cardiomyogenic differentiation of rBM-MSCs. RESULTS: Immunofluorescence staining showed that decellularization of the heart was performed effectively and successfully. After decellularization process, the heart extracellular matrix was completely free of cells. It was observed that rBM-MSCs transplanted onto the heart extracellular matrix remained viable and proliferated for 21 days after recellularization. The rBM-MSCs promoted cardiomyogenic differentiation in the conventional differentiation medium but were inversely affected by both VEGF and heart extracellular matrix proteins. Lower expression of connexin43 and cardiac troponin I genes was observed in cells induced by either matrix proteins or VEGF, compared to cells differentiated by chemical agents alone. CONCLUSION: In this study, we investigated the effect of decellularized heart extracellular matrix and VEGF on cardiomyogenic differentiation of rBM-MSCs. On the decellularized cardiac extracellular matrix, rBM-MSCs maintained their viability by adhering to the matrix and proliferating further. The adhesion of the cells to the matrix also produced a physical stimulus that led to the formation of histological structures resembling myocardial layers. Chemical stimulation of the decellularized heart extracellular matrix and cardiomyogenic differentiation supplements resulted in increased expression of cardiomyogenic biomarkers through modulation of the Notch and Hedgehog signaling pathways.

Graphene Oxide-Coated Patterned Silk Fibroin Films Promote Cell Adhesion and Induce Cardiomyogenic Differentiation of Human Mesenchymal Stem Cells.

Cardiac tissue engineering is a promising strategy for the treatment of myocardial damage. Mesenchymal stem cells (MSCs) are extensively used in tissue engineering. However, transformation of MSCs into cardiac myocytes is still a challenge. Furthermore, weak adhesion of MSCs to substrates often results in poor cell viability. Here, we designed a composite matrix based on silk fibroin (SF) and graphene oxide (GO) for improving the cell adhesion and directing the differentiation of MSCs into cardiac myocytes. Specifically, patterned SF films were first produced by soft lithographic. After being treated by air plasma, GO nanosheets could be successfully coated on the patterned SF films to construct the desired matrix (P-GSF). The resultant P-GSF films presented a nano-topographic surface characterized by linear grooves interlaced with GO ridges. The P-GSF films exhibited high protein absorption and suitable mechanical strength. Furthermore, the P-GSF films accelerated the early cell adhesion and directed the growth orientation of MSCs. RT-PCR results and immunofluorescence imaging demonstrated that the P-GSF films significantly improved the cardiomyogenic differentiation of MSCs. This work indicates that patterned SF films coated with GO are promising matrix in the field of myocardial repair tissue engineering.

Comprehensive transcriptome-wide analysis of spliceopathy correction of myotonic dystrophy using CRISPR-Cas9 in iPSCs-derived cardiomyocytes.

CTG repeat expansion (CTGexp) is associated with aberrant alternate splicing that contributes to cardiac dysfunction in myotonic dystrophy type 1 (DM1). Excision of this CTGexp repeat using CRISPR-Cas resulted in the disappearance of punctate ribonuclear foci in cardiomyocyte-like cells derived from DM1-induced pluripotent stem cells (iPSCs). This was associated with correction of the underlying spliceopathy as determined by RNA sequencing and alternate splicing analysis. Certain genes were of particular interest due to their role in cardiac development, maturation, and function (TPM4, CYP2J2, DMD, MBNL3, CACNA1H, ROCK2, ACTB) or their association with splicing (SMN2, GCFC2, MBNL3). Moreover, while comparing isogenic CRISPR-Cas9-corrected versus non-corrected DM1 cardiomyocytes, a prominent difference in the splicing pattern for a number of candidate genes was apparent pertaining to genes that are associated with cardiac function (TNNT, TNNT2, TTN, TPM1, SYNE1, CACNA1A, MTMR1, NEBL, TPM1), cellular signaling (NCOR2, CLIP1, LRRFIP2, CLASP1, CAMK2G), and other DM1-related genes (i.e., NUMA1, MBNL2, LDB3) in addition to the disease-causing DMPK gene itself. Subsequent validation using a selected gene subset, including MBNL1, MBNL2, INSR, ADD3, and CRTC2, further confirmed correction of the spliceopathy following CTGexp repeat excision. To our knowledge, the present study provides the first comprehensive unbiased transcriptome-wide analysis of the differential splicing landscape in DM1 patient-derived cardiac cells after excision of the CTGexp repeat using CRISPR-Cas9, showing reversal of the abnormal cardiac spliceopathy in DM1.

Cardiomyogenic Differentiation Potential of Human Dilated Myocardium-Derived Mesenchymal Stem/Stromal Cells: The Impact of HDAC Inhibitor SAHA and Biomimetic Matrices.

Dilated cardiomyopathy (DCM) is the most common type of nonischemic cardiomyopathy characterized by left ventricular or biventricular dilation and impaired contraction leading to heart failure and even patients' death. Therefore, it is important to search for new cardiac tissue regenerating tools. Human mesenchymal stem/stromal cells (hmMSCs) were isolated from post-surgery healthy and DCM myocardial biopsies and their differentiation to the cardiomyogenic direction has been investigated in vitro. Dilated hmMSCs were slightly bigger in size, grew slower, but had almost the same levels of MSC-typical surface markers as healthy hmMSCs. Histone deacetylase (HDAC) activity in dilated hmMSCs was 1.5-fold higher than in healthy ones, which was suppressed by class I and II HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) showing activation of cardiomyogenic differentiation-related genes alpha-cardiac actin (ACTC1) and cardiac troponin T (TNNT2). Both types of hmMSCs cultivated on collagen I hydrogels with hyaluronic acid (HA) or 2-methacryloyloxyethyl phosphorylcholine (MPC) and exposed to SAHA significantly downregulated focal adhesion kinase (PTK2) and activated ACTC1 and TNNT2. Longitudinal cultivation of dilated hmMSC also upregulated alpha-cardiac actin. Thus, HDAC inhibitor SAHA, in combination with collagen I-based hydrogels, can tilt the dilated myocardium hmMSC toward cardiomyogenic direction in vitro with further possible therapeutic application in vivo.

Silver nanoparticles induce the cardiomyogenic differentiation of bone marrow derived mesenchymal stem cells via telomere length extension.

Finding new strategies for the treatment of heart failures using stem cells has attracted a lot of attention. Meanwhile, nanotechnology-based approaches to regenerative medicine hypothesize a possible combination of stem cells and nanotechnology in the treatment of diseases. This study aims to investigate the in vitro effect of silver nanoparticles (Ag-NPs) on the cardiomyogenic differentiation of bone marrow-derived mesenchymal stem cells (BM-MSCs) through detection of cardiac markers. For this purpose, MSCs were isolated from bone marrow resident and differentiated to the cardiac cells using a dedicated medium with Ag-NPs. Also, the cardiomyogenic differentiation of BM-MSCs was confirmed using immunocytochemistry. Then, real-time PCR and western blotting assay were used for measuring absolute telomere length (TL) measurement, and gene and protein assessment of the cells, respectively. It was found that 2.5 µg/mL Ag-NPs caused elongation of the telomeres and altered VEGF, C-TnI, VWF, SMA, GATA-4, TERT, and cyclin D protein and gene expression in the cardiomyogenically differentiated BM-MSCs. Also, there was a significant increase in the protein and gene expression of Wnt3 and ß-catenin as main components of pathways. We concluded that Ag-NPs could change the in vitro expression of cardiac markers of BM-MSCs via the Wnt3/ß-catenin signaling pathway.

Uniformly-aligned gelatin/polycaprolactone fibers promote proliferation in adipose-derived stem cells and have distinct effects on cardiac cell differentiation.

Cardiac tissue engineering is a promising technique to regenerate cardiac tissue and treat cardiovascular disease. Here we applied a modified method to generate ultrafine uniformly-aligned composite gelatin/polycaprolactone fibers that mimic functional heart tissue. We tested the physical properties of these fibers and analyzed how these composite fibrous scaffolds affected growth and cardiac lineage differentiation in rat adipose-derived stem cells (rADSCs). We found that uniformly aligned composite fiber scaffolds had an anisotropic arrangement, functional mechanical properties, and strong hydrophilicity. The anisotropic scaffolds improved cell attachment, viability, and proliferative capacity of ADSCs over randomly-aligned scaffolds. Furthermore, uniformly aligned composite fiber scaffolds increased the efficiency of cardiomyogenic differentiation, but might reduce the efficiency of cardiac conduction system cell differentiation in ADSCs compared to randomly-oriented scaffolds and tissue culture polystyrene. However, the randomly-oriented composite scaffolds showed no obviously facilitated effects over tissue culture polystyrene on the two cells' differentiation process. The above results indicate that the scaffold fiber alignment has a greater effect on cell differentiation than the composition of the scaffold. Together, the uniformly-aligned composite fibers displayed excellent physical and biocompatible properties, promoted ADSC proliferation, and played distinct roles in the differentiation of cardiomyogenic cells and cardiac conduction system cells from ADSCs. These results provide new insight for the application of anisotropic fibrous scaffolds in cardiac tissue engineering for both in vitro and in vivo research.

Novel approach to enhance aggregate migration-driven epigenetic memory which induces cardiomyogenic differentiation on a dendrimer-immobilized surface.

The dynamic migratory behavior of human mesenchymal stem cells (hMSCs) has a significant impact on the epigenetic profiles that determine fate choice and lineage commitment during differentiation. Here we report a novel approach to enhance repeated migration-driven epigenetic memory which induces cardiomyogenic differentiation on a dendrimer surface with fifth generation (G5). Cells exhibited the formation of cell aggregates on the G5 surface through active migration with morphological changes, and these aggregates showed strong expression of the cardiac-specific marker cardiac troponin T (cTnT) at 10 days. When cell aggregates were passaged onto a fresh G5 surface over three passages of 40 days, the expression levels of the multiple cardiac-specific markers including GATA4, NKX2.5, MYH7, and TNNT2 were higher compared to those passaged as single cells. To investigate whether cardiomyogenic differentiation of hMSCs was enhanced by repeated aggregate migration-driven epigenetic memory, cells on the G5 surface were reseeded onto a fresh G5 surface during three passages using aggregate-based and single cell-based passage methods. Analyses of global changes in H3 histone modifications exhibited pattern of increased H3K9ac and H3K27me3, and decreased H3K9me3 in aggregate-based passage cultures during three passages. However, the pattern of their histone modification on the PS surface was repeated after the initialization and reformation during three passages in single cell-based passage cultures. Thus, repetitive aggregate migratory behavior during aggregate-based passage led to a greater degree of histone modification, as well as gene expression changes suggestive of cardiomyogenic differentiation.

Metabolic maturation of differentiating cardiosphere-derived cells.

Cardiosphere-derived cells (CDCs) can be expanded in vitro and induced to differentiate along the cardiac lineage. To recapitulate the phenotype of an adult cardiomyocyte, differentiating progenitors need to upregulate mitochondrial glucose and fatty acid oxidation. Here we cultured and differentiated CDCs using protocols aimed to maintain stemness or to promote differentiation, including triggering fatty acid oxidation using an agonist of peroxisome proliferator-activated receptor alpha (PPARα). Metabolic changes were characterised in undifferentiated CDCs and during differentiation towards a cardiac phenotype. CDCs from rat atria were expanded on fibronectin or collagen IV via cardiosphere formation. Differentiation was assessed using flow cytometry and qPCR and substrate metabolism was quantified using radiolabelled substrates. Collagen IV promoted proliferation of CDCs whereas fibronectin primed cells for differentiation towards a cardiac phenotype. In both populations, treatment with 5-Azacytidine induced a switch towards oxidative metabolism, as shown by changes in gene expression, decreased glycolytic flux and increased oxidation of glucose and palmitate. Addition of a PPARα agonist during differentiation increased both glucose and fatty acid oxidation and expression of cardiac genes. We conclude that oxidative metabolism and cell differentiation act in partnership with increases in one driving an increase in the other.

Differentiation of cardiomyocyte-like cells from human amniotic fluid mesenchymal stem cells by combined induction with human platelet lysate and 5-azacytidine.

Human amniotic fluid mesenchymal stem cells (hAF-MSCs) have been shown to be effective in the treatment of many diseases. Platelet lysate (PL) contains multiple growth and differentiation factors; therefore, it can be used as a differentiation inducer. In this study, we attempted to evaluate the efficiency of human platelet lysate (hPL) on cell viability and the effects on cardiomyogenic differentiation of hAF-MSCs. When treating the cells with hPL, the result showed an increase in cell viability. Expressions of cardiomyogenic specific genes, including GATA4, cTnT, Cx43 and Nkx2.5, were higher in the combined treatment groups of 5-azacytidine (5-aza) and hPL than the expressions of cardiomyogenic specific genes in the control group and in the 5-aza treatment group. In terms of the results of immunofluorescence and immunoenzymatic staining, the highest expressions of cardiomyogenic specific proteins were revealed in combined treatment groups. It can be summarized that hPL may be an effective supporting cardiomyogenic supplementary factor for cardiomyogenic differentiation in hAF-MSCs.

Histone Deacetylase Inhibitor Suberoylanilide Hydroxamic Acid Improves Energetic Status and Cardiomyogenic Differentiation of Human Dilated Myocardium-Derived Primary Mesenchymal Cells.

BACKGROUND: In this study the effect of histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) on the energetic status and cardiomyogenic differentiation of human healthy and dilated myocardium-derived mesenchymal stromal cells (hmMSC) have been investigated. METHODS: The hmMSC were isolated from the healthy and dilated post-operation heart biopsies by explant outgrowth method. Cell proliferation, HDAC activity, mitochondrial membrane potential, and level of adenosine triphosphate (ATP) were evaluated. The effect of SAHA on mitochondrial parameters has been investigated also by Seahorse XF analyzer and cardiomyogenic differentiation was confirmed by the expression of transcription factor NK2 Homeobox 5 (Nkx2.5), cardiac troponin T and alpha cardiac actin at gene and protein levels. RESULTS: Dilated myocardium-derived hmMSC had almost 1.5 folds higher HDAC activity compared to the healthy cells and significantly lower mitochondrial membrane potential and ATP level. HDAC class I and II inhibitor SAHA improved energetic status of mitochondria in dilated myocardium-isolated hmMSC and increased expression of cardiac specific proteins during 14 days of exposure of cells to SAHA. CONCLUSIONS: HDAC inhibitor SAHA can be a promising therapeutic for dilated cardiomyopathy (DCM). Dilated hmMSC exposed to SAHA improved energetic status and, subsequently, cardiomyogenic differentiation. Data suggest that human dilated myocardium-derived MSC still have cardio tissue regenerative potential, which might be stimulated by HDAC inhibitors.

Crataegus Extract WS®1442 Stimulates Cardiomyogenesis and Angiogenesis From Stem Cells: A Possible New Pharmacology for Hawthorn?

Extracts from the leaves and flowers of Crataegus spp. (i.e., hawthorn species) have been traditionally used with documented preclinical and clinical activities in cardiovascular medicine. Based on reported positive effects on heart muscle after ischemic injury and the overall cardioprotective profile, the present study addressed potential contributions of Crataegus extracts to cardiopoietic differentiation from stem cells. The quantified Crataegus extract WS®1442 stimulated cardiomyogenesis from murine and human embryonic stem cells (ESCs). Mechanistically, this effect was found to be induced by promoting differentiation of cardiovascular progenitor cell populations but not by proliferation. Bioassay-guided fractionation, phytochemical and analytical profiling suggested high-molecular weight ingredients as the active principle with at least part of the activity due to oligomeric procyanidines (OPCs) with a degree of polymerization between 3 and 6 (DP3-6). Transcriptome profiling in mESCs suggested two main, plausible mechanisms: These were early, stress-associated cellular events along with the modulation of distinct developmental pathways, including the upregulation of brain-derived neurotrophic factor (BDNF) and retinoic acid as well as the inhibition of transforming growth factor ß/bone morphogenetic protein (TGFß/BMP) and fibroblast growth factor (FGF) signaling. In addition, WS®1442 stimulated angiogenesis ex vivo in Sca-1+ progenitor cells from adult mice hearts. These in vitro data provide evidence for a differentiation promoting activity of WS®1442 on distinct cardiovascular stem/progenitor cells that could be valuable for therapeutic heart regeneration after myocardial infarction. However, the in vivo relevance of this new pharmacological activity of Crataegus spp. remains to be investigated and active ingredients from bioactive fractions will have to be further characterized.

Effect of ascorbic acid on differentiation of human amniotic fluid mesenchymal stem cells into cardiomyocyte-like cells.

The aim of this study was to evaluate the efficiency of ascorbic acid (AA) on cell viability, cytotoxicity and the effects on cardiomyogenic differentiation of the human amniotic fluid mesenchymal stem cells (hAF-MSCs). The results of methylthiazole tetrazolium (MTT) assay and cell apoptosis assay indicated that after 24, 48 and 72 h of treatment, AA had no effect on cells viability and cytotoxicity. After treating the hAF-MSCs with 5-azacytidine (5-aza) and a combination of AA and 5-aza, the alamar blue cells proliferation assay showed the normal growth characteristic similar to control group. Especially, the morphological changes were observed between day 0 and day 21, and it was revealed that the hAF-MSCs exhibited myotube-like morphology after 7 days of cell culturing. Moreover, the treatment with a combination of AA and 5-aza was able to up-regulate the cardiomyogenic specific gene levels, which are known to play an important role in cardiomyogenesis. This was specifically notable with the results of immunofluorescence and immunoenzymatic staining in the AA combined with 5-aza treatment group, the highest expression of cardiomyogenic specific proteins was revealed including for GATA4, cTnT, Cx43 and Nkx2.5. It could be concluded that AA may be a good alternative cardiomyogenic inducing factor for hAF-MSCs and may open new insights into future biomedical applications for a clinically treatment.

Biomimetic electrical stimulation induces rat bone marrow mesenchymal stem cells to differentiate into cardiomyocyte-like cells via TGF-beta 1 in vitro.

Electrical conductance is one of the factors of the microenvironment of cardiomyocytes, and electrical stimulation (ES) has been shown to modulate the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) toward a cardiomyogenic fate. Transforming growth factor-beta 1 (TGF-ß1) stimulates the cardiomyogenic marker expression in BMSCs. Herein, we promoted the differentiation of BMSCs into cardiomyocyte-like cells using ES to confirm if TGF-ß1 mediates this event in vitro. ES increased protein levels of TGF-ß1 in BMSCs, and this effect was better than that observed with 5-azacytidine (5-Aza). The effect of ES on promoting cardiomyogenic marker expression in BMSCs was enhanced by TGF-ß1. Furthermore, the protein expression levels of Connexin43 (Cx43) and Alpha-actinin 2 (ACTN2) induced by ES in BMSCs were significantly decreased by pirfenidone. These results show that ES promotes cardiomyocyte-like cells differentiation in rat BMSCs and is possibly mediated by TGF-ß1 in vitro.

Effect of equiaxial cyclic strain on cardiomyogenic induction in mesenchymal stem cells.

Differentiation of stem cells and functionality of target cells are regulated by microenvironmental stimuli to which the cells are exposed. Chemical agents such as growth factors and physical parameters including mechanical loadings are among major stimuli. In this study, equiaxial cyclic strain with two amplitudes was applied on rat adipose-derived mesenchymal stem cells (rAMSCs) with or without 5-azacytidine. The mRNA expression of cardiac-related genes was investigated through RT-PCR (polymerase chain reaction) method. Moreover, morphological features and the actin structure of the cells were studied. Results were indications of significant increase in mRNA expression among four target genes, which marked the increase in two principal cardiac markers of GATA4 and α-cardiac actin, and lesser increase in two other genes (NKX2-5, ßMHC) in all experimental groups treated chemically and/or mechanically. Such effect was maximal when both treatments were applied describing the synergistic effect of combined stimuli. All treatments caused significant increase in cell area and cell shape index. The well spreading of cells was accompanied by enhanced actin structure, especially among samples subjected to mechanical stimulus. Both effects were among required features for functional muscle cells such as cardiac cells. It was concluded that the cyclic equiaxial strain enhanced cardiomyogenic induction among rat adipose-derived mesenchymal stem cells and such effect was strengthened when it was accompanied by application of chemical factor. Results can be considered among strategies for cardiomyogenic differentiation and can be employed in cardiac tissue engineering for production of functional cardiomyocytes to repair of damaged myocardium.

Differentiation of brown adipose-derived stem cells into cardiomyocyte-like cells is regulated by a combination of low 5-azacytidine concentration and bone morphogenetic protein 4.

Adipose-derived stem cells (ADSCs) could be an ideal candidate for seed cells to regenerate damaged heart tissue. This study examined and compared the cardio-myogenic differentiation efficacy of neonatal rat brown ADSCs (rbADSCs) treated with either 5-azacytidine (5-AZA), bone morphogenetic protein 4 (BMP4), or lower doses of both molecules. Briefly, by investigating the protein expression of cardiac-specific markers (i.e., cardiac troponin-I, α-sarcomeric actinin, sarcoplasmic reticulum Ca2+-ATPase, and connexin 43), our data indicated that rbADSCs could be differentiated into cardiomyocyte-like cells by all three treatments. By quantitatively measuring the number of cells with positive staining for the above markers, we found that the low-dose combined treatment showed higher differentiation efficiency compared to standard dose 5-AZA and BMP4 treatment. Similarly, the expression levels of these proteins as determined by western blotting were higher in the low-dose combination group than in the standard dose 5-AZA and BMP4 groups. Also, the combined strategy maintained the decreased cell viability caused by cytotoxicity of 5-AZA, probably through reducing the ratio of apoptotic rbADSCs. Furthermore, the extracellular regulated protein kinase (ERK) signaling pathways participate in the differentiation process, but the observed effects between the BMP4 and 5-Aza treatments are quite different.

Stretchable Piezoelectric Substrate Providing Pulsatile Mechanoelectric Cues for Cardiomyogenic Differentiation of Mesenchymal Stem Cells.

Ex vivo induction of cardiomyogenic differentiation of mesenchymal stem cells (MSCs) before implantation would potentiate therapeutic efficacy of stem cell therapies for ischemic heart diseases because MSCs rarely undergo cardiomyogenic differentiation following implantation. In cardiac microenvironments, electric pulse and cyclic mechanical strain are sequentially produced. However, no study has applied the pulsatile mechanoelectric cues (PMEC) to stimulate cardiomyogenic differentiation of MSCs ex vivo. In this study, we developed a stretchable piezoelectric substrate (SPS) that can provide PMEC to human MSCs (hMSCs) for cardiomyogenic differentiation ex vivo. Our data showed that hMSCs subjected to PMEC by SPS underwent promoted cardiac phenotype development: cell alignment and the expression of cardiac markers (i.e., cardiac transcription factors, structural proteins, ion channel proteins, and gap junction proteins). The enhanced cardiac phenotype development was mediated by the upregulation of cardiomyogenic differentiation-related autocrine factor expression, focal adhesion kinase, and extracellular signal-regulated kinases signaling pathways. Thus, SPS providing electrical and mechanical regulation of stem cells may be utilized to potentiate hMSC therapies for myocardial infarction and provide a tool for the study of stem cell biology.

Characteristics and Cardiomyogenic Potential of Rat Fetal Cardiac Progenitor Cells at Different Developmental Stage.

In recent years, several kinds of cardiac progenitor cells have been identified and isolated from heart tissue. These cells showed differentiation potential into cardiomyocytes, smooth muscle cells, and endothelial cells in vitro and in vivo. Morphogenetic events are tightly regulated during development to determine cell destiny and reshape the embryonic lineage. In this study, we directly compared the characteristics of rat fetal cardiac progenitor cells (rFCPCs) isolated from the chamber formation stage at embryonic day 12 (E12) and at the septation stage of E15. Both kinds of rFCPCs expressed mesenchymal stem cell markers (CD105, CD73, and CD29) but not CD34 and CD45. The E12 rFCPCs expressed a high level of Oct4 compared to E15 until passage 5 and showed a steep decline of Nkx2.5 expression at passage 5. However, Nkx2.5 expression at E15 was maintained until passage 5 and Oct4 expression slightly increased at passage 5. We also detected an intense staining for Oct4 antibody in E12 heart tissue sections. The average doubling time of the E12 rFCPCs from passage 3 to passage 15 was about 5 hours longer than E15. These cells could also be induced into cardiomyocytes expressing α-MHC, cTnT, cTnC, and Cx43 under cardiomyogenic culture conditions and rFCPCs at E15 showed more intense staining of α-MHC than cells at E12 by immunocytochemistry. Taken together, our results show that developmental differences between E12 and E15 may influence their properties and differentiation. Furthermore those differences should be considered when deciding on the optimal cell source for cell replacement therapy in cardiovascular regeneration.