Differentiation Of Cardiac Stem Cells Research Articles

Dynamic links between the cardiac intermediate filaments of the cytoskeleton and nucleoskeleton for proper cardiomyocyte differentiation, maturation and reprogramming

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): 1) ESPA 09SYN-21-966 2) ELIDEK 1594 Accumulating evidence suggests that mechanical forces are integral to the commitment and differentiation of cardiac stem cells. Studies have proposed that the cardiac cytoskeleton and specifically the desmin intermediate filament (IF) network and its association with nuclear lamins through the LINC complex, link the contractile apparatus to both the extracellular and nuclear matrix, thus regulating trafficking processes and mechanotransduction during development and adulthood, governing myocyte homeostasis and survival(1-3). Desmin, in addition to being one of the earliest myogenic markers both in heart and somites(4,5), regulates nuclear shape and positioning(6,7), as well as the expression of myogenic (MyoD, Myogenin)(8) and cardiogenic (MEF2, Nkx2.5) transcription regulators, during embryonic stem cell differentiation(4,5). Consistent with the above findings, desmin deficiency has negative impact in the proper regeneration of skeletal muscle cells by hematopoietic stem cells(9). Despite these overwhelming data linking desmin to the establishment and maintenance of myogenesis and cardiogenesis, the precise underlying mechanisms remain elusive. To dissect the exact mechanism of cardiogenic regulation we took advantage of the fibroblast-to-cardiomyocyte direct reprogramming, where fibroblasts trans-differentiate into functional induced cardiomyocytes (iCM) through ectopic expression of three transcription factors, GATA4, Mef2C and Tbx5 (GMT)(10). We hypothesized that desmin could enhance the reprogramming of fibroblasts into cardiomyocytes. Indeed, we have found that expression of desmin together with the GMT factors increases reprogramming by 40-60%. Involvement of a cytoskeletal protein such as desmin in cell differentiation and potentially regeneration, raises intriguing questions and exciting possibilities. To unravel the mechanism of this enhancement we focused on desmin serving as direct mechanotransducer (through the LINC complex) to chromatin. Toward this goal, we investigate the changes of the genetic profile induced by the GMT cocktail during reprogramming in the presence or absence of desmin. RNA sequence analysis has shown that the Notch developmental pathway is regulated by desmin. With the use of Chromosome Conformation Capture, Hi-C and Chip-sequence analysis, we demonstrated the importance of desmin cytoskeleton on the genome 3D organization. In addition, we show that desmin is expressed in most of the cardiac progenitor cells population declaring its role in proper cardiac formation. Moreover, immunofluorescence, calcium influx and electron microscopy studies establish desmin as a major player for cardiac maturity, a critical subject in cardiac regenerative medicine, since its absence substantial reduces expression and proper localization of cardiac specific proteins, delays myofibril formation and impairs function of the induced cardiomyocytes driving most of them to senescence. All the above data will be presented.

Assessment of the feasibility of human amniotic membrane stem cell-derived cardiomyocytes in vitro

Myocardial infarction (MI) is a leading cause of death worldwide, resulting in extensive loss of cardiomyocytes and subsequent heart failure. Inducing cardiac differentiation of stem cells is a potential approach for myocardial regeneration therapy to improve post-MI prognosis. Mesenchymal stem cells (MSCs) have several advantages, including immune privilege and multipotent differentiation potential. This study aimed to explore the feasibility of chemically inducing human amniotic membrane MSCs (hAMSCs) to differentiate into cardiomyocytes in vitro. Human amniotic membrane (AM) samples were obtained from routine cesarean sections at Far Eastern Memorial Hospital. The isolated cells exhibited spindle-shaped morphology and expressed surface antigens CD73, CD90, CD105, and CD44, while lacking expression of CD19, CD11b, CD19, CD45, and HLA-DR. The SSEA-1, SSEA-3, and SSEA-4 markers were also positive, and the cells displayed the ability for tri-lineage differentiation into adipocytes, chondrocytes, and osteoblasts. The expression levels of MLC2v, Nkx2.5, and MyoD were analyzed using qPCR after applying various protocols for chemical induction, including BMP4, ActivinA, 5-azacytidine, CHIR99021, and IWP2 on hAMSCs. The group treated with 5 ng/ml BMP4, 10 ng/ml Activin A, 10 μM 5-azacytidine, 7.5 μM CHIR99021, and 5 μM IWP 2 expressed the highest levels of these genes. Furthermore, immunofluorescence staining demonstrated the expression of α-actinin and Troponin T in this group. In conclusion, this study demonstrated that hAMSCs can be chemically induced to differentiate into cardiomyocyte-like cells in vitro. However, to improve the functionality of the differentiated cells, further investigation of inductive protocols and regimens is needed.

The impact of diabetic glucose concentration on viability and cardiac differentiation of mesenchymal stem cells

IntroductionHyperglycemia may be a stumbling block for delivery of regenerative benefits of mesenchymal stem cells (MSCs) to diabetic patients with cardiovascular diseases. Our study aims to assess the viability and cardiac differentiation potential of MSCs after being exposed to diabetic glucose concentration. MethodsMSCs were extracted from rat bone marrow. Cells were characterized based on morphology, differentiation potential, and expression of mesenchymal specific markers. MTT assay was done to evaluate the viability of MSCs after treatment with different glucose concentrations. Case group was MSCs treated with diabetic concentration of glucose versus cells treated with PBS as the control group. Growth curve and population doubling time were calculated in both groups. Expression of GATA4 and troponin, as the early and late markers during cardiac differentiation, were measured following 5-azacytidine exposure. ResultsProliferated cells at passage three had fibroblastic-shape, was able to differentiate into adipocytes or osteocytes, and expressed CD73 and CD90. MSCs viability was gradually decreased by increasing glucose concentration. Irrespective of nicotine concentration, three-day exposure imposed more severe detrimental effects on viability compared with one-day treatment. Proliferation rate of the MSCs was lower in the case group, and they need more time for population doubling. Expression of both cardiac markers were downregulated in the case group at day three. However, their expression became higher at day seven. ConclusionDiabetic glucose concentration inhibits normal proliferation and cardiac differentiation of MSCs. This effect should be considered in stem cell therapy of cardiovascular patients who are concurrently affected by hyperglycemia, a common comorbidity in such individuals.Why carry out this study?•Stem cell therapy has opened a promising window to reduce great burden imposed by cardiovascular disease.•Despite substantial benefits observed in early studies, clinical translation of such treatment approaches have been hampered.•Hyperglycemic condition may be one of the hurdles responsible for reduced beneficial effects of stem cell therapy in cardiovascular patients.What was learned from the study? Findings•of our study revealed that diabetic glucose concentration inhibits normal proliferation and cardiac differentiation of mesenchymal stem cells.•Since diabetes is one of the common comorbidity in patients with cardiovascular diseases, glycemic status of such patients should be considered in the time of stem cell transplantation.•Moreover, these patients may need some pretreatments or augmented cells for transplantation in order to observe maximum benefits.

Identification of miR-20b-5p as an inhibitory regulator in cardiac differentiation via TET2 and DNA hydroxymethylation

BackgroundCongenital heart disease (CHD) is a prevalent congenital cardiac malformation, which lacks effective early biological diagnosis and intervention. MicroRNAs, as epigenetic regulators of cardiac development, provide potential biomarkers for the diagnosis and treatment of CHD. However, the mechanisms underlying miRNAs-mediated regulation of cardiac development and CHD malformation remain to be further elucidated. This study aimed to explore the function of microRNA-20b-5p (miR-20b-5p) in cardiac development and CHD pathogenesis.Methods and resultsmiRNA expression profiling identified that miR-20b-5p was significantly downregulated during a 12-day cardiac differentiation of human embryonic stem cells (hESCs), whereas it was markedly upregulated in plasma samples of atrial septal defect (ASD) patients. Our results further revealed that miR-20b-5p suppressed hESCs-derived cardiac differentiation by targeting tet methylcytosine dioxygenase 2 (TET2) and 5-hydroxymethylcytosine, leading to a reduction in key cardiac transcription factors including GATA4, NKX2.5, TBX5, MYH6 and cTnT. Additionally, knockdown of TET2 significantly inhibited cardiac differentiation, which could be partially restored by miR-20b-5p inhibition.ConclusionsCollectively, this study provides compelling evidence that miR-20b-5p functions as an inhibitory regulator in hESCs-derived cardiac differentiation by targeting TET2, highlighting its potential as a biomarker for ASD.

Unsupervised analysis of whole transcriptome data from human pluripotent stem cells cardiac differentiation

The main objective of the present work was to highlight differences and similarities in gene expression patterns between different pluripotent stem cell cardiac differentiation protocols, using a workflow based on unsupervised machine learning algorithms to analyse the transcriptome of cells cultured as a 2D monolayer or as 3D aggregates. This unsupervised approach effectively allowed to portray the transcriptomic changes that occurred throughout the differentiation processes, with a visual representation of the entire transcriptome. The results allowed to corroborate previously reported data and also to unveil new gene expression patterns. In particular, it was possible to identify a correlation between low cardiomyocyte differentiation efficiencies and the early expression of a set of non-mesodermal genes, which can be further explored as predictive markers of differentiation efficiency. The workflow here developed can also be applied to analyse other stem cell differentiation transcriptomic datasets, envisaging future clinical implementation of cellular therapies.

Design-Driven Translational Tools to Manage Cardiac Regeneration as Applicable to Clinical Practice

Cardiovascular disease is the leading cause of death, with 80% of cases occurring in developing countries. Innovative therapies are required to reduce mortality and limit or abolish the necessity for cardiac transplantation. Over the last decade, stem cells have been a promise for the cure of several diseases not only due to their plasticity but also to their capacity to act in a paracrine manner and influence the affected tissue. Human SC-based therapy derivatives are extremely attractive for therapeutic development because they have direct pharmacologic utility in clinical applications, unlike any other adult cells. Moreover, stem cell-derived paracrine factors have been shown to suppress inflammation and apoptosis, stimulate angiogenesis, and amplify the proliferation and differentiation of resident cardiac stem cells (CSCs). And SC therapies are thus viable alternatives to conventional treatments with substantial therapeutic potential; market opportunities are huge, as multiple product candidates are expected to be approved over the coming decade.

The PERK Branch of the Unfolded Protein Response Safeguards Protein Homeostasis and Mesendoderm Specification of Human Pluripotent Stem Cells.

Cardiac development involves large-scale rearrangements of the proteome. How the developing cardiac cells maintain the integrity of the proteome during the rapid lineage transition remains unclear. Here it is shown that proteotoxic stress visualized by the misfolded and/or aggregated proteins appears during early cardiac differentiation of human pluripotent stem cells and is resolved by activation of the PERK branch of unfolded protein response (UPR). PERK depletion increases misfolded and/or aggregated protein accumulation, leading to pluripotency exit defect and impaired mesendoderm specification of human pluripotent stem cells. Mechanistically, it is found that PERK safeguards mesendoderm specification through its conserved downstream effector ATF4, which subsequently activates a novel transcriptional target WARS1, to cope with the differentiation-induced proteotoxic stress. The results indicate that protein quality control represents a previously unrecognized core component of the cardiogenic regulatory network. Broadly, these findings provide a framework for understanding how UPR is integrated into the developmental program by activating the PERK-ATF4-WARS1 axis.

Embryonic exposure of polystyrene nanoplastics affects cardiac development

Micro- and nanoplastics have recently been detected in human blood and placentas, indicating inevitable embryonic exposure to these particles. However, their influence on human embryogenesis and the underlying mechanisms are still unknown. In this study, the effects of polystyrene nanoplastics (PS-NPs) exposure on cardiac differentiation of human embryonic stem cells (hESCs) were evaluated. Uptake of PS-NPs not only caused cellular injury, but also regulated cardiac-related pathways as revealed by RNA-sequencing. Consequently, the efficiency of cardiomyocyte differentiation from hESCs was compromised, leading to immature of cardiomyocytes and smaller cardiac organoids with impaired contractility. Mechanistically, PS-NPs promoted mitochondrial oxidative stress, activated P38/Erk MAPK signaling pathway, blocked autophagy flux, and eventually reduced the pluripotency of hESCs. Consistently, in vivo exposure of PS-NPs from cleavage to gastrula period of zebrafish embryo led to reduced cardiac contraction and blood flow. Collectively, this study suggests that PS-NPs is a risk factor for fetal health, especially for heart development.

Abstract GS.102: Cd206 + Il-4rα + Macrophages Are Drivers Of Adverse Cardiac Remodeling In Ischemic Cardiomyopathy

Background: Cardiac macrophages are significantly expanded in chronic heart failure (HF); however, their role is unclear. We tested the hypothesis that CD206 + macrophages expressing interleukin (IL)-4Rα in the failing heart are key drivers of adverse left ventricular (LV) remodeling and ischemic cardiomyopathy. Methods and Results: Adult male C57BL/6 (WT) mice underwent non-reperfused myocardial infarction (MI) to induce HF, or sham operation, and cardiac macrophages were profiled using flow cytometry. As compared to sham hearts, both the frequency and absolute number of CD206 + macrophages steadily increased in post-MI hearts during the progression of LV remodeling and HF, such that at 8 w post-MI they comprised ~85% of macrophages. These macrophages were exclusively proliferative and predominantly C-C motif chemokine receptor (CCR2) - with nearly half expressing IL-4Rα, and correlated with LV dysfunction and fibrosis. IL-4-polarized bone marrow derived CD206 + macrophages exhibited marked upregulation of found in inflammatory zone (FIZZ)1 and induced FIZZ1-dependent myofibroblast differentiation of cardiac mesenchymal stem cells (cMSCs), in part related to DLL-4/Jagged1-Notch1 signaling. Intramyocardial adoptive transfer of M[IL-4], but not M[IL-10], macrophages induced progressive LV dilation and dysfunction and augmented cardiac fibrosis over 4 weeks in naïve mice. In vivo IL-4Rα gene silencing in mice with established HF effectively depleted cardiac CD206 + IL-4Rα + macrophages and induced reverse LV remodeling, improving fibrosis, neovascularization, and dysfunction, and suppressing local and systemic inflammation. Lastly, alternatively activated CD206 + and CD163 + macrophages were significantly expanded in human failing hearts and correlated with cardiac fibrosis, with the majority of CD163 + macrophages expressing IL-4Rα and FIZZ3, the human homolog of FIZZ1. Conclusion: CD206 + IL-4Rα + macrophages proliferate and expand in the failing heart and are key mediators of pathological remodeling and fibrosis, in part through the secretion of FIZZ1. Inhibition of CD206 + macrophage IL-4Rα signaling may be a fruitful therapeutic approach for alleviating LV remodeling in HF.

Ascorbic acid and salvianolic acid B enhance the valproic acid and 5-azacytidinemediated cardiac differentiation of mesenchymal stem cells.

Cardiovascular diseases remain a major cause of death globally. Cardiac cells once damaged, cannot resume the normal functioning of the heart. Bone marrow derived mesenchymal stem cells (BM-MSCs) have shown the potential to differentiate into cardiac cells. Epigenetic modifications determine cell identity during embryo development via regulation of tissue specific gene expression. The major epigenetic mechanisms that control cell fate and biological functions are DNA methylation and histone modifications. However, epigenetic modifiers alone are not sufficient to generate mature cardiac cells. Various small molecules such as ascorbic acid (AA) and salvianolic acid B (SA) are known for their cardiomyogenic potential. Therefore, this study is aimed to examine the synergistic effects of epigenetic modifiers, valproic acid (VPA) and 5-azacytidine (5-aza) with cardiomyogenic molecules, AA and SA in the cardiac differentiation of MSCs. BM-MSCs were isolated, propagated, characterized, and then treated with an optimized dose of VPA or 5-aza for 24h. MSCs were maintained in a medium containing AA and SA for 21 days. All groups were assessed for the expression of cardiac genes and proteins through q-PCR and immunocytochemistry, respectively. Results show that epigenetic modifiers VPA or 5-aza in combination with AA and SA significantly upregulate the expression of cardiac genes MEF2C, Nkx2.5, cMHC, Tbx20, and GATA-4. In addition, VPA or 5-aza pretreatment along with AA and SA enhanced the expression of the cardiac proteins connexin-43, GATA-4, cTnI, and Nkx2.5. These findings suggest that epigenetic modifiers valproic acid and 5-azacytidine in combination with ascorbic acid and salvianolic acid B promote cardiac differentiation of MSCs. This pretreatment strategy can be exploited for designing future stem cell based therapeutic strategies for cardiovascular diseases.

Stage-specific requirement for m6A RNA methylation during cardiac differentiation of pluripotent stem cells

Precise spatiotemporal control of gene expression patterns is critical for normal development. Pluripotent stem cells, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), with the ability of unlimited self-renewal and differentiation into any cell type, provide a unique tool for understanding the underlying mechanism of development and disease in a dish. N6-methyl-adenosine (m6A) modification is the most extensive internal mRNA modification, which regulates almost all aspects of mRNA metabolism and thus extensively participates in gene expression regulation. However, the role of m6A during cardiogenesis still needs to be fully elucidated. Here, we found that core components of m6A methyltransferase decreased during cardiomyocyte differentiation. Impeding m6A deposition, by either deleting the m6A methyltransferase Mettl3 or overexpressing m6A demethylase alkB homolog 5 (Alkbh5), at early stages of cardiac differentiation of mouse pluripotent stem cells, led to inhibition of cardiac gene activation and retardation of the outgrowth of embryoid bodies, whereas interfering m6A modification at later stages of differentiation had minimal effects. Consistently, stage-specific inhibition of METTL3 with METTL3 inhibitor STM2457 during human ESCs (hESCs) cardiac differentiation demonstrated a similarly pivotal role of METTL3 for the induction of mesodermal cells while dispensable function for later stages. In summary, our study reveals a stage-specific requirement of m6A on the cardiac differentiation of pluripotent stem cells and demonstrates that precise tuning of m6A level is critical for cardiac differentiation.

Evaluation of the effect of boron derivatives on cardiac differentiation of mouse pluripotent stem cells

BackgroundThe heart is one of the first organs to form during embryonic development and has a very important place. So much that the formation of a functional heart is completed on the 55th day of human development and the 15th day of mouse development. Myocardial, endocardial and epicardial cells, which are derived from the mesoderm layer, are the cells that form the basis of the heart. Cardiac development, like other embryonic developments, is tightly controlled and regulated by various signaling pathways. The WNT signaling pathway is the most studied of these signaling pathways and the one with the clearest relationship with heart development. It is known that boron compounds and the Wnt/β-catenin pathway are highly correlated. Therefore, this study aimed to investigate the role of boron compounds in heart development as well as its effect on pluripotency of mouse embryonic stem cells for the first time in the literature. MethodsToxicity of boron compounds was evaluated by using MTS analysis and obtained results were supported by morphological pictures, Trypan Blue staining and Annexin V staining. Additionally, the possible boron-related change in pluripotency of embryonic stem cells were analyzed with alkaline phosphatase activity and immunocytochemical staining of Oct4 protein as well as gene expression levels of pluripotency related OCT4, SOX2 and KLF4 genes. The alterations in the embryonic body formation capacity of mouse embryonic stem cells due to the application boron derivatives were also evaluated. Three linage differentiation was conducted to clarify the real impact of boron compounds on embryonic development. Lastly, cardiac differentiation of mESCs was investigated by using morphological pictures, cytosolic calcium measurement, gene expression and immunocytochemical analysis of cardiac differentiation related genes and in the presence of boron compounds. ResultsObtained results show that boron treatment maintains the pluripotency of embryonic stem cells at non-toxic concentrations. Additionally, endodermal, and mesodermal fate was found to be triggered after boron treatment. Also, initiation of cardiomyocyte differentiation by boron derivative treatments caused an increased gene expression levels of cardiac differentiation related TNNT2, Nkx2.5 and ISL-1 gene expression levels. ConclusionThis study indicates that boron application, which is responsible for maintaining pluripotency of mESCs, can be used for increased cardiomyocyte differentiation of mESCs.

Tissue Engineering and Targeted Drug Delivery in Cardiovascular Disease: The Role of Polymer Nanocarrier for Statin Therapy

Atherosclerosis-related coronary artery disease (CAD) is the leading cause of mortality and morbidity worldwide. This requires effective primary and secondary prevention in reducing the complications related to CAD; the regression or stabilization of the pathology remains the mainstay of treatment. Statins have proved to be the most effective treatment in reducing adverse effects, but there are limitations related to the administration and achievement of effective doses as well as side effects due to the lack of target-related molecular specificity. The implemented technological steps are polymers and nanoparticles for the administration of statins, as it has been seen how the conjugation of drug delivery systems (DDSs) with statins increases bioavailability by circumventing the hepatic-renal filter and increases the related target specificity, enhancing their action and decreasing side effects. Reduction of endothelial dysfunction, reduced intimal hyperplasia, reduced ischemia-reperfusion injury, cardiac regeneration, positive remodeling in the extracellular matrix, reduced neointimal growth, and increased reendothelialization are all drug-related effects of statins enhanced by binding with DDSs. Recent preclinical studies demonstrate how the effect of statins stimulates the differentiation of endogenous cardiac stem cells. Poly-lactic-co-glycolic acid (PLGA) seems to be the most promising DDS as it succeeds more than the others in enhancing the effect of the bound drug. This review intends to summarize the current evidence on polymers and nanoparticles for statin delivery in the field of cardiovascular disease, trying to shed light on this topic and identify new avenues for future studies.

Polycomb protein RYBP activates transcription factor Plagl1 during in vitro cardiac differentiation of mouse embryonic stem cells.

RING1 and YY1 binding protein (RYBP) is primarily known to function as a repressor being a core component of the non-canonical polycomb repressive complexes 1 (ncPRC1s). However, several ncPRC1-independent functions of RYBP have also been described. We previously reported that RYBP is essential for mouse embryonic development and that Rybp null mutant embryonic stem cells cannot form contractile cardiomyocytes (CMCs) in vitro. We also showed that PLAGL1, a cardiac transcription factor, which is often mutated in congenital heart diseases (CHDs), is not expressed in Rybp-null mutant CMCs. However, the underlying mechanism of how RYBP regulates Plagl1 expression was not revealed. Here, we demonstrate that RYBP cooperated with NKX2-5 to transcriptionally activate the P1 and P3 promoters of the Plagl1 gene and that this activation is ncPRC1-independent. We also show that two non-coding RNAs residing in the Plagl1 locus can also regulate the Plagl1 promoters. Finally, PLAGL1 was able to activate Tnnt2, a gene important for contractility of CMCs in transfected HEK293 cells. Our study shows that the activation of Plagl1 by RYBP is important for sarcomere development and contractility, and suggests that RYBP, via its regulatory functions, may contribute to the development of CHDs.

FAM122A Is Required for Mesendodermal and Cardiac Differentiation of Embryonic Stem Cells.

Mesendodermal specification and cardiac differentiation are key issues for developmental biology and heart regeneration medicine. Previously, we demonstrated that FAM122A, a highly conserved housekeeping gene, is an endogenous inhibitor of protein phosphatase 2A (PP2A) and participates in multifaceted physiological and pathological processes. However, the in vivo function of FAM122A is largely unknown. In this study, we observed that Fam122 deletion resulted in embryonic lethality with severe defects of cardiovascular developments and significantly attenuated cardiac functions in conditional cardiac-specific knockout mice. More importantly, Fam122a deficiency impaired mesendodermal specification and cardiac differentiation from mouse embryonic stem cells but showed no influence on pluripotent identity. Mechanical investigation revealed that the impaired differentiation potential was caused by the dysregulation of histone modification and Wnt and Hippo signaling pathways through modulation of PP2A activity. These findings suggest that FAM122A is a novel and critical regulator in mesendodermal specification and cardiac differentiation. This research not only significantly extends our understanding of the regulatory network of mesendodermal/cardiac differentiation but also proposes the potential significance of FAM122A in cardiac regeneration.

Binary Colloidal Crystals Promote Cardiac Differentiation of Human Pluripotent Stem Cells via Nuclear Accumulation of SETDB1

Biophysical cues can facilitate the cardiac differentiation of human pluripotent stem cells (hPSCs), yet the mechanism is far from established. One of the binary colloidal crystals, composed of 5 μm Si and 400 nm poly(methyl methacrylate) particles named 5PM, has been applied as a substrate for hPSCs cultivation and cardiac differentiation. In this study, cell nucleus, cytoskeleton, and epigenetic states of human induced pluripotent stem cells on the 5PM were analyzed using atomic force microscopy, molecular biology assays, and the assay for transposase-accessible chromatin sequencing (ATAC-seq). Cells were more spherical with stiffer cell nuclei on the 5PM compared to the flat control. ATAC-seq revealed that chromatin accessibility decreased on the 5PM, caused by the increased entry of histone lysine methyltransferase SETDB1 into the cell nuclei and the amplified level of histone H3K9me3 modification. Reducing cytoskeleton tension using a ROCK inhibitor attenuated the nuclear accumulation of SETDB1 on the 5PM, indicating that the effect is cytoskeleton-dependent. In addition, the knockdown of SETDB1 reversed the promotive effects of the 5PM on cardiac differentiation, demonstrating that biophysical cue-induced cytoskeletal tension, cell nucleus deformation, and then SETDB1 accumulation are critical outside-in signal transformations in cardiac differentiation. Human embryonic stem cells showed similar results, indicating that the biophysical impact of the 5PM surfaces on cardiac differentiation could be universal. These findings contribute to our understanding of material-assistant hPSC differentiation, which benefits materiobiology and stem cell bioengineering.

Overexpression of GATA binding protein 4 and myocyte enhancer factor 2C induces differentiation of mesenchymal stem cells into cardiac-like cells.

BACKGROUNDHeart diseases are the primary cause of death all over the world. Following myocardial infarction, billions of cells die, resulting in a huge loss of cardiac function. Stem cell-based therapies have appeared as a new area to support heart regeneration. The transcription factors GATA binding protein 4 (GATA-4) and myocyte enhancer factor 2C (MEF2C) are considered prominent factors in the development of the cardiovascular system.AIMTo explore the potential of GATA-4 and MEF2C for the cardiac differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs).METHODShUC-MSCs were characterized morphologically and immunologically by the presence of specific markers of MSCs via immunocytochemistry and flow cytometry, and by their potential to differentiate into osteocytes and adipocytes. hUC-MSCs were transfected with GATA-4, MEF2C, and their combination to direct the differentiation. Cardiac differentiation was confirmed by semiquantitative real-time polymerase chain reaction and immunocytochemistry.RESULTShUC-MSCs expressed specific cell surface markers CD105, CD90, CD44, and vimentin but lack the expression of CD45. The transcription factors GATA-4 and MEF2C, and their combination induced differentiation in hUC-MSCs with significant expression of cardiac genes i.e., GATA-4, MEF2C, NK2 homeobox 5 (NKX2.5), MHC, and connexin-43, and cardiac proteins GATA-4, NKX2.5, cardiac troponin T, and connexin-43.CONCLUSIONTransfection with GATA-4, MEF2C, and their combination effectively induces cardiac differentiation in hUC-MSCs. These genetically modified MSCs could be a promising treatment option for heart diseases in the future.

CTCF acetylation at lysine 20 is required for the early cardiac mesoderm differentiation of embryonic stem cells

The CCCTC-binding factor (CTCF) protein and its modified forms regulate gene expression and genome organization. However, information on CTCF acetylation and its biological function is still lacking. Here, we show that CTCF can be acetylated at lysine 20 (CTCF-K20) by CREB-binding protein (CBP) and deacetylated by histone deacetylase 6 (HDAC6). CTCF-K20 is required for the CTCF interaction with CBP. A CTCF point mutation at lysine 20 had no effect on self-renewal but blocked the mesoderm differentiation of mouse embryonic stem cells (mESCs). The CTCF-K20 mutation reduced CTCF binding to the promoters and enhancers of genes associated with early cardiac mesoderm differentiation, resulting in diminished chromatin accessibility and decreased enhancer-promoter interactions, impairing gene expression. In summary, this study reveals the important roles of CTCF-K20 in regulating CTCF genomic functions and mESC differentiation into mesoderm.

Pioglitazone Mediates Cardiac Progenitor Formation through Increasing ROS Levels.

In order to achieve a sufficient population of cardiac-committed progenitor cells, it is crucial to know the mechanisms of cardiac progenitor formation. Previous studies suggested ROS effect on cardiac commitment events to play a key role in the cell signaling and activate cardiac differentiation of pluripotent stem cells. We previously reported that PPARγ activity is essential for cardiac progenitor cell commitment. Although several studies have conducted the involvement of PPARγ-related signaling pathways in cardiac differentiation, so far, the regulatory mechanisms of these signaling pathways have not been discussed and cleared. In this study, we focus on the role of PPARγ agonist in ROS generation and its further effects on the differentiation of cardiac cells from mESCs. The results of this study show that the presence of ROS is necessary for heart differentiation in the precursor stage of cardiac cells, and the coenzyme Q10 antioxidant precludes proper cardiac differentiation. In addition, this antioxidant prevents the action of pioglitazone in increasing oxygen radicals as well as beating cardiomyocyte differentiation properties. In this case, it can be concluded that PPARγ is required to modulate ROS levels during cardiac differentiation.

Mitochondrial energy metabolic transcriptome profiles during cardiac differentiation from mouse and human pluripotent stem cells.

Simultaneous myofibril and mitochondrial development is crucial for the cardiac differentiation of pluripotent stem cells (PSCs). Specifically, mitochondrial energy metabolism (MEM) development in cardiomyocytes is essential for the beating function. Although previous studies have reported that MEM is correlated with cardiac differentiation, the process and timing of MEM regulation for cardiac differentiation remain poorly understood. Here, we performed transcriptome analysis of cells at specific stages of cardiac differentiation from mouse embryonic stem cells (mESCs) and human induced PSCs (hiPSCs). We selected MEM genes strongly upregulated at cardiac lineage commitment and in a time-dependent manner during cardiac maturation and identified the protein-protein interaction networks. Notably, MEM proteins were found to interact closely with cardiac maturation-related proteins rather than with cardiac lineage commitment-related proteins. Furthermore, MEM proteins were found to primarily interact with cardiac muscle contractile proteins rather than with cardiac transcription factors. We identified several candidate MEM regulatory genes involved in cardiac lineage commitment (Cck, Bdnf, Fabp4, Cebpα, and Cdkn2a in mESC-derived cells, and CCK and NOS3 in hiPSC-derived cells) and cardiac maturation (Ppargc1α, Pgam2, Cox6a2, and Fabp3 in mESC-derived cells, and PGAM2 and SLC25A4 in hiPSC-derived cells). Therefore, our findings show the importance of MEM in cardiac maturation.