Partial reprogramming as a therapeutic approach for heart disease: A state-of-the-art review.

Heart disease such as myocardial infarction is the first cause of mortality in all countries. Today, cardiac cell-based therapy using de novo produced cardiac cells is considered as a novel approach for cardiac regenerative medicine. Recently, an alchemy-like approach, known as direct reprogramming or direct conversion, has been developed to directly convert somatic cells to cardiac cells in vitro and in vivo. This cellular alchemy is a short-cut and safe strategy for generating autologous cardiac cells, and it can be accomplished through activating cardiogenesis- or pluripotency-related factors in noncardiac cells. Importantly, pluripotency factors-based direct cardiac conversion, known as partial reprogramming, is shorter and more efficient for cardiomyocyte generation in vitro. Today, this strategy is achievable for direct conversion of mouse and human somatic cells to cardiac lineage cells (cardiomyocytes and cardiac progenitor cells), using transgene free, chemical-based approaches. Although, heart-specific partial reprogramming seems to be challenging for in vivo conversion of cardiac fibroblasts to cardiac cells, but whole organism-based in vivo partial reprogramming ameliorates cellular and physiological hallmarks of aging and prolongs lifespan in mouse. Notably, cardiac cells produced using partial reprogramming strategy can be a useful platform for disease modeling, drug screening and cardiac cell-based therapy, once the safety issues are overcome. Herein, we discuss about all progresses in de novo production of cardiac cells using partial reprogramming-based direct conversion, as well as give an overview about the potential applications of this strategy in vivo and in vitro.

Developmental heterogeneity of cardiac fibroblasts does not predict pathological proliferation and activation.

RATIONALE: Fibrosis is mediated partly by extracellular matrix-depositing fibroblasts in the heart. Although these mesenchymal cells are reported to have multiple embryonic origins, the functional consequence of this heterogeneity is unknown. OBJECTIVE: We sought to validate a panel of surface markers to prospectively identify cardiac fibroblasts. We elucidated the developmental origins of cardiac fibroblasts and characterized their corresponding phenotypes. We also determined proliferation rates of each developmental subset of fibroblasts after pressure overload injury. METHODS AND RESULTS: We showed that Thy1(+)CD45(-)CD31(-)CD11b(-)Ter119(-) cells constitute the majority of cardiac fibroblasts. We characterized these cells using flow cytometry, epifluorescence and confocal microscopy, and transcriptional profiling (using reverse transcription polymerase chain reaction and RNA-seq). We used lineage tracing, transplantation studies, and parabiosis to show that most adult cardiac fibroblasts derive from the epicardium, a minority arises from endothelial cells, and a small fraction from Pax3-expressing cells. We did not detect generation of cardiac fibroblasts by bone marrow or circulating cells. Interestingly, proliferation rates of fibroblast subsets on injury were identical, and the relative abundance of each lineage remained the same after injury. The anatomic distribution of fibroblast lineages also remained unchanged after pressure overload. Furthermore, RNA-seq analysis demonstrated that Tie2-derived and Tbx18-derived fibroblasts within each operation group exhibit similar gene expression profiles. CONCLUSIONS: The cellular expansion of cardiac fibroblasts after transaortic constriction surgery was not restricted to any single developmental subset. The parallel proliferation and activation of a heterogeneous population of fibroblasts on pressure overload could suggest that common signaling mechanisms stimulate their pathological response.

Ascorbic acid promotes the direct conversion of mouse fibroblasts into beating cardiomyocytes.

Recent advances in the direct conversion of fibroblasts to cardiomyocytes suggest this process as a novel promising approach for cardiac cell-based therapies. Here, by screening the effects of 10 candidate small molecules along with transient overexpression of Yamanaka factors, we show ascorbic acid (AA), also known as vitamin C, enhances reprogramming of mouse fibroblasts into beating cardiomyocytes. Immunostaining and gene expression analyses for pluripotency and cardiac lineage markers confirmed beating patches were derived from non-cardiac lineage cells without passing through a pluripotent intermediate. Further analysis revealed that AA also increased the size of the beating areas and the number of cardiac progenitors. Immunostaining for cardiac markers, as well as electrophysiological analysis confirmed the functionality of directly converted cardiomyocytes. These results illustrate the importance of AA in direct conversion of fibroblasts to cardiomyocytes and may open new insights into future biomedical applications for induced cardiomyocytes.

5-Azacytidine incorporated skeletal muscle-derived hydrogel promotes rat skeletal muscle regeneration.

Decellularized skeletal muscle is a promising biomaterial for muscle regeneration due to the mimicking of the natural microenvironment. Previously, it has been reported that 5-Azacytidine (5-Aza), a DNA methyltransferase inhibitor, induces myogenesis in different types of stem cells. In the current study, we investigated the effect of 5-Aza incorporated muscle-derived hydrogel on the viability and proliferation of muscle-derived stem cells (MDSCs) in vitro and muscle regeneration in vivo. Wistar rat skeletal muscles were decellularized using a physico-chemical protocol. The decellularized tissue was analyzed using SEM, histological staining and evaluation of DNA content. Then, muscle-derived hydrogel was made from Pepsin-digested decellularized muscle tissues. 5-Aza was physically adsorbed in prepared hydrogels. Then, MDSCs were cultured on hydrogels with/without 5-Aza, and their proliferation and cell viability were determined using LIVE/DEAD and DAPI staining. Moreover, myectomy lesions were done in rat femoris muscles, muscle-derived hydroges with/without 5-Aza were injected to the myectomy sites, and histological evaluation was performed after three weeks. The analysis of decellularized muscle tissues showed that they maintained extracellular matrix components of native muscles, while they lacked DNA. LIVE/DEAD and DAPI staining showed that the hydrogel containing 5-Aza supported MDSCs viability. Histological analysis of myectomy sites showed an improvement in muscle regeneration after administration of 5-Aza incorporated hydrogel. These findings suggest that the combination of 5-Aza with skeletal muscle hydrogel may serve as an alternative treatment option to improve the regeneration of injured muscle tissue.

Alpha-ketoglutarate as a potent regulator for lifespan and healthspan: Evidences and perspectives.

Aging is a natural process that determined by a functional decline in cells and tissues as organisms are growing old, resulting in an increase at risk of disease and death. To this end, many efforts have been made to control aging and increase lifespan and healthspan. These efforts have led to the discovery of several anti-aging drugs and compounds such as rapamycin and metformin. Recently, alpha-ketoglutarate (AKG) has been introduced as a potential anti-aging metabolite that can control several functions in organisms, thereby increases longevity and improves healthspan. Unlike other synthetic anti-aging drugs, AKG is one of the metabolites of the tricarboxylic acid (TCA) cycle, also known as the Krebs cycle, and synthesized in the body. It plays a crucial role in the cell energy metabolism, amino acid/protein synthesis, epigenetic regulation, stemness and differentiation, fertility and reproductive health, and cancer cell behaviors. AKG exerts its effects through different mechanisms such as inhibiting mTOR and ATP-synthase, modulating DNA and histone demethylation and reducing ROS formation. Herein, we summarize the recent findings of AKG-related lifespan and healthspan studies and discuss AKG associated cell and molecular mechanisms involved in increasing longevity, improving reproduction, and modulating stem cells and cancer cells behavior. We also discuss the promises and limitations of AKG for delaying aging and other potential applications.

Identification of hub genes and microRNAs with prognostic values in esophageal cancer by integrated analysis.

Esophageal cancer (EC) is the eighth most common cancer in the world, and the sixth most common cause of cancer-related mortality. The aim of the present study was to identify cell and molecular mechanisms involved in EC, and to provide the potential targets for diagnosis and treatment. Here, a microarray dataset (GSE20347) was screened to find differentially expressed genes (DEGs). Different bioinformatic methods were used to analyze the identified DEGs. The up-regulated DEGs were significantly involved in different biological processes and pathways including extracellular matrix organization and ECM-receptor interaction. FN1, CDK1, AURKA, TOP2A, FOXM1, BIRC5, CDC6, UBE2C, TTK, and TPX2 were identified as the most important genes among the up-regulated DEGs. Our analysis showed that has-miR-29a-3p, has-miR-29b-3p, has-miR-29c-3p, and has-miR-767-5p had the largest number of common targets among the up-regulated DEGs. These findings strengthen the understanding of EC development and progression, as well as representing potential markers for EC diagnosis and treatment.

Diagnostic potential and pathogenic performance of circulating miR-146b, miR-194, and miR-214 in liver fibrosis.

Liver fibrosis is the excessive accumulation of extracellular matrix proteins. Due to the lack of an accurate test for an early diagnosis of liver fibrosis and the invasiveness of the liver biopsy procedure, there is an urgent need for effective non-invasive biomarkers for screening the patients. we aimed to evaluate the diagnostic performance of circulating miRNAs (miR-146b, -194, -214) and their related mechanisms in the pathogenesis of liver fibrosis. The expression levels of miR-146b, -194, and -214 were quantified in whole blood samples from NAFLD patients using real-time PCR. The competing endogenous RNA (ceRNA) network was constructed and a gene set enrichment analysis (GSEA) was performed for HSC activation-related genes. Also, the transcription factor (TF)-miR co-regulatory network and the survival plot for three miRNAs and core genes were illustrated. The qPCR results showed that the relative expression of miR-146b and miR-214 significantly increased in NAFLD patients, while miR-194 showed significant down-regulation. The ceRNA network analysis implicated NEAT1 and XIST as sponge candidates for these miRNAs. The GSEA results identified 15 core genes involved in HSC activation, primarily enriched in NF-κB activation and autophagy pathways. STAT3, TCF3, RELA, and RUNX1 were considered potential transcription factors connected to miRNAs in the TF-miR network. Our study elucidated three candidate circulating miRNAs differentially expressed in NAFLD that could serve as a promising non-invasive diagnostic tool for early detection strategies. Also, NF-κB activation, autophagy, and negative regulation of the apoptotic process are the main potential underlying mechanisms regulated by these miRNAs in liver fibrosis pathogenesis.

Identification of potential microRNA diagnostic panels and uncovering regulatory mechanisms in breast cancer pathogenesis.

Early diagnosis of breast cancer (BC), as the most common cancer among women, increases the survival rate and effectiveness of treatment. MicroRNAs (miRNAs) control various cell behaviors, and their dysregulation is widely involved in pathophysiological processes such as BC development and progress. In this study, we aimed to identify potential miRNA biomarkers for early diagnosis of BC. We also proposed a consensus-based strategy to analyze the miRNA expression data to gain a deeper insight into the regulatory roles of miRNAs in BC initiation. Two microarray datasets (GSE106817 and GSE113486) were analyzed to explore the differentially expressed miRNAs (DEMs) in serum of BC patients and healthy controls. Utilizing multiple bioinformatics tools, six serum-based miRNA biomarkers (miR-92a-3p, miR-23b-3p, miR-191-5p, miR-141-3p, miR-590-5p and miR-190a-5p) were identified for BC diagnosis. We applied our consensus and integration approach to construct a comprehensive BC-specific miRNA-TF co-regulatory network. Using different combination of these miRNA biomarkers, two novel diagnostic models, consisting of miR-92a-3p, miR-23b-3p, miR-191-5p (model 1) and miR-92a-3p, miR-23b-3p, miR-141-3p, and miR-590-5p (model 2), were obtained from bioinformatics analysis. Validation analysis was carried out for the considered models on two microarray datasets (GSE73002 and GSE41922). The model based on similar network topology features, comprising miR-92a-3p, miR-23b-3p and miR-191-5p was the most promising model in the diagnosis of BC patients from healthy controls with 0.89 sensitivity, 0.96 specificity and area under the curve (AUC) of 0.98. These findings elucidate the regulatory mechanisms underlying BC and represent novel biomarkers for early BC diagnosis.

Identification of key regulators associated with colon cancer prognosis and pathogenesis.

Colon cancer (CC) is the fourth deadliest cancer in the world. New insights into prognostication might be helpful to define the optimal adjuvant treatments for patients in routine clinical practice. Here, a microarray dataset with 30 primary tumors and 30 normal samples was analyzed using GEO2R to find differentially expressed genes (DEGs). Then, DAVID, KEGG, ChEA and X2K were used to analyze DEGs-related Gene Ontology, pathways, transcription factors (TFs) and kinases, respectively. Protein-protein interaction (PPI) networks were constructed using the STRING database and Cytoscape. The modules and hub genes of DEGs was determined through MCODE and CytoHubba plugins, and the expression of hub genes was verified using GEPIA. To find microRNAs and metabolites associated with DEGs, miRTarBase and HMDB were used, respectively. It was found that 233 and 373 genes were upregulated and downregulated in CC, respectively. GO analysis showed that the upregulated DEGs were mainly involved in mitotic nuclear division and cell division. Top 10 hub genes were identified, including AURKB, CDK1, DLGAP5, AURKA, CCNB2, CCNB1, BUB1B, CCNA2, KIF20A and BUB1. Whereas, FOMX1, E2F7, E2F1, E2F4 and AR were identified as top 5 TFs in CC. Moreover, CDK1, CDC2, MAPK14, ATM and CK2ALPHA was identified as top 5 kinases in CC. miRNAs analysis showed that Hsa-miR-215-5p hsa-miR-193b-3p, hsa-miR-192-5p and hsa-miR-16-5p could target the largest number of CC genes. Taken together, CC-related genes, especially the hub genes, TFs, and metabolites might be used as novel biomarkers for CC, as well as for diagnosis and guiding therapeutic strategies for CC.

Granulosa Cell Conditioned Medium Enhances The Rate of Mouse Oocyte In Vitro Maturation and Embryo Formation.

OBJECTIVE: In vitro maturation (IVM) and cryopreservation of oocytes are two important parts of assisted reproductive technology (ART), but their efficacy is low. This study aimed to improve the quality of in vitro vitrified-warmed maturated oocytes using granulosa cell conditioned medium (GCCM). MATERIALS AND METHODS: In the experimental study, fresh/non-vitrified and vitrified-warmed mouse germinal vesicle (GV) oocytes (as F and V) were In vitro maturated using basal medium (BM) and also BM supplemented with 50% GCCM as treated groups (GM), and categorized as FBM, FGM, VBM and VGM groups, respectively. The rate of successful IVM (MII oocyte formation), mitochondrial membrane potential and the viability of MII oocytes were determined using inverted microscopy, JC-1 and trypan blue staining. Then, the rate of In vitro fertilization (IVF) and subsequent two-cell embryo formation was calculated. Finally, the expression levels of Oct4, Sox2, Cdk-2, Gdf9, Integrin beta1 and Igf2 were analyzed using real-time polymerase chain reaction (PCR) in MII oocytes and two-cell embryos. RESULTS: These analyses showed that GCCM significantly increased the IVM rate, oocyte meiotic resumption and mitochondrial membrane potential (P<0.05). In addition, the rate of IVF and two-cell embryo formation was significantly higher in FGM and VGM compared to FBM and VBM (P<0.05). Interestingly, GCCM significantly affected the expression of the studied genes. CONCLUSION: Our findings suggest that GCCM might be useful for improving the efficiency of IVM and the subsequent IVF outcomes.

Bioinformatics analysis reveals molecular connections between non-alcoholic fatty liver disease (NAFLD) and COVID-19.

The ongoing coronavirus disease 2019 (COVID-19) pandemic caused by SARS-CoV-2 has devastatingly impacted people's lives. Non-alcoholic fatty liver disease (NAFLD) is fatal comorbidity of COVID-19 seen with potential risk factors to develop severe symptoms. This research focuses on determining and elucidating the molecular factors and connections that might contribute to the severity of SARS-CoV-2 infection in NAFLD patients. Here, we comprehensively inspected the genes involved in NAFLD and SARS-CoV-2 entry factors (SCEFs) found by searching through the DisGeNet database and literature review, respectively. Further, we identified the SCEFs-related proteins through protein-protein interaction (PPI) network construction, MCODE, and Cytohubba. Next, the shared genes involved in NAFLD and SARS-CoV-2 entry, and hub gene were determined, followed by the GO and KEGG pathways analysis. X2K database was used to construct the upstream regulatory network of hub genes, as well as to identify the top ten candidates of transcription factors (TFs) and protein kinases (PKs). PPI analysis identified connections between 4 top SCEFs, including ACE, ADAM17, DPP4, and TMPRSS2 and NAFLD-related genes such as ACE, DPP4, IL-10, TNF, and AKT1. GO and KEGG analysis revealed the top ten biological processes and pathways, including cytokine-mediated signaling, PI3K-Akt, AMPK, and mTOR signaling pathways. The upstream regulatory network revealed that AKT1 and MAPK14 as important PKs and HIF1A and SP1 as important TFs associated with AKT1, IL-10, and TNF. The molecular connections identified between COVID-19 and NAFLD may shed light on discovering the causes of the severity of SARS-CoV-2 infected NAFLD patients.

Identification of potential key genes and miRNAs involved in Hepatoblastoma pathogenesis and prognosis.

Hepatoblastoma (HB) is one of the most common liver malignancies in children, while the molecular basis of the disease is largely unknown. Therefore, this study aims to explore the key genes and molecular mechanisms of the pathogenesis of HB using a bioinformatics approach. The gene expression dataset GSE131329 was used to find differentially expressed genes (DEGs). Functional and enrichment analyses of the DEGs were performed by the EnrichR. Then, the protein-protein interaction (PPI) network of the up-regulated genes was constructed and visualized using STRING database and Cytoscape software, respectively. MCODE was used to detect the significant modules of the PPI network, and cytoHubba was utilized to rank the important nodes (genes) of the PPI modules. Overall, six ranking methods were employed and the results were validated by the Oncopression database. Moreover, the upstream regulatory network and the miRNA-target interactions of the up-regulated DEGs were analyzed by the X2K web and the miRTarBase respectively. A total of 594 DEGs, including 221 up- and 373 down-regulated genes, were obtained, which were enriched in different cellular and metabolic processes, human diseases, and cancer. Furthermore, 15 hub genes were screened, out of which, 11 were validated. Top 10 transcription factors, kinases, and miRNAs were also determined. To the best of our knowledge, the association of RACGAP1, MKI67, FOXM1, SIN3A, miR-193b, and miR-760 with HB was reported for the first time. Our findings may be used to shed light on the underlying mechanisms of HB and provide new insights for better prognosis and therapeutic strategies.

Comprehensive transcriptomic analysis identifies novel regulators of lung adenocarcinoma.

Lung adenocarcinoma (LA) is a subtype of lung cancer that accounts for about 40% of all lung cancers. Analysis of molecular mechanisms controlling this cancer can help scientists to detect, control and treat LA. Here, a microarray dataset (GSE118370) containing six normal lung (NL) and six LA samples was screened using GEO2R to find differentially expressed genes (DEGs). Then, DAVID, KEGG and ChEA were used to analyze DEGs-related gene ontology, pathways and transcription factors (TFs), respectively. The Protein-protein interaction network for DEGs and TFs was constructed by STRING and Cytoscape. To find microRNAs and metabolites associated with DEGs, miRTarBase and HMDB were used, respectively. It was found that 350 genes were upregulated and 608 genes were downregulated in LA. The upregulated genes or LA-related gens were enriched in biological process and pathways such as extracellular matrix disassembly and p53 signaling pathway, whereas the downregulated genes or NL-related genes were enriched in cell adhesion and cell-surface receptor signaling pathway. ESR1, KIF18B, BIRC5, CHEK1, CCNB1 and AURKA were determined as hub genes for LA. FOXA1 and TFAP2A had the highest number of connectivity in LA-related TFs. hsa-miR-192-5p and hsa-miR-215-5p could target the highest number of LA-related genes. Metabolite analysis showed that Estrone and NADPH were among the top ten metabolites associated with LA-related genes. Taken together, LA-related genes, especially the hub genes, TFs, and metabolites might be used as novel markers for LA, as well as for diagnosis and guiding therapeutic strategies of LA.

Design and characterization of an electroconductive scaffold for cardiomyocytes based biomedical assays.

Cardiovascular diseases (CVD) are a major cause of mortality worldwide. Accessibility to heart tissue is limited due to sampling issues and lack of appropriate culture conditions. In addition, animal models are not an ideal choice for physiological, pharmacological, and fundamental evaluations in the cardiovascular field due to interspecies differences. Hence, there is an inevitable need for functional in vitro cardiac models. In this study, we have synthesized a novel electroconductive scaffold comprised of cardiac extracellular matrix (ECM) derived pre-cardiogel (pCG) blended with polypyrrole (Ppy). Our data revealed that 2.5% (w/v) pyrrole (Py) had the highest possible Py ratio that provided pCG-Ppy gel formation. The prepared mixture was fabricated into a scaffold by using the freeze-dried method. The scaffolds had open interconnected pores that ranged from 55 ± 24 µm for the cardiogel (CG)-Ppy to 74 ± 26 µm for the CG scaffolds, with no alterations in vital ECM components of collagen, polysaccharides, and glycosaminoglycans (GAGs). Incorporation of Ppy increased the CG stiffness with a final complex modulus from 80 pa to 140 pa. The CG-Ppy group had significantly greater electrical conductivity than the CG group. Scaffolds supported neonatal mouse cardiomyocyte (NMCM) adhesion, viability, cardiac-specific gene expression, and spontaneous beating up to 14 days after seeding. Among the fabricated hydrogels, the CG-Ppy group resulted in the synchronous beating of cardiomyocyte clusters and upregulation of cardiac genes involved in cardiac muscle contraction (cardiac troponin T [cTNT]) and cardiomyocyte electrical coupling (connexin 43 [Cx43]). Thus, this ECM-based electro-conductive scaffold might provide a promising substrate for constructing in vitro cardiac models for drug testing, disease modeling, developmental studies, and cardiac regenerative approaches.

In vivo administration of G9a inhibitor A366 decreases osteogenic potential of bone marrow-derived mesenchymal stem cells.

Epigenetic mechanisms such as histone methylation are considered as one of the most important mediators that control stem cell behaviors such as proliferation, senescence and differentiation. G9a, a histone methyltransferase, has recently generated intense attention as potential target for controlling many diseases such as cancers. The aim of the present study was to evaluate the effect of in vivo administration of A366, a G9a inhibitor, on proliferative and differentiation potential of bone marrow-derived mesenchymal stem cells (BM-MSCs). We inhibited G9a using intraperitoneally administration of A366, and we evaluated BM-MSC proliferation and differentiation behaviors in vitro. Colony formation assay of BM-MSCs at primary culture showed that in vivo administration of A366 reduced the colony forming capacity of BM-MSCs. Moreover, PDT of BM-MSC isolated from A366-treated rats was higher than control, especially in the early passages. BM-MSC isolated from A366-treated rats showed higher adipogenic potential compared to the control at the early passages as determined by gene expression and Oil Red staining. Whereas, osteogenic potential of BM-MSC isolated from A366-treated rats was lower than control, especially at early passages. Our results suggest that the epigenetic modifier such as A366, which seems to be a therapeutic approach for controlling diseases such as cancer, might also influence the proliferation and differentiation capacity of MSCs both in vitro and in vivo. Moreover, epigenetic modifying chemicals seem to be a strategy to manipulate MSC expansion capacity and differentiation propensity, as well as to efficiently involvement of MSCs in tissue homeostasis, cell-based therapy and tissue engineering.

Global transcriptomic analysis of induced cardiomyocytes predicts novel regulators for direct cardiac reprogramming.

Heart diseases are the most significant cause of morbidity and mortality in the world. De novo generated cardiomyocytes (CMs) are a great cellular source for cell-based therapy and other potential applications. Direct cardiac reprogramming is the newest method to produce CMs, known as induced cardiomyocytes (iCMs). During a direct cardiac reprogramming, also known as transdifferentiation, non-cardiac differentiated adult cells are reprogrammed to cardiac identity by forced expression of cardiac-specific transcription factors (TFs) or microRNAs. To this end, many different combinations of TFs (±microRNAs) have been reported for direct reprogramming of mouse or human fibroblasts to iCMs, although their efficiencies remain very low. It seems that the investigated TFs and microRNAs are not sufficient for efficient direct cardiac reprogramming and other cardiac specific factors may be required for increasing iCM production efficiency, as well as the quality of iCMs. Here, we analyzed gene expression data of cardiac fibroblast (CFs), iCMs and adult cardiomyocytes (aCMs). The up-regulated and down-regulated genes in CMs (aCMs and iCMs) were determined as CM and CF specific genes, respectively. Among CM specific genes, we found 153 transcriptional activators including some cardiac and non-cardiac TFs that potentially activate the expression of CM specific genes. We also identified that 85 protein kinases such as protein kinase D1 (PKD1), protein kinase A (PRKA), calcium/calmodulin-dependent protein kinase (CAMK), protein kinase C (PRKC), and insulin like growth factor 1 receptor (IGF1R) that are strongly involved in establishing CM identity. CM gene regulatory network constructed using protein kinases, transcriptional activators and intermediate proteins predicted some new transcriptional activators such as myocyte enhancer factor 2A (MEF2A) and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A), which may be required for qualitatively and quantitatively efficient direct cardiac reprogramming. Taken together, this study provides new insights into the complexity of cell fate conversion and better understanding of the roles of transcriptional activators, signaling pathways and protein kinases in increasing the efficiency of direct cardiac reprogramming and maturity of iCMs.

Boosters and barriers for direct cardiac reprogramming.

Heart disease is currently the most significant cause of morbidity and mortality worldwide, which accounts for approximately 33% of all deaths. Recently, a promising and alchemy-like strategy has been developed called direct cardiac reprogramming, which directly converts somatic cells such as fibroblasts to cardiac lineage cells such as cardiomyocytes (CMs), termed induced CMs or iCMs. The first in vitro cardiac reprogramming study, mediated by cardiac transcription factors (TFs)-Gata4, Tbx5 and Mef2C-, was not enough efficient to produce an adequate number of fully reprogrammed, functional iCMs. As a result, numerous combinations of cardiac TFs exist for direct cardiac reprogramming of mouse and human fibroblasts. However, the efficiency of direct cardiac reprogramming remains low. Recently, a number of cellular and molecular mechanisms have been identified to increase the efficiency of direct cardiac reprogramming and the quality of iCMs. For example, microgrooved substrate, cardiogenic growth factors [VEGF, FGF, BMP4 and Activin A], and an appropriate stoichiometry of TFs boost the direct cardiac reprogramming. On the other hand, serum, TGFß signaling, activators of epithelial to mesenchymal transition, and some epigenetic factors (Bmi1 and Ezh2) are barriers for direct cardiac reprogramming. Manipulating these mechanisms by the application of boosters and removing barriers can increase the efficiency of direct cardiac reprogramming and possibly make iCMs reliable for cell-based therapy or other potential applications. In this review, we summarize the latest trends in cardiac TF- or miRNA-based direct cardiac reprogramming and comprehensively discuses all molecular and cellular boosters and barriers affecting direct cardiac reprogramming.

Transient Activation of Reprogramming Transcription Factors Using Protein Transduction Facilitates Conversion of Human Fibroblasts Toward Cardiomyocyte-Like Cells.

Derivation of cardiomyocytes directly from patients' own fibroblasts could offer a new therapeutic approach for those with ischemic heart disease. An essential step toward clinical application is to establish safe conversion of human fibroblasts into a cardiac fate. Here we aimed to efficiently and safely generate cardiomyocytes from human fibroblasts by direct delivery of reprogramming recombinant cell permeant form of reprogramming proteins followed by cardio-inductive signals. Human fetal and adult fibroblasts were transiently exposed to transactivator of transcription-fused recombinant OCT4, SOX2, KLF4 and c-MYC for 2 weeks and then were directly differentiated toward protein-induced cardiomyocyte-like cells (p-iCLCs) in a cardiac fate niche, carried out by treatment with a set of cardiogenic small molecules (sequential treatment of Chir, and IWP-2, SB431542 and purmorphamine). The cells showed cardiac phenotype over a period of 3 weeks without first undergoing reprogramming into or through a pluripotent intermediate, shown by lack of expression of key pluripotency markers. p-iCLCs exhibited cardiac features at both the gene and protein levels. Our study provides an alternative method for the generation of p-iCLCs which shortcut reprogramming toward allogeneic cardiomyocytes in a safe and efficient manner and could facilitate generation of genetic material-free cardiomyocytes.

Human cardiomyocyte generation from pluripotent stem cells: A state-of-art.

The human heart is considered a non-regenerative organ. Worldwide, cardiovascular diseases continue to be the leading cause of death. Despite advances in cardiac treatment, myocardial repair remains severely limited by the lack of an appropriate source of viable cardiomyocytes (CMs) to replace damaged tissue. Human pluripotent stem cells (hPSCs), embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can efficiently be differentiated into functional CMs necessary for cell replacement therapy and other potential applications. The number of protocols that derive CMs from hPSCs has increased exponentially over the past decade following observation of the first human beating CMs. A number of highly efficient, chemical based protocols have been developed to generate human CMs (hCMs) in small-scale and large-scale suspension systems. To reduce the heterogeneity of hPSC-derived CMs, the differentiation protocols were modulated to exclusively generate atrial-, ventricular-, and nodal-like CM subtypes. Recently, remarkable advances have been achieved in hCM generation including chemical-based cardiac differentiation, cardiac subtype specification, large-scale suspension culture differentiation, and development of chemically defined culture conditions. These hCMs could be useful particularly in the context of in vitro disease modeling, pharmaceutical screening and in cellular replacement therapies once the safety issues are overcome. Herein we review recent progress in the in vitro generation of CMs and cardiac subtypes from hPSCs and discuss their potential applications and current limitations.