Induce Cardiomyocyte Differentiation Research Articles

Extracellular vesicles and microRNAs in the regulation of cardiomyocyte differentiation and proliferation

Cardiomyocyte differentiation and proliferation are essential processes for the regeneration of an injured heart. In recent years, there have been several reports highlighting the involvement of extracellular vesicles (EVs) in cardiomyocyte differentiation and proliferation. These EVs originate from mesenchymal stem cells, pluripotent stem cells, and heart constituting cells (cardiomyocytes, cardiac fibroblasts, cardiac progenitor cells, epicardium). Numerous reports also indicate the involvement of microRNAs (miRNAs) in cardiomyocyte differentiation and proliferation. Among them, miRNA-1, miRNA-133, and miRNA-499, recently demonstrated to promote cardiomyocyte differentiation, and miRNA-199, shown to promote cardiomyocyte proliferation, were found effective in various studies. MiRNA-132 and miRNA-133 have been identified as cargo in EVs and are reported to induce cardiomyocyte differentiation. Similarly, miRNA-30a, miRNA-100, miRNA-27a, miRNA-30e, miRNA-294 and miRNA-590 have also been identified as cargo in EVs and are shown to have a role in the promotion of cardiomyocyte proliferation. Regeneration of the heart by EVs or artificial nanoparticles containing functional miRNAs is expected in the future. In this review, we outline recent advancements in understanding the roles of EVs and miRNAs in cardiomyocyte differentiation and proliferation. Additionally, we explore the related challenges when utilizing EVs and miRNAs as a less risky approach to cardiac regeneration compared to cell transplantation.

Polycaprolactone-co-polylactic acid nanofiber scaffold in combination with 5-azacytidine and transforming growth factor-β to induce cardiomyocyte differentiation of adipose-derived mesenchymal stem cells.

Adipose-derived mesenchymal stem cells (Ad-MSCs) are promising candidates for cardiac repair/regeneration. The application of copolymer nanoscaffolds has received great attention in tissue engineering to support differentiation and functional tissue organization toward effective tissue regeneration. The objective of the current study was to develop functional and bioactive scaffolds by combining polycaprolactone (PCL) and polylactic acid (PLA) for cardiomyocyte differentiation of human Ad-MSC (hAd-MSCs) in the absence or presence of 5-azacytidine and transforming growth factor-β (TGF-β). To that end, the human MSCs were extracted from human adipose tissue (AD). The cardiomyocyte differentiation potency of hAd-MSCs was evaluated on the novel synthetic PCL/PLA nanofiber scaffolds prepared in the absence and presence of 5-azacytidine and TGF-β supplements. A PCL/PLA nanofibrous scaffold was fabricated using the electrospinning method and its nanotopography and porous structure werecharacterized using scanning electron microscopy. In addition, the attachment of hAd-MSCs on the PCL/PLA scaffolds was semiquantitatively investigated. Compared with other treatments, the PCL/PLA nanofibrous scaffold supplemented with both 5-azacytidine and TGF-β was observed to differentiate hAd-MSCs into cardiomyocytes at Day 21 as evidenced by real-time PCR for cardiac-specific genes including cardiac troponin I (cTnI), GATA4, MYH7, and NKX2.5. In addition, flow cytometric analysis of cTnI-positive cells demonstrated that the cardiomyocytedifferentiation of hAd-MSCs was more efficient on the PCL/PLA nanofibrous scaffold supplemented with both 5-azacytidine and TGF-β than it was in the other treatment groups. Generally speaking, the results show that PCL/PLA nanofibrous scaffolds may be applied as a platform for efficient differentiation of hAd-MSCs into functional cardiomyocytes.

The nuclear receptor ERR cooperates with the cardiogenic factor GATA4 to orchestrate cardiomyocyte maturation

Estrogen-related receptors (ERR) α and γ were shown recently to serve as regulators of cardiac maturation, yet the underlying mechanisms have not been delineated. Herein, we find that ERR signaling is necessary for induction of genes involved in mitochondrial and cardiac-specific contractile processes during human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) differentiation. Genomic interrogation studies demonstrate that ERRγ occupies many cardiomyocyte enhancers/super-enhancers, often co-localizing with the cardiogenic factor GATA4. ERRγ interacts with GATA4 to cooperatively activate transcription of targets involved in cardiomyocyte-specific processes such as contractile function, whereas ERRγ-mediated control of metabolic genes occurs independent of GATA4. Both mechanisms require the transcriptional coregulator PGC-1α. A disease-causing GATA4 mutation is shown to diminish PGC-1α/ERR/GATA4 cooperativity and expression of ERR target genes are downregulated in human heart failure samples suggesting that dysregulation of this circuitry may contribute to congenital and acquired forms of heart failure.

Induction of Stem-Cell-Derived Cardiomyogenesis by Fibroblast Growth Factor 10 (FGF10) and Its Interplay with Cardiotrophin-1 (CT-1)

Simple SummaryCardiovascular diseases are worldwide one of the leading contributors of mortality, and among multiple therapeutic approaches, stem cell therapy has been introduced as a robust therapeutic strategy to alleviate related symptoms and restore cardiac functions. Prior to this, however, for successful cell therapy, an adequate number of functional and safe cardiac cells needs to be generated. For this purpose, our approach was boosting the proliferative capacity of stem cell-derived cardiomyocytes using growth factors, such as fibroblast growth factor 10 (FGF10) and cardiotrophin-1 (CT-1). Our results demonstrated that FGF10 and CT-1 substantially increased the number of cardiac cells that originated from stem cells. In molecular assays, to assess RNA and protein level alterations, the enhanced presence of specific markers for cardiac cells after treatment of stem cells with FGF10 and/or CT-1 was confirmed. This inducing potential of cardiac cells can particularly be applicable in the cell replacement-based therapies of cardiac infarction. This research sheds light on the putative effect of FGF10 and CT-1 in the transition of stem cells to cardiac cells, leading to the repair and survival of the heart.For heart regeneration purposes, embryonic stem cell (ES)-based strategies have been developed to induce the proliferation of cardiac progenitor cells towards cardiomyocytes. Fibroblast growth factor 10 (FGF10) contributes to cardiac development and induces cardiomyocyte differentiation in vitro. Yet, among pro-cardiogenic factors, including cardiotrophin-1 (CT-1), the hyperplastic function of FGF10 in cardiomyocyte turnover remains to be further characterized. We investigated the proliferative effects of FGF10 on ES-derived cardiac progenitor cells in the intermediate developmental stage and examined the putative interplay between FGF10 and CT-1 in cardiomyocyte proliferation. Mouse ES cells were treated with FGF10 and/or CT-1. Differential expression of cardiomyocyte-specific gene markers was analyzed at transcript and protein levels. Substantial upregulation of sarcomeric α-actinin was detected by qPCR, flow cytometry, Western blot and immunocytochemistry. FGF10 enhanced the expression of other structural proteins (MLC-2a, MLC-2v and TNNT2), transcriptional factors (NKX2-5 and GATA4), and proliferation markers (Aurora B and YAP-1). FGF10/CT-1 co-administration led to an upregulation of proliferation markers, suggesting the synergistic potential of FGF10 + CT-1 on cardiomyogenesis. In summary, we provided evidence that FGF10 and CT-1 induce cardiomyocyte structural proteins, associated transcription factors, and cardiac cell proliferation, which could be applicable in therapies to replenish damaged cardiomyocytes.

Icariin Promotes In Vitro Cardiomyocyte Proliferation and Differentiation in Human Bone Marrow-Derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are the excellent candidates in myocardial regeneration given their easy accessibility, low immunogenicity and high potential for cardiomyocyte differentiation. This work focused on investigating the role of icariin, a main active component of the Traditional Chinese herb epimedium, in human bone marrow-derived MSCs (BMSCs) proliferation and differentiation into cardiomyocytes In Vitro. Human BMSCs were cultivated In Vitro, and MTT assay was conducted to measure their proliferation. On this basis, we selected the optimal icariin dose for promoting the proliferation to induce cardiomyocyte differentiation of MSCs, which were pretreated with or without 5-azacytidine (5-Aza). Cardiac-specific cardiac troponin I (cTnI) and connexin 43 (Cx43)-positive cells were detected by immunofluorescent staining. The differentiation ratio of MSCs was examined by flow cytometry. This study measured early cardiac transcription factors (TFs) Nkx2.5 and GATA4 levels through RT-PCR and Western blotting (WB). As a result, icariin increased MSC proliferation dependent on its dose, and the optimal dose was determined to be 80 μg/l. Furthermore, MSCs showed minimal cardiomyogenic differentiation when induced by icariin alone as confirmed by the expression of cardiac-related markers. Moreover, a synergic interaction was observed when icariin and 5-Aza cooperated to induce cardiomyocyte differentiation of MSCs. In conclusion, Icariin stimulates proliferation and facilitates cardiomyocyte differentiation of MSCs In Vitro and may be potentially used as a new method for enhancing the MSCs efficacy in cardiovascular disease.

The effect of shear stress on cardiac differentiation of mesenchymal stem cells.

Stem cell therapy is developing as a valuable therapeutic trend for heart diseases. Most recent studies are aimed to find the most appropriate types of stem cells for the treatment of myocardial infarction (MI). The animal models have shown that bone marrow-derived mesenchymal stem cells (BMSCs) are a possible, safe, and efficient type of stem cell used in MI. The previous study demonstrated that 5-Azacytidine (5-Aza) could promote cardiac differentiation in stem cells. This study used 5-Aza to induce cardiomyocyte differentiation in BMSCs both in static and microfluidic cell culture systems. For this purpose, we investigated the differentiation by using real-time PCR and Immunocytochemistry (ICC) Analysis. Our results showed that 5-Aza could cause to express cardiac markers in BMSCs as indicated by real-time PCR and immunocytochemistry (ICC). However, BMSCs are exposed to both 5-Aza and shear stress, and their synergistic effects could significantly induce cardiac gene expressions in BMSCs. This level of gene expression was observed neither in 5-Aza nor in shear stress groups only. These results demonstrate that when BMSCs expose to 5-Aza as well as mechanical cues such as shear stress, the cardiac gene expression can be increased dramatically.

Human Engineered Heart Tissue Patches Remuscularize the Injured Heart in a Dose-Dependent Manner

Human engineered heart tissue (EHT) transplantation represents a potential regenerative strategy for patients with heart failure and has been successful in preclinical models. Clinical application requires upscaling, adaptation to good manufacturing practices, and determination of the effective dose. Cardiomyocytes were differentiated from 3 different human induced pluripotent stem cell lines including one reprogrammed under good manufacturing practice conditions. Protocols for human induced pluripotent stem cell expansion, cardiomyocyte differentiation, and EHT generation were adapted to substances available in good manufacturing practice quality. EHT geometry was modified to generate patches suitable for transplantation in a small-animal model and perspectively humans. Repair efficacy was evaluated at 3 doses in a cryo-injury guinea pig model. Human-scale patches were epicardially transplanted onto healthy hearts in pigs to assess technical feasibility. We created mesh-structured tissue patches for transplantation in guinea pigs (1.5×2.5 cm, 9-15×106 cardiomyocytes) and pigs (5×7 cm, 450×106 cardiomyocytes). EHT patches coherently beat in culture and developed high force (mean 4.6 mN). Cardiomyocytes matured, aligned along the force lines, and demonstrated advanced sarcomeric structure and action potential characteristics closely resembling human ventricular tissue. EHT patches containing ≈4.5, 8.5, 12×106, or no cells were transplanted 7 days after cryo-injury (n=18-19 per group). EHT transplantation resulted in a dose-dependent remuscularization (graft size: 0%-12% of the scar). Only high-dose patches improved left ventricular function (+8% absolute, +24% relative increase). The grafts showed time-dependent cardiomyocyte proliferation. Although standard EHT patches did not withstand transplantation in pigs, the human-scale patch enabled successful patch transplantation. EHT patch transplantation resulted in a partial remuscularization of the injured heart and improved left ventricular function in a dose-dependent manner in a guinea pig injury model. Human-scale patches were successfully transplanted in pigs in a proof-of-principle study.

Pilot study on cardiogenic differentiation capability of rabbit mesenchymal stem cells

Cardiovascular diseases are still one of the most common reasons for mortality in humans. Mesenchymal stem cells (MSCs) are preferable in cardiac regeneration cell-based therapies because of their allogeneic and high proliferative potential. The electrophysiological properties of the rabbit heard is closer to human than the mouse. The current study aimed to trace mRNA expression changes of two stemness/cardiogenic differentiation ability-related transcriptionala factors OCT4 and GATA4 in rabbit MSCs during early stages of induced cardiomyocyte differentiation in vitro. The mesenchymal stem cell originated from different anatomical areas-subcutaneous, visceral, bone marrow and pericardial tissue. The cardiac differentiation protocol for mouse embryonic stem cells in hanging drop was adopted in rabbit MSCs. The best formed embryonal bodies (EBs) like structures were collected and cultivated on gelatin-coated plates. The total mRNA was obtained before cardiac differentiation and on the 6th day after it. SYBER based real-time PCR was performed to evaluate the mRNA expression fold-changes of OCT4 and GATA4. The cultivation of MSCs in hanging drops during cardiac differentiation induced EBs formation, without any contractile activity up to the 6th day of the differentiation in all cell types. The applied differentiation protocol significantly downregulated GATA4 expression in ADSCs - EBs, while in BMSCs, both target genes were significantly upregulated. In conclusion, the adopted cardiac differentiation protocol from mouse embryonic stem cells could be a useful approach for rabbit bone marrow mesenchymal stem cells. Since the rest of the cells revealed weak cardiogenic capability at this early stage, some modifications of induction protocols should be considered.

Splice-Junction-Based Mapping of Alternative Isoforms in the Human Proteome

SUMMARYThe protein-level translational status and function of many alternative splicing events remain poorly understood. We use an RNA sequencing (RNA-seq)-guided proteomics method to identify protein alternative splicing isoforms in the human proteome by constructing tissue-specific protein databases that prioritize transcript splice junction pairs with high translational potential. Using the custom databases to reanalyze ~80 million mass spectra in public proteomics datasets, we identify more than 1,500 noncanonical protein isoforms across 12 human tissues, including ~400 sequences undocumented on TrEMBL and RefSeq databases. We apply the method to original quantitative mass spectrometry experiments and observe widespread isoform regulation during human induced pluripotent stem cell cardiomyocyte differentiation. On a proteome scale, alternative isoform regions overlap frequently with disordered sequences and post-translational modification sites, suggesting that alternative splicing may regulate protein function through modulating intrinsically disordered regions. The described approach may help elucidate functional consequences of alternative splicing and expand the scope of proteomics investigations in various systems.

P2571Differentiation of iPS-derived cardiac mesoderm cells into cardiomyocytes using inducible shRNA technology

Abstract Introduction The development of robust and scalable cardiomyocyte differentiation protocols from human pluripotent stem cells (hPSCs) offers the opportunity to derive large number of autologous cardiomyocytes for cell therapy. However, human induced pluripotent stem cells-derived cardiomyocytes (hiPSCs-CM) do not proliferate, are susceptible to ischemia and poorly retained in the recipient myocardium. In addition, arrhythmias are a severe side effect of cell therapy with hiPSC-CM, probably due to their immature phenotype. Based on previous observations in our lab, we speculate that the heart environment might be an important factor to enable full maturation and in situ coupling of the implanted cells in vivo. We therefore speculate that transplantation of robust, proliferative, partially committed, non-tumorigenic cardiac mesoderm cells and subsequent differentiation in situ may overcome these limitations. Purpose We seek to engineer hiPSC-derived cardiac mesoderm cells for implantation in the failing myocardium that, once they have settled, consolidated, and proliferated in situ, can be selectively induced to mature into cardiomyocytes. Methods Wnt signalling induces mesoderm lineage differentiation of hiPSCs and inhibits further maturation towards CM. To induce Wnt signalling inhibition, we designed a customized biomimetic shRNA targeting the Wnt agonist β-catenin fused to a fluorescent reported protein controlled by a Tet-ON promoter. This transgene was transferred to the safe harbour locus AAVS1 of the OPTI-OX hiPS cell line constitutively expressing the reverse tetracycline transactivator protein (rtTA) in the ROSA26 locus. In this configuration, gene expression can be activated in trans upon doxycycline administration. Results We studied the kinetics of gene expression activation on the engineered undifferentiated hiPSCs and observed that a maximal reduction of 80–90% β-catenin correlating with reporter expression was achieved upon a 4-day treatment with doxycycline. Importantly, this activation could be fully reverted 6 days upon withdrawal. Furthermore, in vitro differentiation of cardiac mesoderm cells into cardiomyocytes was enhanced upon doxycycline administration as compared to the untreated cells as assessed by the upregulation of cardiomyocyte specific genes and the increased percentage of cTNT+ cells. Conclusion An inducible system for targeted β-catenin downregulation by shRNA allows for significant, sustained, and reversible Wnt signalling modulation, which is sufficient to induce cardiomyocyte differentiation of cardiac mesoderm cells in vitro. This technology may be implemented to differentiate the transplanted cells in the failing myocardium, opening new prospects to overcome the current limitations of hiPSC-based cardiac cell therapy. Acknowledgement/Funding BMBF funding (FKZ 13GW0098, FKZ 13GW0099) DFG funding through the BSRT GSC 203

Priming with oxytocin and relaxin improves cardiac differentiation of adipose tissue-derived stem cells.

Previous studies have identified the heart as a source and a target tissue for oxytocin and relaxin hormones. These hormones play important roles in the regulation of cardiovascular function and repair of ischemic heart injury. In the current study, we examined the impact of oxytocin and relaxin on the development of cardiomyocytes from mesenchymal stem cells. For this purpose, mouse adipose tissue-derived stem cells (ADSCs) were treated with different concentrations of oxytocin or relaxin for 4 days. Three weeks after initiation of cardiac induction, differentiated ADSCs expressed cardiac-specific genes, Gata4, Mef2c, Nkx2.5, Tbx5, α- and β-Mhc, Mlc2v, Mlc2a and Anp, and cardiac proteins including connexin 43, desmin and α-actinin. 10 -7 M oxytocin and 50 ng/mL relaxin induced the maximum upregulation in the expression of cardiac markers. A combination of oxytocin and relaxin induced cardiomyocyte differentiation more potently than the individual factors. In our experiment, oxytocin-relaxin combination increased the population of cardiac troponin I-expressing cells to 6.84% as compared with 2.36% for the untreated ADSCs, 3.7% for oxytocin treatment and 3.41% for relaxin treatment groups. In summary, the results of this study indicated that oxytocin and relaxin hormones individually and in combination can improve cardiac differentiation of ADSCs, and treatment of the ADSCs and possibly other mesenchymal stem cells with these hormones may enhance their cardiogenic differentiation and survival after transplantation into the ischemic heart tissue.

Probiotic Metabolites Promote Stem Cell Proliferation and Differentiation

Probiotic metabolites (PM) impart numerous health benefits in human and may have a potential to induce cell proliferation and differentiation. We tested the effects of PM released by probiotics found in yogurts on stem cell proliferation and differentiation. Five different brands of yogurts (Oikos, Stonyfield, Yoplait, Chobani and Activia) often sold in the supermarkets were tested in this study. Yogurt cultures analyzed by Gram‐staining and DNA sequencing of 16s rRNA gene identified that these yogurts are not equally supplemented and include different types and various amounts of probiotics. The probiotics were cultured and the condition media containing PMs were collected and used in our study. To evaluate the effect of PM on stem cells, human induced pluripotent stem cells (iPSC) were treated with probiotic conditioned media. We found that probiotic Lactobacillus reuteri conditioned media (LRCM) induced iPSC proliferation at lower doses but inhibited cell proliferation at higher doses. Additionally, expression of the proliferating cell nuclear antigen (PCNA) and pluripotent stem cell factors, OCT4, SOX2 and NANOG transcripts was upregulated in iPSCs treated with LRCM. iPS cells exposed to LRCM during in vitro differentiation led to enhanced expression of myocyte markers, Nkx2.5 and cTnT. Consistently, sodium butyrate, a defined bacterial metabolite (short chain fatty acid), had similar effect on iPSC proliferation and expression of pluripotent markers and myocyte markers. In summary, we have demonstrated that probiotics in different brands of yogurts are diverse in their numbers, viability and composition. Further, our results show that PMs can potentiate iPS proliferation and induce cardiomyocyte differentiation. Our findings have laid a foundation for further investigation of how probiotics and its metabolites from yogurt affect human health and disease.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Evaluation of Hydrogels Presenting Extracellular Matrix-Derived Adhesion Peptides and Encapsulating Cardiac Progenitor Cells for Cardiac Repair.

Cell therapy is an emerging paradigm for the treatment of heart disease. In spite of the exciting and promising preclinical results, the benefits of cell therapy for cardiac repair in patients have been modest at best. Biomaterials-based approaches may overcome the barriers of poor differentiation and retention of transplanted cells. In this study, we prepared and tested hydrogels presenting extracellular matrix (ECM)-derived adhesion peptides as delivery vehicles for c-kit+ cardiac progenitor cells (CPCs). We assessed their effects on cell behavior in vitro as well as cardiac repair in rats undergoing ischemia reperfusion. Hydrogels presenting the collagen-derived GFOGER peptide induced cardiomyocyte differentiation of CPCs as demonstrated by increased expression of cardiomyocyte structural proteins. However, conditioned media obtained from GFOGER hydrogels showed lower levels of secreted reparative factors. Interestingly, following injection in rats undergoing ischemia-reperfusion, treatment with CPCs encapsulated in nonadhesive RDG-presenting hydrogels resulted in the preservation of cardiac contractility and attenuation of postinfarct remodeling whereas the adhesion peptide-presenting hydrogels did not induce any functional improvement. Retention of cells was significantly higher when delivered with nonadhesive hydrogels compared to ECM-derived peptide gels. These data suggest that factors including cell differentiation state, paracrine factors and interaction with biomaterials influence the effectiveness of biomaterials-based cell therapy. A holistic consideration of these multiple variables should be included in cell-biomaterial combination therapy designs.

Pathologic Stimulus Determines Lineage Commitment of Cardiac C-kit+ Cells.

Although cardiac c-kit+ cells are being tested in clinical trials, the circumstances that determine lineage differentiation of c-kit+ cells in vivo are unknown. Recent findings suggest that endogenous cardiac c-kit+ cells rarely contribute cardiomyocytes to the adult heart. We assessed whether various pathological stimuli differentially affect the eventual cell fates of c-kit+ cells. We used single-cell sequencing and genetic lineage tracing of c-kit+ cells to determine whether various pathological stimuli would result in different fates of c-kit+ cells. Single-cell sequencing of cardiac CD45-c-kit+ cells showed innate heterogeneity, indicative of the existence of vascular and mesenchymal c-kit+ cells in normal hearts. Cardiac pressure overload resulted in a modest increase in c-kit-derived cardiomyocytes, with significant increases in the numbers of endothelial cells and fibroblasts. Doxorubicin-induced acute cardiotoxicity did not increase c-kit-derived endothelial cell fates but instead induced cardiomyocyte differentiation. Mechanistically, doxorubicin-induced DNA damage in c-kit+ cells resulted in expression of p53. Inhibition of p53 blocked cardiomyocyte differentiation in response to doxorubicin, whereas stabilization of p53 was sufficient to increase c-kit-derived cardiomyocyte differentiation. These results demonstrate that different pathological stimuli induce different cell fates of c-kit+ cells in vivo. Although the overall rate of cardiomyocyte formation from c-kit+ cells is still below clinically relevant levels, we show that p53 is central to the ability of c-kit+ cells to adopt cardiomyocyte fates, which could lead to the development of strategies to preferentially generate cardiomyocytes from c-kit+ cells.

Differentiation of stem cells isolated from rat skeletal muscles towards cardiomyocytes: The effect of an inhibitor of DNA methylation 5-azacytidine

The effects of the inhibitor of DNA methylation 5-azacytidine on stem (satellite) cells isolated from fetal and definitive skeletal muscles of rats lead to the expression of marker genes of cardiomyogenesis (Gata4, Nkx2.5, connexin-43, n-cadherin, as well as Cacna 1 c encoding the cardiac subunit of the L-type Ca2+-channel). Through comparative analysis of the dynamics of expression of key markers of cardiomyogenesis, it was established that satellite cells isolated from fetal muscles have a more expressive potency to cardiomyocyte differentiation in vitro (expression of specific marker genes) compared to cells from muscles of adult animals. In the process of induced cardiomyocyte differentiation, the expression of MyoD and m-cadherin marker genes of skeletal muscle differentiation was not detected, suggesting that in the experiments presented the program of myogenic differentiation of skeletal muscles is inhibited.

Coculturing with endothelial cells promotes in vitro maturation and electrical coupling of human embryonic stem cell–derived cardiomyocytes

Pluripotent human embryonic stem cells (hESC) are a promising source of repopulating cardiomyocytes. We hypothesized that we could improve maturation of cardiomyocytes and facilitate electrical interconnections by creating a model that more closely resembles heart tissue; that is, containing both endothelial cells (ECs) and cardiomyocytes. We induced cardiomyocyte differentiation in the coculture of an hESC line expressing the cardiac reporter NKX2.5-green fluorescent protein (GFP), and an Akt-activated EC line (E4+ECs). We quantified spontaneous beating rates, synchrony, and coordination between different cardiomyocyte clusters using confocal imaging of Fura Red-detected calcium transients and computer-assisted image analysis. After 8 days in culture, 94% ± 6% of the NKX2-5GFP+ cells were beating when hESCs embryonic bodies were plated on E4+ECs compared with 34% ± 12.9% for controls consisting of hESCs cultured on BD Matrigel (BD Biosciences) without ECs at Day 11 in culture. The spatial organization of beating areas in cocultures was different. The GFP+ cardiomyocytes were close to the E4+ECs. The average beats/min of the cardiomyocytes in coculture was faster and closer to physiologic heart rates compared with controls (50 ± 14 [n = 13] vs 25 ± 9 [n = 8]; p < 0.05). The coculture with ECs led to synchronized beating relying on the endothelial network, as illustrated by the loss of synchronization upon the disruption of endothelial bridges. The coculturing of differentiating cardiomyocytes with Akt-activated ECs but not EC-conditioned media results in (1) improved efficiency of the cardiomyocyte differentiation protocol and (2) increased maturity leading to better intercellular coupling with improved chronotropy and synchrony.

Mitochondrial pyruvate dehydrogenase phosphatase 1 regulates the early differentiation of cardiomyocytes from mouse embryonic stem cells.

Mitochondria are crucial for maintaining the properties of embryonic stem cells (ESCs) and for regulating their subsequent differentiation into diverse cell lineages, including cardiomyocytes. However, mitochondrial regulators that manage the rate of differentiation or cell fate have been rarely identified. This study aimed to determine the potential mitochondrial factor that controls the differentiation of ESCs into cardiac myocytes. We induced cardiomyocyte differentiation from mouse ESCs (mESCs) and performed microarray assays to assess messenger RNA (mRNA) expression changes at differentiation day 8 (D8) compared with undifferentiated mESCs (D0). Among the differentially expressed genes, Pdp1 expression was significantly decreased (27-fold) on D8 compared to D0, which was accompanied by suppressed mitochondrial indices, including ATP levels, membrane potential, ROS and mitochondrial Ca2+. Notably, Pdp1 overexpression significantly enhanced the mitochondrial indices and pyruvate dehydrogenase activity and reduced the expression of cardiac differentiation marker mRNA and the cardiac differentiation rate compared to a mock control. In confirmation of this, a knockdown of the Pdp1 gene promoted the expression of cardiac differentiation marker mRNA and the cardiac differentiation rate. In conclusion, our results suggest that mitochondrial PDP1 is a potential regulator that controls cardiac differentiation at an early differentiation stage in ESCs.

Requirement of IP3 receptor 3 (IP3R3) in nitric oxide induced cardiomyocyte differentiation of mouse embryonic stem cells

Nitric oxide (NO) markedly induces cardiomyocyte (CM) differentiation of embryonic stem (ES) cells. Here we examined the role of the Ca2+ signaling in the NO-induced CM differentiation of mouse ES cells. We found that NO induced intracellular Ca2+ increases in ES cells in a dose-dependent manner, and application of IP3 pathway antagonists not only significantly inhibited this induced Ca2+ increase but also abolished NO-induced CM differentiation of ES cells. Subsequently, all 3 types of inositol 1, 4, 5-trisphosphate (IP3) receptors (IP3Rs) in mouse ES cells were individually or triply knocked down. Interestingly, only knockdown of type 3 IP3R (IP3R3) or triple-knockdown of three types of IP3Rs significantly inhibited the NO-induced Ca2+ increases. Consistently, IP3R3 knockdown blocked the NO-induced CM differentiation of ES cells. CMs derived from IP3R3 knockdown ES cells also showed both structural and functional defects. In summary, our results indicate that the IP3R3-Ca2+ pathway is required for NO-induced CM differentiation of ES cells.

Neonatal Heart-Enriched miR-708 Promotes Differentiation of Cardiac Progenitor Cells in Rats.

Cardiovascular disease is becoming the leading cause of death throughout the world. However, adult hearts have limited potential for regeneration after pathological injury, partly due to the quiescent status of stem/progenitor cells. Reactivation of cardiac stem/progenitor cells to create more myocyte progeny is one of the key steps in the regeneration of a damaged heart. In this study, miR-708 was identified to be enriched in the neonatal cardiomyocytes of rats, but this has not yet been proven in adult humans. A lower level of miR-708 in c-kit(+) stem/progenitor cells was detected compared to non-progenitors. Overexpression of miR-708 induced cardiomyocyte differentiation of cardiac stem/progenitor cells. This finding strengthened the potential of applying miRNAs in the regeneration of injured hearts, and this indicates that miR-708 could be a novel candidate for treatment of heart diseases.

5-azacytidine promotes terminal differentiation of hepatic progenitor cells

5-azacytidine (5-azaC) is known to induce cardiomyocyte differentiation. However, its function in hepatocyte differentiation is unclear. The present study investigated the in vitro capability of 5-azaC to promote maturation and differentiation of mouse embryonic hepatic progenitor cells, with the aim of developing an approach for improving hepatic differentiation. Mouse embryonic hepatic progenitor cells (HP14.5 cells) were treated with 5-azaC at concentrations from 0 to 20 μmol/l, in addition to hepatocyte induction culture medium. Hepatocyte induction medium induces HP14.5 cell differentiation. 5-azaC may enhance the albumin promotor-driven Gaussia luciferase (ALB-GLuc) activity in induced HP14.5 cells. In the present study 2 μmol/l was found to be the optimum concentration with which to achieve this. The expression of hepatocyte-associated factors was not significantly different between the group treated with 5-azaC alone and the control group. The mRNA levels of ALB; cytokeratin 18 (CK18); tyrosine aminotransferase (TAT); and cytochrome p450, family 1, member A1 (CYP1A1); in addition to the protein levels of ALB, CK18 and uridine diphosphate glucuronyltransferase 1A (UGT1A) in the induced group with 5-azaC, were higher than those in the induced group without 5-azaC, although no significant differences were detected in expression of the hepatic stem cell markers, DLK and α-fetoprotein, between the two groups. Treatment with 5-azaC alone did not affect glycogen synthesis or indocyanine green (ICG) metabolic function in HP14.5 cells, although it significantly increased ICG uptake and periodic acid-Schiff-positive cell numbers amongst HP14.5 cells. Therefore, the present study demonstrated that treatment with 5-azaC alone exerted no effects on the maturation and differentiation of HP14.5 cells. However, 5-azaC exhibited a synergistic effect on the terminal differentiation of induced hepatic progenitor cells in association with a hepatic induction medium.