Cardiac Lineage Research Articles

Sexual Dimorphism of the Heart: Genetics, Epigenetics, and Development.

The democratization of genomic technologies has revealed profound sex biases in expression patterns in every adult tissue, even in organs with no conspicuous differences, such as the heart. With the increasing awareness of the disparities in cardiac pathophysiology between males and females, there are exciting opportunities to explore how sex differences in the heart are established developmentally. Although sexual dimorphism is traditionally attributed to hormonal influence, expression and epigenetic sex biases observed in early cardiac development can only be accounted for by the difference in sex chromosome composition, i.e., XX in females and XY in males. In fact, genes linked to the X and Y chromosomes, many of which encode regulatory factors, are expressed in cardiac progenitor cells and at every subsequent developmental stage. The effect of the sex chromosome composition may explain why many congenital heart defects originating before gonad formation exhibit sex biases in presentation, mortality, and morbidity. Some transcriptional and epigenetic sex biases established soon after fertilization persist in cardiac lineages, suggesting that early epigenetic events are perpetuated beyond early embryogenesis. Importantly, when sex hormones begin to circulate, they encounter a cardiac genome that is already functionally distinct between the sexes. Although there is a wealth of knowledge on the effects of sex hormones on cardiac function, we propose that sex chromosome-linked genes and their downstream targets also contribute to the differences between male and female hearts. Moreover, identifying how hormones influence sex chromosome effects, whether antagonistically or synergistically, will enhance our understanding of how sex disparities are established. We also explore the possibility that sexual dimorphism of the developing heart predicts sex-specific responses to environmental signals and foreshadows sex-biased health-related outcomes after birth.

Adherens junction engagement regulates functional patterning of the cardiac pacemaker cell lineage.

Cardiac pacemaker cells (CPCs) rhythmically initiate the electrical impulses that drive heart contraction. CPCs display the highest rate of spontaneous depolarization in the heart despite being subjected to inhibitory electrochemical conditions that should theoretically suppress their activity. While several models have been proposed to explain this apparent paradox, the actual molecular mechanisms that allow CPCs to overcome electrogenic barriers to their function remain poorly understood. Here, we have traced CPC development at single-cell resolution and uncovered a series of cytoarchitectural patterning events that are critical for proper pacemaking. Specifically, our data reveal that CPCs dynamically modulate adherens junction (AJ) engagement to control characteristics including surface area, volume, and gap junctional coupling. This allows CPCs to adopt a structural configuration that supports their overall excitability. Thus, our data have identified a direct role for local cellular mechanics in patterning critical morphological features that are necessary for CPC electrical activity.

Cardiac Differentiation of Mesenchymal Stem Cells: Impact of Biological and Chemical Inducers.

Cardiovascular disorders (CVDs) are the leading cause of global death, widely occurs due to irreparable loss of the functional cardiomyocytes. Stem cell-based therapeutic approaches, particularly the use of Mesenchymal Stem Cells (MSCs) is an emerging strategy to regenerate myocardium and thereby improving the cardiac function after myocardial infarction (MI). Most of the current approaches often employ the use of various biological and chemical factors as cues to trigger and modulate the differentiation of MSCs into the cardiac lineage. However, the recent advanced methods of using specific epigenetic modifiers and exosomes to manipulate the epigenome and molecular pathways of MSCs to modify the cardiac gene expression yield better profiled cardiomyocyte like cells in vitro. Hitherto, the role of cardiac specific inducers triggering cardiac differentiation at the cellular and molecular level is not well understood. Therefore, the current review highlights the impact and recent trends in employing biological and chemical inducers on cardiac differentiation of MSCs. Thereby, deciphering the interactions between the cellular microenvironment and the cardiac inducers will help us to understand cardiomyogenesis of MSCs. Additionally, the review also provides an insight on skeptical roles of the cell free biological factors and extracellular scaffold assisted mode for manipulation of native and transplanted stem cells towards translational cardiac research.

Characterization of IPSCs-Derived Cardiomyocytes

Purpose Functional cardiomyocytes are instrumental to in vitro studies of myocardial diseases, understanding toxicities, and studying new therapies. Because functional cardiomyocytes in tissue culture are scarce and difficult to obtain we sought to differentiate and characterize cardiomyocytes derived from induced pluripotent stem cells (iPSCs) in our lab. Methods Induced pluripotent stem cells (iPSCs) from dermal fibroblasts obtained from a healthy donor were utilized to derive cardiomyocytes. Cells were cultured until 70-85% confluent and differentiated to cardiomyocytes using differentiation kit that contain a high dose of GSK3B inhibitor to induce mesodermal lineage commitment for one day that will result in up-regulation of endogenous BMP4 and activin/NODAL followed by WNT signaling modulation for two days for Cardiac mesodermal induction. Immunocytochemistry for NKX2-5 and TNNT2 were used to characterize differentiated cardiomyocytes. NanoString micro RNA was used to profile commercial and lab differentiated cardiomyocytes. Results Cells start beating on day eight and stayed beating for 30 days in maintenance media. Furthermore, cells were positive for two key markers of the human cardiac lineage: NKX2-5 for early cardiac mesoderm and TNNT2/cTNT for cardiomyocytes. The percentage of differentiation to cardiomyocytes were about 70% of the total iPSCs. Both commercial and lab dedifferentiated cardiomyocytes have similar contractility function. We compared micro RNA profile of our differentiated cardiomyocytes to commercial iPSCs-derived cardiomyocytes and they cluster very close to each other. However, both were clustered differently from miRNA profile of normal adult tissue cardiomyocytes. Conclusion Unlimited iPSCs-derived cardiomyocytes can be obtained in a robust and cost-effective method from fibroblasts. The cells have similar miRNA profile, molecular markers and contractile function in comparison to commercially available cardiomyocytes but have unique patterns of gene expression in comparison to non-failing adult tissue.

Spatiotemporal single-cell RNA sequencing of developing chicken hearts identifies interplay between cellular differentiation and morphogenesis

Single-cell RNA sequencing is a powerful tool to study developmental biology but does not preserve spatial information about tissue morphology and cellular interactions. Here, we combine single-cell and spatial transcriptomics with algorithms for data integration to study the development of the chicken heart from the early to late four-chambered heart stage. We create a census of the diverse cellular lineages in developing hearts, their spatial organization, and their interactions during development. Spatial mapping of differentiation transitions in cardiac lineages defines transcriptional differences between epithelial and mesenchymal cells within the epicardial lineage. Using spatially resolved expression analysis, we identify anatomically restricted expression programs, including expression of genes implicated in congenital heart disease. Last, we discover a persistent enrichment of the small, secreted peptide, thymosin beta-4, throughout coronary vascular development. Overall, our study identifies an intricate interplay between cellular differentiation and morphogenesis.

Genetic and Cellular Interaction During Cardiovascular Development Implicated in Congenital Heart Diseases.

Congenital heart disease (CHD) is the most common life-threatening congenital anomaly. CHD occurs due to defects in cardiovascular development, and the majority of CHDs are caused by a multifactorial inheritance mechanism, which refers to the interaction between genetic and environmental factors. During embryogenesis, the cardiovascular system is derived from at least four distinct cell lineages: the first heart field, second heart field, cardiac neural crest, and proepicardial organ. Understanding the genes involved in each lineage is essential to uncover the genomic architecture of CHD. Therefore, we provide an overview of recent research progress using animal models and mutation analyses to better understand the molecular mechanisms and pathways linking cardiovascular development and CHD. For example, we highlight our recent work on genes encoding three isoforms of inositol 1,4,5-trisphosphate receptors (IP3R1, 2, and 3) that regulate various vital and developmental processes, which have genetic redundancy during cardiovascular development. Specifically, IP3R1 and 2 have redundant roles in the atrioventricular cushion derived from the first heart field lineage, whereas IP3R1 and 3 exhibit redundancy in the right ventricle and the outflow tract derived from the second heart field lineage, respectively. Moreover, 22q11.2 deletion syndrome (22q11DS) is highly associated with CHD involving the outflow tract, characterized by defects of the cardiac neural crest lineage. However, our studies have shown that TBX1, a major genetic determinant of 22q11DS, was not expressed in the cardiac neural crest but rather in the second heart field, suggesting the importance of the cellular interaction between the cardiac neural crest and the second heart field. Comprehensive genetic analysis using the Japanese genome bank of CHD and mouse models revealed that a molecular regulatory network involving GATA6, FOXC1/2, TBX1, SEMA3C, and FGF8 was essential for reciprocal signaling between the cardiac neural crest and the second heart field during cardiovascular development. Elucidation of the genomic architecture of CHD using induced pluripotent stem cells and next-generation sequencing technology, in addition to genetically modified animal models and human mutation analyses, would facilitate the development of regenerative medicine and/or preventive medicine for CHD in the near future.

Bi-allelic MCM10 variants associated with immune dysfunction and cardiomyopathy cause telomere shortening

Minichromosome maintenance protein 10 (MCM10) is essential for eukaryotic DNA replication. Here, we describe compound heterozygous MCM10 variants in patients with distinctive, but overlapping, clinical phenotypes: natural killer (NK) cell deficiency (NKD) and restrictive cardiomyopathy (RCM) with hypoplasia of the spleen and thymus. To understand the mechanism of MCM10-associated disease, we modeled these variants in human cell lines. MCM10 deficiency causes chronic replication stress that reduces cell viability due to increased genomic instability and telomere erosion. Our data suggest that loss of MCM10 function constrains telomerase activity by accumulating abnormal replication fork structures enriched with single-stranded DNA. Terminally-arrested replication forks in MCM10-deficient cells require endonucleolytic processing by MUS81, as MCM10:MUS81 double mutants display decreased viability and accelerated telomere shortening. We propose that these bi-allelic variants in MCM10 predispose specific cardiac and immune cell lineages to prematurely arrest during differentiation, causing the clinical phenotypes observed in both NKD and RCM patients.

Myocyte-specific enhancer factor 2c triggers transdifferentiation of adipose tissue-derived stromal cells into spontaneously beating cardiomyocyte-like cells

Cardiomyocyte regeneration is limited in adults. The adipose tissue-derived stromal vascular fraction (Ad-SVF) contains pluripotent stem cells that rarely transdifferentiate into spontaneously beating cardiomyocyte-like cells (beating CMs). However, the characteristics of beating CMs and the factors that regulate the differentiation of Ad-SVF toward the cardiac lineage are unknown. We developed a simple culture protocol under which the adult murine inguinal Ad-SVF reproducibly transdifferentiates into beating CMs without induction. The beating CMs showed the striated ventricular phenotype of cardiomyocytes and synchronised oscillation of the intracellular calcium concentration among cells on day 28 of Ad-SVF primary culture. We also identified beating CM-fated progenitors (CFPs) and performed single-cell transcriptome analysis of these CFPs. Among 491 transcription factors that were differentially expressed (≥ 1.75-fold) in CFPs and the beating CMs, myocyte-specific enhancer 2c (Mef2c) was key. Transduction of Ad-SVF cells with Mef2c using a lentiviral vector yielded CFPs and beating CMs with ~ tenfold higher cardiac troponin T expression, which was abolished by silencing of Mef2c. Thus, we identified the master gene required for transdifferentiation of Ad-SVF into beating CMs. These findings will facilitate the development of novel cardiac regeneration therapies based on gene-modified, cardiac lineage-directed Ad-SVF cells.

TMT-based quantitative proteome profiles reveal the memory function of a whole heart decellularized matrix for neural stem cell trans-differentiation into the cardiac lineage.

Whole organ or tissue decellularized matrices are a promising scaffold for tissue engineering because they maintain the specific memory of the original organ or tissue. A whole organ or tissue decellularized matrix contains extracellular matrix (ECM) components, and exhibits ultrastructural and mechanical properties, which could significantly regulate the fate of stem cells. To better understand the memory function of whole organ decellularized matrices, we constructed a heart decellularized matrix and seeded cross-embryonic layer stem cells - neural stem cells (NSCs) to repopulate the matrix, engineering cardiac tissue, in which a large number of NSCs differentiated into the neural lineage, but besides that, NSCs showed an obvious tendency of trans-differentiating into cardiac lineage cells. The results demonstrated that the whole heart decellularized microenvironment possesses memory function. To reveal the underlying mechanism, TMT-based quantitative proteomics analysis was used to identify the differently expressed proteins in the whole heart decellularized matrix compared with a brain decellularized matrix. 937 of the proteins changed over 1.5 fold, with 573 of the proteins downregulated and 374 of the proteins upregulated, among which integrin ligands in the ECM serve as key signals in regulating NSC fate. The findings here provide a novel insight into the memory function of tissue-specific microenvironments and pave the way for the therapeutic application of personalized tissues.

Alternative Splicing of a Transposable Element into the Human Long Noncoding RNA <i>CARMEN</i> Is a Switch for Cardiac Precursor Cell Specification

The major cardiac cell types composing the adult heart arise from common multipotent precursor cells. Cardiac lineage decisions are guided by extrinsic and cell-autonomous factors, including recently discovered long noncoding RNAs (lncRNAs). The CARMEN locus, which is known to dictate specification towards the cardiomyocyte (CM) and the smooth muscle cell (SMC) fates, generates a diversity of alternatively spliced lncRNAs. Here, we identify one of these isoforms, CARMEN-201, to be crucial for SMC commitment. CARMEN-201 activity is encoded within an alternatively-spliced exon containing a MIRc short interspersed nuclear element. This element binds the transcriptional repressor REST (RE1 Silencing Transcription Factor), targets it to cardiogenic loci, including ISL1, IRX1, IRX5, and SFRP1, and thereby blocks the CM gene program. In turn, genes regulating SMC differentiation are induced. These data show how a critical physiological switch is wired by alternative splicing and functional transposable elements in a long noncoding RNA.

Analysis of Transcriptional Profiling of Chamber-Specific Human Cardiac Myocytes Derived from Pluripotent Stem Cells.

Differentiation protocols to direct cell fate decision from pluripotent stem cells to cardiac myocytes normally achieve high purity and quality of cells. Nonetheless, the highly specialized anatomy of the heart enables the possibility that acquisition of terminal somatic differentiation from pluripotency might imply heterogeneity of non-desire cell lineages. Directed cardiac differentiation empowers differentiation of pool of cells commonly reported to contain different proportions of ventricular, atrial, and nodal-like cells. RNA sequencing (RNA-Seq) allows a precise transcriptional profiling, ensuring a quality checking of the cell identity our protocol has derived as a main outcome. Here we describe a workflow methodology on how to adapt RNA sequencing analysis for integration into the R analysis pipeline in order to characterize chamber-specific gene signatures of the major cardiac lineages of myocytes in the heart.

AMPKβ1 and AMPKβ2 define an isoform-specific gene signature in human pluripotent stem cells, differentially mediating cardiac lineage specification

AMP-activated protein kinase (AMPK) is a key regulator of energy metabolism that phosphorylates a wide range of proteins in order to maintain cellular homeostasis. AMPK consists of three subunits: α, β and γ. AMPKα and β are encoded by two genes, the γ subunit by three genes, all of which being expressed in a tissue-specific manner. If individual isoforms have different functions is not understood so far. Using RNA-Seq technology, we provide evidence that the loss of AMPKβ1 and AMPKβ2 lead to different gene expression profiles in human induced pluripotent stem cells (hiPSCs), indicating isoform-specific function. The knockout of AMPKβ2 was associated with a higher number of differentially regulated genes than the deletion of AMPKβ1, suggesting that AMPKβ2 has a more comprehensive impact on the transcriptome. Bioinformatics analysis identified cell differentiation as one biological function being specifically associated with AMPKβ2. Correspondingly, the two isoforms differentially affected lineage decision towards a cardiac cell fate. While the lack of PRKAB1 impacted differentiation into cardiomyocytes only at late stages of cardiac maturation, the availability of PRKAB2 was indispensable for mesoderm specification as shown by gene expression analysis and histochemical staining for cardiac lineage markers such as cTnT, GATA4, and NKX2.5. Ultimately, the lack of AMPKβ1 impairs, while deficiency of AMPKβ2 abrogates differentiation into cardiomyocytes. Finally, we demonstrate that AMPK affects cellular physiology by engaging in the regulation of hiPSC transcription in an isoform-specific manner, providing the basis for further investigations elucidating the role of dedicated AMPK subunits in the modulation of gene expression.

Abstract 17503: ETV2 Functions as a Pioneer Factor and Regulates the Endothelial Lineage

Background: Genetic mutations perturb the multipotent progenitors, which results in congenital cardiovascular disease. Therefore, it is essential to decipher the pioneer factors and the regulatory pathways that govern the specification and differentiation of mesodermal progenitors and use this information to develop targeted therapies to promote cardiovascular regeneration. Etv2 as an essential transcription factor for the development of cardiac, endothelial and hematopoietic lineages. In the present study, we used ES/EB differentiation and MEF reprogramming systems, to define Etv2 as a novel pioneer factor that relaxes the closed chromatin and drives endothelial development. Results: Using the iHA-Etv2 ES cell line, we engineered a mouse that inducibly overexpresses ETV2. The bulk RNA-seq, single cell RNA-seq data and ATAC-seq experiments showed that inducing Etv2 in MEFs and ES/EBs activated the downstream endothelial marker genes and promoted the development of endothelial lineages, supporting the notion that Etv2 functioned as a master regulator to drive the endothelial lineage development in different cellular contexts. We found that similar to other known pioneer factors, Etv2 was intrinsically able to target and bind the nucleosomes, and this capability appeared to be independent of the cellular context. To further define the mechanism, we performed Etv2, Brg1 and H3K27ac ChIP-seq analyses during MEF reprogramming and ES/EB differentiation. We found that Brg1 maintains and stabilizes the binding of Etv2 on the nucleosome, and Etv2 requires Brg1 to activate downstream genes during reprograming. Conclusion: In these studies, we defined Etv2 as a novel pioneer factor that relaxed the closed chromatin and promoted the endothelial lineage in both ES/EB differentiation and MEF reprogramming. The definition of these mechanisms will enhance our understanding of cardiovascular development and regeneration and serve as a platform for therapeutic applications for patients with congenital or aging related cardiovascular diseases.

Abstract 13545: Atlas of Exosomal Microrna Secreted From Human Ipsc-derived Cardiac Cell Types

Exosomes are small particles secreted from cells through an endosomal sorting complexes required for transport (ESCRT)-mediated pathway that releases vesicles into the extracellular matrix following fusion of the endosomal multivesicular body to the plasma membrane. Initially thought to be a waste disposal system for the cell, exosomes are now recognized as important mediators in cellular communication, transporting nucleic acid, protein and lipid. We hypothesize that microRNA (miRNA) transcriptomic profiles isolated from exosomes are distinct for each cardiovascular lineage in healthy control patients. Using induced pluripotent stem cells (iPSC), established differentiation protocols and a high-yield mechanical-sorting device (ExoTIC), we characterized the transcriptomic profiles of exosomal secretions derived from cardiomyocytes (CM), cardiac fibroblasts (CF) and endothelial cells (EC). We identified 120 miRNAs from 94 miRNA genes to be secreted at appreciable levels. We discovered 1) let-7 miRNA precursors are depleted in iPSC secretomes; 2) only a subset of total cellular miRNAs are present; 3) a common core of miRNAs are secreted by all three cardiac cell types; and 4) distinct enrichment or depletion of different mature miRNAs in each exosome type. The exosomal transcriptomic profiles of each lineage was compared to a published atlas of miRNA from seventeen tissue types from the human body. miR-302c known to be specific to pluripotent cells and is enriched in iPSC exosomes and found in bone, reflecting hematopoietic stem cells. miR-1, critical for cardiac development and pathology, is secreted by iPSC-CM and enriched in striated muscle and myocardium. miR-151 is secreted by iPSC-CF and is expressed ubiquitously. The relative enrichment of miRNAs in different cardiac tissues could be used to quantifying the cellular content of heterogenous mixtures of iPSC-derived cardiac lineages. We developed a ratiometric cell purity assay that can sensitively detect iPSC contamination in iPSC-CM differentiations.

Lysophosphatidic Acid Receptor 4 Is Transiently Expressed during Cardiac Differentiation and Critical for Repair of the Damaged Heart

Efficient differentiation of pluripotent stem cells (PSCs) into cardiac cells is essential for the development of new therapeutic modalities to repair damaged heart tissue. We identified a novel cell surface marker, the G protein-coupled receptor lysophosphatidic acid receptor 4 (LPAR4), specific to cardiac progenitor cells (CPCs) and determined its functional significance and therapeutic potential. During in vitro differentiation of mouse and human PSCs toward cardiac lineage, LPAR4 expression peaked after 3−7 days of differentiation in cardiac progenitors and then declined. In vivo, LPAR4 was specifically expressed in the early stage of embryonal heart development, and as development progressed, LPAR4 expression decreased and was non-specifically distributed. We identified the effective agonist octadecenyl phosphate and a p38 MAPK blocker as the downstream signal blocker. Sequential stimulation and inhibition of LPAR4 using these agents enhanced the in vitro efficiency of cardiac differentiation from mouse and human PSCs. Importantly, in vivo, this sequential stimulation and inhibition of LPAR4 reduced the infarct size and rescued heart dysfunction in mice. In conclusion, LPAR4 is a novel CPC marker transiently expressed only in heart during embryo development. Modulation of LPAR4-positive cells may be a promising strategy for repairing myocardium after myocardial infarction.

Elimination of Mutant mtDNA by an Optimized mpTALEN Restores Differentiation Capacities of Heteroplasmic MELAS-iPSCs

Various mitochondrial diseases, including mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), are associated with heteroplasmic mutations in mitochondrial DNA (mtDNA). Herein, we refined a previously generated G13513A mtDNA-targeted platinum transcription activator-like effector nuclease (G13513A-mpTALEN) to more efficiently manipulate mtDNA heteroplasmy in MELAS-induced pluripotent stem cells (iPSCs). Introduction of a nonconventional TALE array at position 6 in the mpTALEN monomer, which recognizes the sequence around the m.13513G>A position, improved the mpTALEN effect on the heteroplasmic shift. Furthermore, the reduced expression of the new Lv-mpTALEN(PKLB)/R-mpTALEN(PKR6C) pair by modifying codons in their expression vectors could suppress the reduction in the mtDNA copy number, which contributed to the rapid recovery of mtDNA in mpTALEN-applied iPSCs during subsequent culturing. Moreover, MELAS-iPSCs with a high proportion of G13513A mutant mtDNA showed unusual properties of spontaneous, embryoid body-mediated differentiation in vitro, which was relieved by decreasing the heteroplasmy level with G13513A-mpTALEN. Additionally, drug-inducible, myogenic differentiation 1 (MYOD)-transfected MELAS-iPSCs (MyoD-iPSCs) efficiently differentiated into myosin heavy chain-positive myocytes, with or without mutant mtDNA. Hence, heteroplasmic MyoD-iPSCs controlled by fine-tuned mpTALENs may contribute to a detailed analysis of the relationship between mutation load and cellular phenotypes in disease modeling.

Minimal contribution of IP3R2 in cardiac differentiation and derived ventricular-like myocytes from human embryonic stem cells.

Type 2 inositol 1,4,5-trisphosphate receptor (IP3R2) regulates the intracellular Ca2+ release from endoplasmic reticulum in human embryonic stem cells (hESCs), cardiovascular progenitor cells (CVPCs), and mammalian cardiomyocytes. However, the role of IP3R2 in human cardiac development is unknown and its function in mammalian cardiomyocytes is controversial. hESC-derived cardiomyocytes have unique merits in disease modeling, cell therapy, and drug screening. Therefore, understanding the role of IP3R2 in the generation and function of human cardiomyocytes would be valuable for the application of hESC-derived cardiomyocytes. In the current study, we investigated the role of IP3R2 in the differentiation of hESCs to cardiomyocytes and in the hESC-derived cardiomyocytes. By using IP3R2 knockout (IP3R2KO) hESCs, we showed that IP3R2KO did not affect the self-renewal of hESCs as well as the differentiation ability of hESCs into CVPCs and cardiomyocytes. Furthermore, we demonstrated the ventricular-like myocyte characteristics of hESC-derived cardiomyocytes. Under the α1-adrenergic stimulation by phenylephrine (10 μmol/L), the amplitude and maximum rate of depolarization of action potential (AP) were slightly affected in the IP3R2KO hESC-derived cardiomyocytes at differentiation day 90, whereas the other parameters of APs and the Ca2+ transients did not show significant changes compared with these in the wide-type ones. These results demonstrate that IP3R2 has minimal contribution to the differentiation and function of human cardiomyocytes derived from hESCs, thus provide the new knowledge to the function of IP3R2 in the generation of human cardiac lineage cells and in the early cardiomyocytes.

Maintenance and differentiation of human ES cells on polyvinylidene fluoride scaffolds immobilized with a vitronectin-derived peptide.

Polyvinylidene fluoride (PVDF) is biocompatible, easy to fabricate, and has piezoelectric properties; it has been used for many biomedical applications including stem cell engineering. However, long-term cultivation of human embryonic stem cells (hESCs) and their differentiation toward cardiac lineages on PVDF have not been investigated. Herein, PVDF nanoscaled membrane scaffolds were fabricated by electrospinning; a vitronectin-derived peptide-mussel adhesive protein fusion (VNm) was immobilized on the scaffolds. hESCs cultured on the VNm-coated PVDF scaffold (VNm-PVDF scaffold) were stably expanded for more than 10 passages while maintaining the expression of pluripotency markers and genomic integrity. Under cardiac differentiation conditions, hESCs on the VNm-PVDF scaffold generated more spontaneously beating colonies and showed the upregulation of cardiac-related genes, compared with those cultured on Matrigel and VNm alone. Thus, VNm-PVDF scaffolds may be suitable for the long-term culture of hESCs and their differentiation into cardiac cells, thus expanding their application in regenerative medicine.

Metabolic Maturation of Differentiating Cardiosphere-Derived Cells

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

Biphasic Electrical Pulse by a Micropillar Electrode Array Enhances Maturation and Drug Response of Reprogrammed Cardiac Spheroids.

Direct reprogramming is an efficient strategy to produce cardiac lineage cells necessary for cardiac tissue engineering and drug testing for cardiac toxicity. However, functional maturation of reprogrammed cardiomyocytes, which is of great importance for their regenerative potential and drug response, still remains challenging. In this study, we propose a novel electrode platform to promote direct cardiac reprogramming and improve the functionality of reprogrammed cardiac cells. Nonviral cardiac reprogramming was improved via a three-dimensional spheroid culture of chemically induced cardiomyocytes exposed to a small-molecule cocktail. A micropillar electrode array providing biphasic electrical pulses mimicking the heartbeat further enhanced maturation and electrophysiological properties of reprogrammed cardiac spheroids, leading to proper responses and increased sensitivity to drugs. On the basis of our results, we conclude that our device may have a wider application in the generation of functional cardiac cells for regenerative medicine and screening of novel drugs.