Decellularized Extracellular Matrix Powder Accelerates Metabolic Maturation at Early Stages of Cardiac Differentiation in Human Induced Pluripotent Stem Cell-derived Cardiomyocytes

During fetal development, cardiomyocytes switch from glycolysis to oxidative metabolism to sustain the energy requirements of functional cells. State-of-the-art cardiac differentiation protocols yield phenotypically immature cardiomyocytes, and common methods to improve metabolic maturation require multistep protocols to induce maturation only after cardiac specification is completed. Here, we describe a maturation method using ventricle-derived decellularized extracellular matrix (dECM) that promoted early-stage metabolic maturation of cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs). Chemically and architecturally preserved particles (45-500 µm) of pig ventricular dECM were added to hiPSCs at the start of differentiation. At the end of our maturation protocol (day 15 of cardiac differentiation), we observed an intimate interaction between cardiomyocytes and dECM particles without impairment of cardiac differentiation efficiency (~70% of cTNT+). Compared with control cells (those cultured without pig dECM), 15-day-old dECM-treated cardiomyocytes demonstrated increased expression of markers related to cardiac metabolic maturation, MAPK1, FOXO1, and FOXO3, and a switch from ITGA6 (the immature integrin isoform) to ITGA3 and ITGA7 (those present in adult cardiomyocytes). Electrical parameters and responsiveness to dobutamine also improved in pig ventricular dECM-treated cells. Extending the culture time to 30 days, we observed a switch from glucose to fatty acid metabolism, indicated by decreased glucose uptake and increased fatty acid consumption in cells cultured with dECM. Together, these data suggest that dECM contains endogenous cues that enable metabolic maturation of hiPSC-CMs at early stages of cardiac differentiation.

Identification and characterization of hPSC-derived FOXA2+ progenitor cells with ventricular cardiac differentiation potential

While much progress has been made in understanding early cardiac development, the precise mechanisms that specify the different cardiomyocyte subtypes remain poorly understood. Recent data from our lab have shown that transient Foxa2 expression identifies a progenitor population with exclusive ventricular differentiation potential in the mouse heart. Here we have translated this concept to the human pluripotent stem cell (hPSC) system. Using a FOXA2-GFP reporter cell line we characterized expression of FOXA2 during hPSC cardiac differentiation and found that a subset of cardiac mesoderm precursors transiently expresses FOXA2. Gene expression analysis of FOXA2+ and FOXA2- cardiac mesoderm revealed that both populations similarly express early cardiac specification markers such as PDGFRA, TBX5, and ISL1, while other key candidates including TBX20 and GATA4 are significantly upregulated in the FOXA2+ population. Isolation and subsequent differentiation of FOXA2+ and FOXA2- populations demonstrates their comparable differentiation potential to both cardiomyocytes and epicardial cells. However, cardiomyocytes derived from FOXA2+ precursors showed enhanced differentiation efficiency toward ventricular cardiomyocytes compared to cardiomyocytes derived from FOXA2- precursors. To identify new mechanisms that regulate ventricular specification, we performed small molecule screening and found that inhibition of the EGFR pathway strongly increased the cardiac mesoderm population in general, and the FOXA2+ precursors in particular. Finally, we have identified a combination of cell surface markers to specifically isolate FOXA2+ cardiac precursors. In summary, our results suggest that FOXA2+ cardiac mesoderm harbors ventricular-specific differentiation potential and isolation of these cells permits the generation of cultures enriched for ventricular cardiomyocytes. Generating such enriched cardiac populations will be relevant for regenerative medicine approaches, as well as for disease modeling from induced pluripotent stem cells.

Single-cell transcriptomics defines heterogeneity of epicardial cells and fibroblasts within the infarcted murine heart

In the adult heart, the epicardium becomes activated after injury, contributing to cardiac healing by secretion of paracrine factors. Here, we analyzed by single-cell RNA sequencing combined with RNA in situ hybridization and lineage tracing of Wilms tumor protein 1-positive (WT1+) cells, the cellular composition, location, and hierarchy of epicardial stromal cells (EpiSC) in comparison to activated myocardial fibroblasts/stromal cells in infarcted mouse hearts. We identified 11 transcriptionally distinct EpiSC populations, which can be classified into three groups, each containing a cluster of proliferating cells. Two groups expressed cardiac specification markers and sarcomeric proteins suggestive of cardiomyogenic potential. Transcripts of hypoxia-inducible factor (HIF)-1α and HIF-responsive genes were enriched in EpiSC consistent with an epicardial hypoxic niche. Expression of paracrine factors was not limited to WT1+ cells but was a general feature of activated cardiac stromal cells. Our findings provide the cellular framework by which myocardial ischemia may trigger in EpiSC the formation of cardioprotective/regenerative responses.

Single-cell transcriptomics defines heterogeneity of epicardial cells and fibroblasts within the infarcted heart

In the adult heart, the epicardium becomes activated after injury, contributing to cardiac healing by secretion of paracrine factors. Here we analyzed by single-cell RNA sequencing combined with RNA in situ hybridization and lineage tracing of WT1+ cells the cellular composition, location, and hierarchy of epicardial stromal cells (EpiSC) in comparison to activated myocardial fibroblasts/stromal cells in infarcted mouse hearts. We identified 11 transcriptionally distinct EpiSC populations, that can be classified in three groups each containing a cluster of proliferating cells. Two groups expressed cardiac specification makers and sarcomeric proteins suggestive of cardiomyogenic potential. Transcripts of HIF-1α and HIF-responsive genes were enriched in EpiSC consistent with an epicardial hypoxic niche. Expression of paracrine factors was not limited to WT1+ cells but was a general feature of activated cardiac stromal cells. Our findings provide the cellular framework by which myocardial ischemia may trigger in EpiSC the formation of cardioprotective/regenerative responses.

Specificity, redundancy and dosage thresholds among gata4/5/6 genes during zebrafish cardiogenesis

ABSTRACTThe Gata4/5/6 sub-family of zinc finger transcription factors regulate many aspects of cardiogenesis. However, critical roles in extra-embryonic endoderm also challenge comprehensive analysis during early mouse cardiogenesis, while zebrafish models have previously relied on knockdown assays. We generated targeted deletions to disrupt each gata4/5/6 gene in zebrafish and analyzed cardiac phenotypes in single, double and triple mutants. The analysis confirmed that loss of gata5 causes cardia bifida and validated functional redundancies for gata5/6 in cardiac precursor specification. Surprisingly, we discovered that gata4 is dispensable for early zebrafish development, while loss of one gata4 allele can suppress the bifid phenotype of the gata5 mutant. The gata4 mutants eventually develop an age-dependent cardiomyopathy. By combining combinations of mutant alleles, we show that cardiac specification depends primarily on an overall dosage of gata4/5/6 alleles rather than a specific gene. We also identify a specific role for gata6 in controlling ventricle morphogenesis through regulation of both the first and second heart field, while loss of both gata4/6 eliminates the ventricle. Thus, different developmental programs are dependent on total dosage, certain pairs, or specific gata4/5/6 genes during embryonic cardiogenesis.This article has an associated First Person interview with the first author of the paper.