Transcriptome-Guided Functional Analyses Reveal Novel Biological Properties and Regulatory Hierarchy of Human Embryonic Stem Cell-Derived Ventricular Cardiomyocytes Crucial for Maturation

Abstract

Human (h) embryonic stem cells (ESC) represent an unlimited source of cardiomyocytes (CMs); however, these differentiated cells are immature. Thus far, gene profiling studies have been performed with non-purified or non-chamber specific CMs. Here we took a combinatorial approach of using systems biology to guide functional discoveries of novel biological properties of purified hESC-derived ventricular (V) CMs. We profiled the transcriptomes of hESCs, hESC-, fetal (hF) and adult (hA) VCMs, and showed that hESC-VCMs displayed a unique transcriptomic signature. Not only did a detailed comparison between hESC-VCMs and hF-VCMs confirm known expression changes in metabolic and contractile genes, it further revealed novel differences in genes associated with reactive oxygen species (ROS) metabolism, migration and cell cycle, as well as potassium and calcium ion transport. Following these guides, we functionally confirmed that hESC-VCMs expressed IKATP with immature properties, and were accordingly vulnerable to hypoxia/reoxygenation-induced apoptosis. For mechanistic insights, our coexpression and promoter analyses uncovered a novel transcriptional hierarchy involving select transcription factors (GATA4, HAND1, NKX2.5, PPARGC1A and TCF8), and genes involved in contraction, calcium homeostasis and metabolism. These data highlight novel expression and functional differences between hESC-VCMs and their fetal counterparts, and offer insights into the underlying cell developmental state. These findings may lead to mechanism-based methods for in vitro driven maturation.

Proteomic Analysis of Human Pluripotent Stem Cell–Derived, Fetal, and Adult Ventricular Cardiomyocytes Reveals Pathways Crucial for Cardiac Metabolism and Maturation

“…These results in human cells are reminiscent of those that take place during mouse embryonic development, 30 and are also consistent with those of Birket et al 42 who showed that repression of PPARGC1A decreased mitochondrial content, and thereby compromised the capacity for coping with energetic stress. Indeed, our recent transcriptomic profiling experiment also identifies PPARGC1A as a member of a cardiac transcription factor network, which regulates genes important for heart development and function 26 ; similarly, Xu et al 14 report that PPARGC1A/PPARA signaling regulate genes enriched in CMs relative to undifferentiated hESCs.…”

“…Of the 121 proteins detected here, transcripts for 116 could be detected by microarray. 26 Among proteins that showed differential abundance between hESC-VCMs and hF-VCMs, 37% had significant transcriptomic changes in the same direction. Only 19% of proteins differentially expressed between hF-VCMs and hA-VCMs showed the same trend.…”

Phospholamban as a Crucial Determinant of the Inotropic Response of Human Pluripotent Stem Cell–Derived Ventricular Cardiomyocytes and Engineered 3-Dimensional Tissue Constructs

“…Hearts from healthy adults of 53 to 70 years of age were digested using the Langendorff system at 37°C as we previously reported. 13 …”

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

“…Therefore, interpretation of data regarding cardiomyogenesis efficiency and subtype identity must not only consider the presence and quantity of reference marker levels, but must consider the developmental stage(s) to which the timepoints of differentiation that are analyzed correspond. This is especially important considering that the maturation stage of cardiomyogenic cells generated by in vitro differentiation of hPSCs resembles most closely those of embryonic/fetal development [21][22][23][24][25] . Thus, relying on a marker's spatial expression in the postnatal heart may not be appropriate for the assessment of hPSCderived cells, at least in some cases.…”