SUMMARY
Cardiac differentiation of human pluripotent stem cells (hPSCs) requires orchestration of dynamic gene regulatory networks during stepwise fate transitions, but often generates immature cell types that do not fully recapitulate properties of their adult counterparts, suggesting incomplete activation of key transcriptional networks. We performed extensive single-cell transcriptomic analyses to map fate choices and gene expression programs during cardiac differentiation of hPSCs, and identified strategies to improve in vitro cardiomyocyte differentiation. Utilizing genetic gain- and loss-of-function approaches, we found that hypertrophic signaling is not effectively activated during monolayer-based cardiac differentiation, thereby preventing expression of HOPX and its activation of downstream genes that govern late stages of cardiomyocyte maturation. This study therefore provides a key transcriptional roadmap of in vitro cardiac differentiation at single-cell resolution, revealing fundamental mechanisms underlying heart development and differentiation of hPSC-derived cardiomyocytes.
45Differentiation into diverse cell lineages requires orchestration of gene regulatory networks guiding cell fate choices. Here, we present the dissection of cellular composition and gene networks from transcriptomic data of 43,168 cells across five discrete time points during cardiac-directed differentiation. We utilize unsupervised clustering and implement a lineage trajectory prediction algorithm that integrates transcription factor networks to predict cell fate progression of 15 subpopulations that correlate with germ 50 layer and cardiovascular differentiation in vivo. These data reveal transcriptional networks underlying lineage derivation of mesoderm, definitive endoderm, vascular endothelium, cardiac precursors, and definitive cell types that comprise cardiomyocytes and a previously unrecognized cardiac outflow tract population. Single cell analysis of genetic regulators governing cardiac fate diversification identified the non-DNA binding homeodomain protein, HOPX, as functionally necessary for endothelial specification.
55Our findings further implicate dysregulation of HOPX during in vitro cardiac-directed differentiation underlying the molecular and physiological immaturity of stem cell-derived cardiomyocytes.
65Key words: human pluripotent stem cells, cardiomyocytes, transcriptomic profiling, outflow tract, heart, development, CRISPR, engineered heart tissue, RNA-sequencing, HOPX, scdiff 70
75. CC-BY 4.0 International license peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/229294 doi: bioRxiv preprint first posted online Dec. 7, 2017;
Introduction
80Studies of cardiac development at single-cell resolution have provided valuable new insights into cell diversity and genetic regulation of cell types revealing mechanisms underlying cardiovascular differentiation and morphogenesis. Single-cell analysis of in vivo mouse heart development have revealed chamber-specific and temporal changes in gene expression underlying embryonic heart development from E9.5 to postnatal day 21 establishing anatomical patterns of gene expression in the heart (Li et al., 2016) 85and new insights into transcriptional programs underlying cardiac maturation (DeLaughter et al., 2016). These studies provide a valuable resource by which to understand transcriptional mechanisms underlying diverse fate choices involved in cardiac development and morphogenesis in vivo. Like many single-cell transcriptomic studies, they further highlight the importance of dissecting cell heterogeneity to understand mechanisms underlying the identity and fate of cells in health and disease. 90Human pluripotent stem cells are a key model system to study human cardiovascular developmental biology (Murry and Keller, 2008). However, the fidelity by which cardiac directed differentiation in vitro recapitulates the transcriptional programs governing the diversity of cell fates generated in vivo is not well understood. Analyzing differentiation efficiency...
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