Analysis of cardiac differentiation at single cell resolution reveals a requirement of hypertrophic signaling for HOPX transcription

Abstract

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...

Trajectory reconstruction identifies dysregulation of perinatal maturation programs in pluripotent stem cell-derived cardiomyocytes

“…In our previous study, we found that, similar to mouse PSC-CMs, human PSC-CMs appear to have a maturation state similar to fetal CMs and appear arrested at the onset of perinatal maturation (5). Here, we focused on four published datasets of CMs generated from induced PSCs - Friedman, Nguyen, and Lukowski et al (38) (FNL); Churko et al (39) (C); Gerbin, Grancharova, Donovan-Maye, and Hendershott et al (40) (GGDH); and Ruan and Liao et al (41) (RL). We initially aimed to perform trajectory reconstruction with Monocle 3 as done with the mouse PSC-CMs.…”

“…(38) (FNL); Churko et al (39) (C); Gerbin, Grancharova, Donovan-Maye, and Hendershott et al (40) (GGDH); and Ruan and Liao et al (41) (RL).…”

Human Induced Pluripotent Stem Cell-Derived Non-Cardiomyocytes Modulate Cardiac Electrophysiological Maturation Through Connexin 43-Mediated Cell-Cell Interactions

“…Interestingly, those cells were also found to have enriched expression of genes associated with outflow tract development, suggesting that the non-CMs may be cardiac outflow tract-like cells. However, the authors posited that further studies are still needed to fully elucidate the identity of these noncontractile cell types [22].…”

Coculture of Endothelial Cells with Human Pluripotent Stem Cell‐Derived Cardiac Progenitors Reveals a Differentiation Stage‐Specific Enhancement of Cardiomyocyte Maturation

“…This may be attributed to the fact that ECs and CPCs arise from the same mesodermal population as CPCs, or the differentiation of ISL1 + CPCs to ECs . Indeed, ECs have been found as a byproduct of small molecule‐induced CM differentiation . It is possible that our use of EGM‐2, an EC growth medium, for our cocultures selectively expanded the EC population that arose during CM differentiation.…”