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

Friedman C1, Nguyen Q1, Lukowski S1, Chiu H1, Bar-Joseph Z5, Tam P4, Murry CE3, Ruohola Baker H3, Powell J2 and Palpant NJ1

  1. University of Queensland.
  2. Garvan Institute.
  3. University of Washington.
  4. University of Sydney.
  5. Carnegie Mellon University.

Differentiation into diverse cell lineages requires the orchestration of gene regulatory networks guiding diverse cell fate choices. Utilizing human pluripotent stem cells, we measured expression dynamics of 17,718 genes from 43,168 cells across five time points over a thirty day time-course of in vitro cardiac-directed differentiation. We used unsupervised clustering to identify transcriptional networks underlying lineage derivation of 15 subpopulations including mesoderm, definitive endoderm, vascular endothelium, cardiac precursors, and definitive cardiac fates including contractile cardiomyocytes and non-contractile derivatives. Utilizing customized machine learning algorithms, we analyzed scRNA-seq data to identify transcription factor regulatory networks linking the trajectory of subpopulations in vitro with cell types derived during cardiac development in vivo. We leveraged this data to study gene networks governing cardiomyocyte differentiation in vivo to advance translational applications of stem cells in disease modelling and therapies. Among a network of known genetic drivers of differentiation, we identified dysregulation of the non-DNA binding homeodomain protein, HOPX as a candidate cause for the immature state of in vitro derived cardiomyocytes. While HOPX is expressed in cardiac progenitor cells (CPC) in vivo, we show during in vitro differentiation that HOPX is expressed in only 16% of hPSC-derived cardiomyocytes. Using genetic models we determined the mechanisms underlying transcriptional regulation of HOPX. We show that HOPX is situated downstream from hypertrophic signaling, HOPX directly drives hypertrophic growth, and is required for expression of myofibrillar genes involved in cardiomyocyte maturation. Through genetic dissection underlying the HOPX transcriptional landscape, we show that the distal HOPX transcriptional start site is the primary regulatory driver of HOPX expression underlying hypertrophic stimulation. Taken together, we utilized single cell analysis of cardiac in vitro differentiation to identify mechanisms for activating gene networks in cardiac differentiation as they occur during in vivo heart development that enhance the utility of hPSCs for cardiac translational applications.