Cardiac Directed Differentiation Using Small Molecule WNT Modulation at Single-Cell Resolution

Abstract

Differentiation 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 cardiacdirected 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 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. Our findings further implicate dysregulation of HOPX during in vitro cardiac-directed differentiation underlying the molecular and physiological immaturity of stem cell-derived cardiomyocytes.