Establishment of an in vitro second heart field platform with human induced pluripotent stem cells

Abstract

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can be readily generated from human induced pluripotent stem cells via temporal modulation of canonical Wnt pathway with chemical means. Resulting hiPSC-CMs harbour structural, functional and electrophysiological properties of native human embryonic ventricular cardiomyocytes. Attempts to advance the hiPSC-CM phenotype beyond the embryonic stage and as a biological model for the native human adult ventricle has only been met with limited success due to the extensive structural development and remodelling of cardiomyocytes during gestation, in which we have a rudimentary understanding of the molecular processes underlying such cellular changes. Furthermore, it is still unknown which anatomical or temporal stage of the in vivo human heart hiPSC-CMs correspond to in its current stage. The in vivo heart is derived from two distinct cell populations derived from the lateral plate mesoderm (LPM), which are the first and second heart field (SHF). Transient expression of markers indicative of both progenitor populations has been identified in in vitro cardiac differentiation, but a distinct cardiac progenitor nor its specification trajectory has yet to be identified.

In this study, I utilized single-cell RNA sequencing (scRNA-seq) on different stages of in vitro cardiac specification and identified a multipotent SHF progenitor with the propensity to specify into cardiomyocyte, cardiac mesenchymal and endothelial lineage. Apelin receptor (APLNR) was identified as a surface marker for multipotent SHF progenitors and isolated APLNR+ cells were enriched for SHF markers and culturing of progenitors results in autonomous specification into predominantly cardiomyocyte with cardiac mesenchyme lineages. Consecutive RNA sequencing of APLNR+ SHF progenitors at 0, 24, 48 and 72 hours post-isolation have revealed a differentiation trajectory analogous with in vivo SHF sub-specification to the anterior heart field and further specification into right ventricular lineage cardiomyocytes. Furthermore, an isolated culture of APLNR+ in vitro SHF progenitors has led to the expression of sarcomeric isoforms and cardiac transcription factors cardiomyocytes crucial for ventricular morphogenesis but absent from conventional in vitro cardiac differentiation. Combined analysis of scRNA-seq and RNA sequencing results has suggested and demonstrated the paracrine effect of canonical notch pathway mediated by JAG1-NOTCH2 interaction in promoting the proliferation of the cardiomyocyte lineage APLNR+ in vitro SHF progenitors. Inhibition of canonical notch pathway enhances cardiac specification of APLNR+ in vitro SHF progenitors via preferential MAML1 interaction with MEF2C to enhance its transcriptional activity.

In summary, this study has identified in vitro cardiac differentiation is analogous with in vivo SHF cell specification and APLNR as a surface marker for genetic label-free isolation of in vitro multipotent SHF progenitors. This platform allows for a better understanding of SHF development and a consistent source for the generation of cardiac lineages with applications in bioengineering, drug screening and cardiac regeneration.