Activin-A and Bmp4 Levels Modulate Cell Type Specification during CHIR-Induced Cardiomyogenesis

The use of human pluripotent cell progeny for cardiac disease modeling, drug testing and therapeutics requires the ability to efficiently induce pluripotent cells into the cardiomyo-genic lineage. Although direct activation of the Activin-A and/or Bmp pathways with growth factors yields context-dependent success, recent studies have shown that induction of Wnt signaling using low molecular weight molecules such as CHIR, which in turn induces the Activin-A and Bmp pathways, is widely effective. To further enhance the reproducibility of CHIR-induced cardiomyogenesis, and to ultimately promote myocyte maturation, we are using exogenous growth factors to optimize cardiomyogenic signaling downstream of CHIR induction. As indicated by RNA-seq, induction with CHIR during Day 1 (Days 0-1) was followed by immediate expression of Nodal ligands and receptors, followed later by Bmp ligands and receptors. Co-induction with CHIR and high levels of the Nodal mimetic ActivinA (50-100 ng/ml) during Day 0-1 efficiently induced definitive endoderm, whereas CHIR supplemented with Activin-A at low levels (10 ng/ml) consistently improved cardiomyogenic efficiency, even when CHIR alone was ineffective. Moreover, co-induction using CHIR and low levels of Activin-A apparently increased the rate of cardiomyogenesis, as indicated by the initial appearance of rhythmically beating cells by Day 6 instead of Day 8. By contrast, co-induction with CHIR plus low levels (3-10 ng/ml) of Bmp4 during Day 0-1 consistently and strongly inhibited cardiomyogenesis. These findings, which demonstrate that cardiomyogenic efficacy is improved by optimizing levels of CHIR-induced growth factors when applied in accord with their sequence of endogenous expression, are consistent with the idea that Nodal (Activin-A) levels toggle the entry of cells into the endodermal or mesodermal lineages, while Bmp levels regulate subsequent allocation into mesodermal cell types.