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

Abstract

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 cardiomyogenic 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 Activin-A (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.

Impact of MYH6 variants in hypoplastic left heart syndrome

“…5 D . As anticipated (21), MYH7 transcription was first detected on differentiation day 5 , at which stage similar levels of MYH7 transcripts were seen in cardiomyocytes representing the proband and its unaffected parent. On day 8 , although substantial increases in MYH7 expression were seen in cardiomyocytes from both individuals, the level of expression in proband cardiomyocytes was ∼2.5-fold greater than that observed in myocytes from the unaffected parent.…”

“…Following cellular expansion and verification of pluripotency, iPSCs representing the proband and an unaffected parent were induced to undergo cardiomyogenic differentiation as recently described (21) (depicted in Fig. 5 A ), during which cultures of cardiomyogenic cells were removed for RNA-Seq determinations on the differentiation days shown in Fig.…”

“…S3), percentages of cells exhibiting positive Oct4 immunostaining (99–100%), being karyotypically normal (Supplemental Fig. S3), and the ability to differentiate into multiple lineages (definitive endoderm and cardiomyogenic mesoderm) (21). Experimenters were blinded as to the identity of family members from whom each line was generated.…”

Activin A Modulates CRIPTO-1/HNF4α+Cells to Guide Cardiac Differentiation from Human Embryonic Stem Cells

“…Consistent with these studies, we demonstrated that the addition of ActA in combination with BMP4 and bFGF increased the expression of the early endodermal marker SOX17 and the definitive endodermal marker HNF4α in human ESCs. More recently, a monolayer-based cardiac differentiation protocol has been published, applying the Wnt signaling agonist CHIR in combination with high or low levels of ActA [48]. This study is not in contrast with our data.…”

Small molecules and human cardiomyogenesis: Is there a bottleneck in current research?

“…Additionally, the regulation of cardiac differentiation in stem cell via metabolic oxidation is also of key research interest [38,39] as well as the role of mitochondria [40]. Furthermore, the interplay between small-molecule compounds and signaling molecules has been found to have a profound effect on cell type specification [6,41]. Therefore the structure-activity relationship (SAR) and pharmacological properties (e.g.…”