Activin-A and Bmp4 levels modulate cell type specification during CHIR-induced cardiomyogenesis

Area of Science:

• Cardiovascular Biology
• Stem Cell Biology
• Developmental Biology

Background:

• Efficient cardiomyogenesis from human pluripotent stem cells is crucial for cardiac disease modeling, drug testing, and regenerative therapies.
• While Wnt signaling (e.g., CHIR) is effective for inducing cardiomyogenesis, optimizing downstream signaling pathways is key for reproducibility and maturation.
• Endogenous Nodal and Bmp pathways are sequentially activated following CHIR induction, suggesting a temporal requirement for specific growth factors.

Purpose of the Study:

• To enhance the efficiency and reproducibility of CHIR-induced cardiomyogenesis by optimizing exogenous growth factor supplementation.
• To investigate the role of Activin-A and Bmp4 in modulating CHIR-driven cardiac cell differentiation.
• To promote myocyte maturation by fine-tuning cardiomyogenic signaling downstream of Wnt activation.

Main Methods:

• Human pluripotent cells were treated with CHIR (CHIR99021) alone or in combination with varying concentrations of Activin-A or Bmp4 during specific differentiation windows (Day 0-1).
• RNA sequencing (RNA-seq) was performed to analyze gene expression patterns following CHIR induction.
• Cardiomyogenic efficiency and the rate of differentiation were assessed by monitoring the appearance of rhythmically beating cells.

Main Results:

• Co-induction with CHIR and high levels of Activin-A (50-100 ng/ml) favored definitive endoderm formation.
• Low levels of Activin-A (10 ng/ml) significantly improved cardiomyogenic efficiency, even when CHIR alone was ineffective.
• Co-induction with CHIR and low levels of Bmp4 (3-10 ng/ml) strongly inhibited cardiomyogenesis.
• Low-level Activin-A accelerated cardiomyogenesis, with beating cells appearing by Day 6 compared to Day 8 with CHIR alone.
• RNA-seq confirmed sequential endogenous expression of Nodal and Bmp ligands and receptors following CHIR induction.

Conclusions:

• Optimizing the timing and levels of exogenous growth factors, particularly low-level Activin-A, downstream of CHIR induction significantly enhances cardiomyogenic efficiency and rate.
• The findings support a model where Nodal (Activin-A) levels determine lineage entry (endoderm vs. mesoderm), and Bmp levels regulate subsequent mesodermal cell fate.
• This optimized approach holds promise for improving the generation of cardiomyocytes for therapeutic and modeling applications.