Ginkgolide A Enhances Cardiomyocyte Differentiation from Pluripotent Stem Cells by Targeting Cytochrome c to Attenuate Intrinsic Apoptosis

Efficient differentiation of pluripotent stem cells (PSCs) into functional cells is critical for regenerative medicine and biomanufacturing, yet is often hampered by apoptosis. Ginkgolide A (GA) is a diterpene lactone derived from Ginkgo biloba leaves and a member of the ginkgolide family, compounds known for diverse biological activities including neuroprotection and cardiovascular regulation. However, the mechanism by which ginkgolides influence the directed differentiation of stem cells remains unclear. Here, using a CRISPR-Cas9-engineered TNNT2-mCherry reporter PSC line and a defined cardiac differentiation protocol, we screened ginkgolides for their effects on cardiomyocyte production. Results demonstrated that GA significantly enhanced cardiomyocyte induction efficiency and accelerated the onset of spontaneous beating. Concurrently, GA effectively inhibited apoptosis during differentiation and RNA-seq results also revealed that GA orchestrates stage-specific upregulation of anti-apoptotic genes (e.g., MCL1, XIAP) and core cardiogenic transcription factors (NKX2-5, GATA4). Molecular docking predictions suggested a high binding potential between GA and cytochrome c, suggesting GA might inhibit the intrinsic mitochondrial apoptosis cascade by interfering with cytochrome c's binding to Apoptotic Peptidase Activating Factor-1 (APAF-1). These data demonstrate that GA enhances the differentiation of pluripotent stem cells into cardiomyocytes, potentially through its anti-apoptotic effect. This mechanism highlights its potential as a safe culture additive to boost cell survival and yield for large-scale biomanufacturing and tissue engineering applications.