A Universal System for Highly Efficient Cardiac Differentiation of Human Induced Pluripotent Stem Cells That Eliminates Interline Variability

TLDR

Cardiomyocyte production from human induced pluripotent stem cells (hiPSC) promises patient‑specific cardiotoxicity testing, disease modeling, and cardiac regeneration, yet current differentiation protocols are inefficient and highly variable. The study presents a highly efficient system for differentiating hESC and hiPSC into cardiomyocytes. The method uses forced‑aggregation embryoid bodies in a chemically defined medium with staged 5 % oxygen and optimized BMP4, FGF2, polyvinyl alcohol, serum, and insulin, after systematic optimization of over 45 variables, achieving 94.7 % efficiency in nine days across multiple hESC and hiPSC lines. The resulting contracting embryoid bodies contain 64–89 % cardiac troponin I+ cells with functional ultrastructure and uniform electrophysiology, demonstrating a scalable, cost‑effective platform for drug testing, disease modeling, and future regenerative therapies.

Abstract

Background The production of cardiomyocytes from human induced pluripotent stem cells (hiPSC) holds great promise for patient-specific cardiotoxicity drug testing, disease modeling, and cardiac regeneration. However, existing protocols for the differentiation of hiPSC to the cardiac lineage are inefficient and highly variable. We describe a highly efficient system for differentiation of human embryonic stem cells (hESC) and hiPSC to the cardiac lineage. This system eliminated the variability in cardiac differentiation capacity of a variety of human pluripotent stem cells (hPSC), including hiPSC generated from CD34+ cord blood using non-viral, non-integrating methods. Methodology/Principal Findings We systematically and rigorously optimized >45 experimental variables to develop a universal cardiac differentiation system that produced contracting human embryoid bodies (hEB) with an improved efficiency of 94.7±2.4% in an accelerated nine days from four hESC and seven hiPSC lines tested, including hiPSC derived from neonatal CD34+ cord blood and adult fibroblasts using non-integrating episomal plasmids. This cost-effective differentiation method employed forced aggregation hEB formation in a chemically defined medium, along with staged exposure to physiological (5%) oxygen, and optimized concentrations of mesodermal morphogens BMP4 and FGF2, polyvinyl alcohol, serum, and insulin. The contracting hEB derived using these methods were composed of high percentages (64–89%) of cardiac troponin I+ cells that displayed ultrastructural properties of functional cardiomyocytes and uniform electrophysiological profiles responsive to cardioactive drugs. Conclusion/Significance This efficient and cost-effective universal system for cardiac differentiation of hiPSC allows a potentially unlimited production of functional cardiomyocytes suitable for application to hPSC-based drug development, cardiac disease modeling, and the future generation of clinically-safe nonviral human cardiac cells for regenerative medicine.