Extracellular Matrix Promotes Highly Efficient Cardiac Differentiation of Human Pluripotent Stem Cells

TLDR

Cardiomyocytes derived from human pluripotent stem cells are increasingly used for cardiovascular research and potential clinical applications, yet current differentiation protocols vary in success and rely mainly on growth factors. The study aimed to develop a more effective cardiac differentiation protocol for human PSCs by combining extracellular matrix with cardiogenic growth factors. Human PSCs were cultured as monolayers on Matrigel and then overlaid with Matrigel, creating a matrix sandwich that was combined with sequential growth factor application. The matrix sandwich induced epithelial‑mesenchymal transition and, when combined with sequential growth factors, produced highly pure (up to 98%) and abundant (up to 11 CMs per PSC) cardiomyocytes that matured over 30 days, exhibited action potentials of nodal, atrial, and ventricular phenotypes, and formed electrically coupled monolayers, demonstrating robust cardiac differentiation.

Abstract

Cardiomyocytes (CMs) differentiated from human pluripotent stem cells (PSCs) are increasingly being used for cardiovascular research, including disease modeling, and hold promise for clinical applications. Current cardiac differentiation protocols exhibit variable success across different PSC lines and are primarily based on the application of growth factors. However, extracellular matrix is also fundamentally involved in cardiac development from the earliest morphogenetic events, such as gastrulation.We sought to develop a more effective protocol for cardiac differentiation of human PSCs by using extracellular matrix in combination with growth factors known to promote cardiogenesis.PSCs were cultured as monolayers on Matrigel, an extracellular matrix preparation, and subsequently overlayed with Matrigel. The matrix sandwich promoted an epithelial-to-mesenchymal transition as in gastrulation with the generation of N-cadherin-positive mesenchymal cells. Combining the matrix sandwich with sequential application of growth factors (Activin A, bone morphogenetic protein 4, and basic fibroblast growth factor) generated CMs with high purity (up to 98%) and yield (up to 11 CMs/input PSC) from multiple PSC lines. The resulting CMs progressively matured over 30 days in culture based on myofilament expression pattern and mitotic activity. Action potentials typical of embryonic nodal, atrial, and ventricular CMs were observed, and monolayers of electrically coupled CMs modeled cardiac tissue and basic arrhythmia mechanisms.Dynamic extracellular matrix application promoted epithelial-mesenchymal transition of human PSCs and complemented growth factor signaling to enable robust cardiac differentiation.