Generation and purification of human stem cell-derived cardiomyocytes

Abstract

Efficient and reproducible generation and purification of human stem cell-derived cardiomyocytes (CMs) is crucial for regenerative medicine, disease modeling, drug screening and study of developmental events during cardiac specification. Established methods to generate CMs from human pluripotent stem cells (hPSCs) include the Spin-embryoid body (Spin-EB) and monolayer-based differentiation protocol. In the presence of an optimized cocktail of growth factors under defined conditions, hPSCs differentiate efficiently into functional contracting CMs within 10 days. Nevertheless, despite high efficiencies, cardiac-directed differentiations of hPSCs typically result in heterogeneous populations comprised of both CMs and uncharacterized non-cardiac cell-types. Therefore, generation of pure populations of stem cell-derived CMs is of fundamental importance for basic cardiac research and pre-clinical and possible clinical applications. For the purification of CMs from heterogeneous populations, fluorescent activated cell sorting (FACS) is a widely appreciated method. Nonetheless, FACS-based isolation of CMs comes along with several disadvantages, such as undesired contaminations and low viability of target cells. Here, we describe a convenient and rapid procedure for the purification of hPSCs-derived CMs under sterile culture conditions, resulting in high purity and viability of sorted CMs. Purification with VCAMI-coupled magnetic Dynabeads led to robust enrichment of CMs, which will especially be important for cardiac differentiations of cell lines with poor differentiation efficiencies. In addition, this will also be beneficial for the standardization and reproducibility of human stem cell-derived assays in the fields of cardiac disease modeling, drug discovery and disease modeling.

Keywords: Cardiac monolayer differentiation; Cardiac spin-embryoid body differentiation; Cardiomyocyte purification; Human pluripotent stem cells; Magnetic bead-based cell separation.