A Massive Suspension Culture System With Metabolic Purification for Human Pluripotent Stem Cell-Derived Cardiomyocytes

Abstract

Cardiac regenerative therapy with human pluripotent stem cells (hPSCs), such as human embryonic stem cells and induced pluripotent stem cells, has been hampered by the lack of efficient strategies for expanding functional cardiomyocytes (CMs) to clinically relevant numbers. The development of the massive suspension culture system (MSCS) has shed light on this critical issue, although it remains unclear how hPSCs could differentiate into functional CMs using a MSCS. The proliferative rate of differentiating hPSCs in the MSCS was equivalent to that in suspension cultures using nonadherent culture dishes, although the MSCS provided more homogeneous embryoid bodies (EBs), eventually reducing apoptosis. However, pluripotent markers such as Oct3/4 and Tra-1-60 were still expressed in EBs 2 weeks after differentiation, even in the MSCS. The remaining undifferentiated stem cells in such cultures could retain a strong potential for teratoma formation, which is the worst scenario for clinical applications of hPSC-derived CMs. The metabolic purification of CMs in glucose-depleted and lactate-enriched medium successfully eliminated the residual undifferentiated stem cells, resulting in a refined hPSC-derived CM population. In colony formation assays, no Tra-1-60-positive colonies appeared after purification. The nonpurified CMs in the MSCS produced teratomas at a rate of 60%. However, purified CMs never induced teratomas, and enriched CMs showed proper electrophysiological properties and calcium transients. Overall, the combination of a MSCS and metabolic selection is a highly effective and practical approach to purify and enrich massive numbers of functional CMs and provides an essential technique for cardiac regenerative therapy with hPSC-derived CMs.

Assessing Cardiac Metabolism

“…246 Furthermore, the inherent flexibility in energy substrate metabolism underlies the cells’ ability to prioritize certain metabolic pathways in support of stage-specific demands. 248 The combination of a massive suspension culture system with metabolic selection holds promise as an effective and practical approach to purify and enrich the large numbers of cardiomyocytes found in iPSCs. 248 …”

“…248 The combination of a massive suspension culture system with metabolic selection holds promise as an effective and practical approach to purify and enrich the large numbers of cardiomyocytes found in iPSCs. 248 …”

Signaling Pathways and Gene Regulatory Networks in Cardiomyocyte Differentiation

“…155 Similar enrichment of hPSC-derived cardiomyocytes has been demonstrated in spinner flask cultures. 156 Another vexing issue is the phenotypic immaturity of hPSC-derived cardiomyocytes including the lack of fully formed sacromeric elements and multinucleation, and their suboptimal Ca + 2 handling. For their in vivo maturation, cardiomyocytes experience mechanical loads and electrical activity influencing contractile function, protein synthesis, cell size, and overall tissue remodeling.…”

A Universal and Robust Integrated Platform for the Scalable Production of Human Cardiomyocytes From Pluripotent Stem Cells

“…However, these techniques have limited differentiation potential, scalability (microwell-mediated control, hanging drop, and microprinting technologies), universality, and/or reproducibility because of the differentiation protocol itself or the low throughput of the methods used such as forced aggregation techniques before transferring cells to dynamic culture conditions. In addition, most of the protocols depend on using expensive and complex media or reagents (mTeSR1; StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com; or StemPro-34; Thermo Fisher Scientific, Waltham, MA, http:// www.thermofisher.com) or microcarriers for expansion of hPSCs and their directed differentiation to cardiomyocytes [24,25].…”