Scalable manufacturing of clinical-grade differentiated cardiomyocytes derived from human-induced pluripotent stem cells for regenerative therapy

Abstract

Basic research on human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) for cardiac regenerative therapy is one of the most active and complex fields to achieve this alternative to heart transplantation and requires the integration of medicine, science, and engineering. Mortality in patients with heart failure remains high worldwide. Although heart transplantation is the sole strategy for treating severe heart failure, the number of donors is limited. Therefore, hPSC-derived CM (hPSC-CM) transplantation is expected to replace heart transplantation. To achieve this goal, for basic research, various issues should be considered, including how to induce hPSC proliferation efficiently for cardiac differentiation, induce hPSC-CMs, eliminate residual undifferentiated hPSCs and non-CMs, and assess for the presence of residual undifferentiated hPSCs in vitro and in vivo. In this review, we discuss the current stage of resolving these issues and future directions for realizing hPSC-based cardiac regenerative therapy.

FIGURE 1

Scalable manufacturing of clinical‐grade hiPSC‐CMs. Tryptophan‐fortified media promotes the proliferation of hiPSCs. Large numbers of hiPSC‐CMs are induced in a multilayer culture plate. Cardiac differentiation efficiency is evaluated by cell count, flow cytometry, and immunostaining. Orlistat treatment selectively eliminates residual undifferentiated hPSCs. Then, hiPSC‐CMs were metabolically selected with glucose‐ and glutamine‐depleted lactate‐supplemented media. After purification, hiPSC‐CMs are isolated, harvested, and cryopreserved. The purity of hiPSC‐CMs and the contamination rate of residual undifferentiated hPSCs are assessed. After thawing, cardiac spheroids are produced. Beating profiles of cardiac spheroids are evaluated before transplantation. Cardiac spheroids are transplanted using our developed spheroids transplantation device.

FIGURE 2

Overview of metabolic hallmarks in hPSCs and hPSC‐CMs. (A) hPSCs depend on glucose and glutamine metabolism for ATP and biomass production. They also show activated fatty acids synthesis for survival and maintenance of pluripotency. (B) hPSC‐CMs utilize glucose, glutamine, and lactate for ATP production via oxidative phosphorylation. (C) hPSCs cannot survive under glucose‐ and glutamine‐depleted with lactate‐supplemented conditions because they cannot utilize lactate efficiently. (D) hPSC‐CMs can survive under glucose‐ and glutamine‐depleted with lactate‐supplemented conditions because they can utilize lactate efficiently via oxidative phosphorylation. (E) Glutamine‐derived GSH, methionine‐derived SAM, and tryptophan‐derived KYN play key roles in the maintenance of pluripotency.