Massive expansion and cryopreservation of functional human induced pluripotent stem cell-derived cardiomyocytes

Abstract

Summary Since the discovery of human induced pluripotent stem cells (hiPSCs), numerous strategies have been established to efficiently derive cardiomyocytes from hiPSCs (hiPSC-CMs). Here, we describe a cost-effective strategy for the subsequent massive expansion (>250-fold) of high-purity hiPSC-CMs relying on two aspects: removal of cell-cell contacts and small-molecule inhibition with CHIR99021. The protocol maintains CM functionality, allows cryopreservation, and the cells can be used in downstream assays such as disease modeling, drug and toxicity screening, and cell therapy. For complete details on the use and execution of this protocol, please refer to Buikema (2020) .

Sarcomere Disassembly and Transfection Efficiency in Proliferating Human iPSC-Derived Cardiomyocytes

“…We and others demonstrated that a third phase of Wnt signal modulation in immature CMs results in a maturation block while prolongating the window for CM proliferation [3,[19][20][21][22]. Using this strategy, it now becomes feasible to maintain 30-50% of Ki67 positive cells and up to 10% of mitotic CMs after day 12 of hiPSC-CM differentiation [3,21]. Here we utilized live imaging in a hiPSC-CM culture system to follow the sequence of sarcomere breakdown during the mitotic phases of CM cell division.…”

“…hiPSC obtained from the Stanford Cardiovascular Institute Biobank (SCVI-111 and SCVI-273) were non-enzymatically passaged using 0.5 mM EDTA (Thermo Fisher, Waltham, MA, USA) every 4 days as previously described [3]. In brief, cells were incubated with 0.5 mM EDTA-PBS at room temperature for 5-8 min until cells began to separate uniformly throughout the colonies.…”

“…Proper contraction of CMs is dictated by the architecture of the sarcomeres and appears together with polyploidy inversely correlated with self-renewal. Higher sarcomere architectural organization is related to maturity of the heart [2], while immature organization of sarcomeres correlates to the capacity of CMs to proliferate [3]. Previous studies in rodent hearts have shown that during embryonic and early postnatal hyperplastic growth phases the CMs gradually disassemble their sarcomeres during mitosis to ensure cytokinesis [4][5][6].…”

“…Currently, hiPSC-CMs are widely used in mechanistic studies, drug testing and tissue engineering [17,18]. We and others demonstrated that a third phase of Wnt signal modulation in immature CMs results in a maturation block while prolongating the window for CM proliferation [3,[19][20][21][22]. Using this strategy, it now becomes feasible to maintain 30-50% of Ki67 positive cells and up to 10% of mitotic CMs after day 12 of hiPSC-CM differentiation [3,21].…”

Protocol to measure contraction, calcium, and action potential in human-induced pluripotent stem cell-derived cardiomyocytes

“…Human-induced pluripotent stem cells (hiPSCs) can be requested from Stanford Cardiovascular Institute iPSC Biobank ( https://med.stanford.edu/scvibiobank.html ) or obtained from various commercial sources. For maintenance of hiPSCs, differentiation of hiPSCs into cardiomyocytes (hiPSC-CMs), expansion and cryopreservation of hiPSC-CMs, please refer to other STAR protocols ( Lin and Zou, 2020 ; Lyra-Leite et al., 2021 ; Maas et al., 2021 ). Here, we used a small molecule-based cardiomyocyte differentiation method as described in Zhang et al.…”