Efficient and reproducible generation of human iPSC-derived cardiomyocytes and cardiac organoids in stirred suspension systems

Human iPSC-derived cardiomyocytes (hiPSC-CMs) have proven invaluable for cardiac disease modeling and cardiac regeneration. Challenges with quality, inter-batch consistency, cryopreservation and scale remain, reducing experimental reproducibility and clinical translation. Here, we report a robust stirred suspension cardiac differentiation protocol with careful functional characterization of the resulting hiPSC-CMs. In a bioreactor, the protocol produced 1.2E6/mL hiPSC-CMs with ～94% purity from 14 iPSC lines. Bioreactor-differentiated CMs (bCMs) showed high viability after cryo-recovery (>90%) and predominantly ventricular identity. Compared to standard monolayer-differentiated CMs (mCMs), bCMs had greater reproducibility and more mature functional properties, including pacing capture to 4 Hz and greater force production in 3D engineered heart tissues. In more readily available magnetically stirred spinner flasks, the protocol yielded 1.8E6/mL spinner-differentiated CMs (sCMs) with 94% purity. Differentiation scaled readily in spinner flasks, as a 3.8-fold increase in cultured volume yielded 3.4E6/ml sCMs. sCMs had intermediate functional properties between mCMs and bCMs. Minor protocol modifications generated the first bioreactor-derived cardiac organoids (bCOs) fully generated in suspension. These reproducible, scalable, and resource efficient approaches to generate cardiac cells and organoids with well-characterized properties will expand the applications of hiPSC-CMs. We performed scRNAseq of 15-day-old bioreactor-derived hiPSC-cardiomyocytes (bCMs; 2 biological replicates), monolayer-derived hiPSC-cardiomyocytes (mCMs; 2 biological replicates) and bioreactor-derived cardiac organoids (bCOs; 1 biological replicate).

人类诱导多能干细胞衍生心肌细胞（hiPSC-CMs）已被证实为心脏疾病建模与心脏再生研究提供了不可或缺的研究工具。目前该类细胞仍面临质量管控、批次间一致性、冷冻保存及规模化生产等多重挑战，制约了实验可重复性与临床转化进程。本研究报道了一种稳定的悬浮搅拌心脏分化方案，并对所得hiPSC-CMs开展了严谨的功能表征。在生物反应器中，该方案可从14株诱导多能干细胞系中，以约94%的纯度每毫升培养体系获得1.2×10^6个hiPSC-CMs。经生物反应器分化的心肌细胞（bCMs）在冷冻复苏后仍保持高活性（存活率＞90%），且主要呈现心室肌细胞表型。与传统单层培养分化的心肌细胞（mCMs）相比，bCMs具有更高的实验可重复性与更成熟的功能特性，包括可实现4Hz的起搏捕获，以及在三维工程心脏组织中产生更大的收缩力。在更易获取的磁力搅拌转瓶体系中，该方案同样可获得每毫升培养体系1.8×10^6个转瓶分化心肌细胞（sCMs），纯度达94%。该分化方案可轻松实现规模化扩增：当培养体积提升3.8倍时，每毫升体系可获得3.4×10^6个sCMs。sCMs的功能特性介于mCMs与bCMs之间。通过对方案进行小幅优化，我们首次获得了完全悬浮培养的生物反应器来源心脏类器官（bCOs）。这些具备良好可重复性、可规模化且资源高效的心肌细胞与类器官制备方案，及其经过充分表征的细胞特性，将进一步拓展hiPSC-CMs的应用场景。本研究对15天龄的生物反应器来源hiPSC-CMs（bCMs；2次生物学重复）、单层培养来源hiPSC-CMs（mCMs；2次生物学重复）以及生物反应器来源心脏类器官（bCOs；1次生物学重复）进行了单细胞RNA测序（scRNAseq）。