A complete workflow for the differentiation and the dissociation of hiPSC-derived cardiospheres

Abstract

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) are an invaluable tool for both basic and translational cardiovascular research. The potential that these cells hold for therapy, disease modeling and drug discovery is hampered by several bottlenecks that currently limit both the yield and the efficiency of cardiac induction. Here, we present a complete workflow for the production of ready-to-use hiPSC-CMs in a dynamic suspension bioreactor. This includes the efficient and highly reproducible differentiation of hiPSCs into cardiospheres, which display enhanced physiological maturation compared to static 3D induction in hanging drops, and a novel papain-based dissociation method that offers higher yield and viability than the broadly used dissociation reagents TrypLE and Accutase. Molecular and functional analyses of the cardiomyocytes reseeded after dissociation confirmed both the identity and the functionality of the cells, which can be used in downstream applications, either as monolayers or spheroids.

A novel human pluripotent stem cell-based assay to predict developmental toxicity

“…Cardiac differentiation was performed as published (Fischer et al 2018) with slight modifications. Briefly, hiPSC were harvested from culture dishes with good colony morphology (day 5-6 after split) by removing medium and adding Gibco™ TrypLE™ Select (Thermo Fisher Scientific) for 1-2 min.…”

“…With this, we obtained a set of male and female cell lines of different donor ages. We implemented a 3D protocol for cardiomyocyte differentiation to retrieve spheres with contracting cardiomyocytes as a simple readout in conical bottom 96-well plates (Zhang et al 2015;Fischer et al 2018). An outline of the assay is depicted in Fig.…”

“…We optimized the concentration of CHIR99021 for our three cell lines, because the optimal level of this factor is known to be cell line dependent. We tested concentrations from 1 µM (Fischer et al 2018) to 3.5 µM, and chose 2.5 µM as the lowest concentration with significantly improved differentiation efficiency in all three cell lines (Fig. 1d).…”

“…Importantly, we used three different hiPSC lines to investigate inter-line variability as well as male and female cells to exclude any gender-specific effects. We optimized an advanced and recently developed differentiation method for use with these three cell lines (Fischer et al 2018), and differentiated them into beating cardiomyocytes in only 8 days. Importantly, we used the 3D culture of hiPSC in EBs and showed that these indeed undergo key developmental changes similar to early mammalian embryos.…”

Concise review: Harnessing iPSC-derived cells for ischemic heart disease treatment

“…IPSCs are pluripotent stem cells that are produced from adult somatic cells or blood cells over a genetic reprogramming process back into an embryonic-like pluripotent state. [12,13] Table 1 summarizes some advantages and disadvantages/ concerns as compared to the use of other stem cells (like MSCs and ESCs). hiPSCs have been successfully demonstrated to be capable of differentiating into beating cardiomyocytes through various protocols.…”