Human iPSC Line

Generation and Maturation of Human Ipsc-Derived 3D Organotypic Cardiac Microtissues in Long-Term Culture

Cardiovascular diseases are a major global health concern, with existing models failing to fully replicate human heart complexity. Using induced pluripotent stem cells (iPSCs), Ergir et al. established a scaffold-free protocol to generate 3D human cardiac microtissues (hOCMTs).

Since 2D monolayer differentiation of iPSC-derived cardiomyocytes is well established, and these monolayers tend to delaminate into beating clusters, they first differentiated hiPSCs in a 2D system and tested their ability to form 3D cardiac aggregates without an external ECM scaffold. Cardiac differentiation was induced in confluent 2D hiPSC monolayers by modulating the WNT pathway with small molecules, as previously described (Fig. 1A). Briefly, mesoderm specification was achieved by transient WNT activation, followed by cardiac mesoderm differentiation through WNT inhibition. On day 7, insulin was added to support cell survival. On day 15, the monolayer was dissociated into single cells and seeded in ultra-low attachment plates to induce aggregate formation in the presence of a ROCK inhibitor. We used 2D monolayer cultures as controls. One day after switching to 3D culture, they observed spontaneous 3D microtissue formation without an ECM scaffold, which began beating after 48 h. To monitor this process, they repeated the experiment with hiPSCs expressing GFP-tagged cardiac troponin I. GFP-positive cardiomyocytes and non-GFP non-myocytes rearranged over time from a random distribution to more discrete regions (Fig. 1B). The microtissues' diameter increased from 0.9 ± 0.04 mm on day 21 to 1 ± 0.09 mm on day 42 (Fig. 1C). They continued to beat spontaneously in long-term culture, lasting at least until day 100.

Fig. 1. Long-term human cardiac microtissues can be spontaneously generated by scaffold-free conditions in 3D (Ergir E, Oliver-De La Cruz J, et al., 2022).

Efficient and Reproducible Generation of Human iPSC-Derived Cardiomyocytes and Cardiac Organoids in Stirred Suspension Systems

Human iPSC-derived cardiomyocytes (hiPSC-CMs) are crucial for cardiac disease modeling and regeneration, but challenges such as quality control, inter-batch consistency, cryopreservation, and scalability limit their reproducibility and clinical translation. Prondzyski et al. introduce a stirred suspension protocol using bioreactors and spinner flasks to produce hiPSC-CMs and cardiac organoids.

They first observed contraction in bCMs at differentiation day 5, compared to dd7 in mCMs and previously reported hiPSC-CM differentiations. To validate this, they differentiated hiPSCs with GFP fused to TNNI1 in the bioreactor and saw GFP expression in bCMs on dd5, in contracting areas at the edges of EBs (Fig. 2g). mCMs showed GFP expression on day 6 but did not visibly contract until dd7. RT-qPCR showed higher ACTN2 expression in dd5 bCMs than in mCMs (Fig. 2h). bCMs also had less inter-batch variation in ACTN2 levels at day 15 compared to mCMs (Fig. 2i). Vimentin (VIM) was lower in dd15 bCMs, though not significantly due to mCM variation (Fig. 2j). bCMs had higher expression of ventricular markers MYH7, MYL2, and MYL3 (Fig. 1k–m) and 83.4% MLC2v-positive cells by flow cytometry. Atrial markers MYL4 and MYL7 were higher or unchanged in bCMs (Fig. 2n, o). HCN4 was higher in some mCM batches with high variability (Fig. 2p). TNNT2 protein levels were significantly higher in dd15 bCMs than in mCMs.

Fig. 2. Optimized stirred bioreactor cardiac differentiation protocol (Prondzynski M, Berkson P, et al., 2024).