307 Modeling and Rescue of PLN-R14del Cardiomyopathy Phenotype in Human iPSC-Derived Cardiac Spheroids 12 ◀Figure 1. Comparison of Ca2+ handling properties between hCSs from wild type (CTR lines) and PLN-R14del patients (R14del lines). (A) Schematic representation of Wnt-based directed cardiac differentiation, the subsequent expansion of hiPSC-CMs, and the generation pipeline for hCSs. Created with biorender.com (B) Time-lapse Ca2+ imaging of spontaneous Ca2+ release in hCSs. Scale bar: 200 μm (C) Representative recordings of time-lapse Ca2+ transients. (D) Measurement of Ca2+ handling parameters from automated recorded data. Comparison of (E) Decay time (Tau), (F) Ca2+ transient amplitude, (G) Rise time, (H) transient duration 10% (CTD10), (I) transient duration 90% (CTD90), and (J) beating rate in 1CiCTR (biological replicates=3, n=120), 273iCTR (biological replicates=5, n=217), C31iCTR (biological replicates =3, n=138), D4iR14del (biological replicates=5, n=287), 6BiR14del (biological replicates=3, n=89), and 10BiR14del (biological replicates=3, n=60) hCSs. **** P < 0.0001 vs. control lines by One-Way ANOVA. Each dot represents one individual spheroid and 3 biological (=b) per condition were included in this analysis. Morphological characterization of PLN-R14del hCSs Notably, when culturing the hCS, we observed an overall increase in the size of the hCSs, both in the controls and PLN-R14del-derived lines. Remarkably, spheroid size in PLN-R14del was massively increased when compared to controls (Figure 2A, Supplementary Figure 3A), already after 7 days of culturing. PLN-R14del hCSs showed a significantly larger diameter compared to controls at days 7, 14, and 21 (Figure 2B), with a final average size of 1092.87 µM (± 597.28) vs 422.94 µM (± 251.97) of 21 days old hCSs (Figure 2C). The number of nuclei per spheroid was significantly increased compared to controls, with an average number of 460 (± 306) vs 163 (± 101) of 21-day-old hCSs (Figure 2D). To ensure comparable CM numbers for initial spheroid generation, we enriched hiPSC-CMs to >80% purity through metabolic selection before hCS generation for all lines. The purity of the hiPSC-CMs used for the generation of hCSs was not significantly different between cell lines when hCSs spheroids were generated (78.04 ± 10.31 % alpha-actin positive cells) (Figure 2E, Supplementary Figure 3B). However, after 3 weeks of spheroid culturing, the PLN-R14del hCS showed a significant 50% reduction in alpha-actinin positive cells (43.33 ± 9.10) compared to healthy control spheroids (70.71 ± 3.38) (Figure 2F, Supplementary Figure 3C). The increased spheroid size, nuclei number, and reduced purity indicate an increase in PLN-R14del spheroid cell number. The observed increase in cell number was confirmed by a significant increase in expression of the proliferation marker ki67 from 0.21 (±0.47) to 37.46 (±76.3) fold (Figure 2G). Effect of hiPSC-CF and hiPSC-CM ratios in PLN-R14del vs isogenic control hCSc size Next, we controlled and gradually changed the ratio of hiPSC-derived cardiac fibroblasts to hiPSC-CMs (100%-0%, 90%-10%, 70%-30%, and 50%-50%, per hCS respectively) derived from the same donor. Interestingly, we observed again an increase in the PLN-R14del spheroid size after 14-21 days in culture (Figure 3A). The size of the isogenic control hCSs was not affected by the addition of different ratios with CFs (Figure 3B), whereas a significant increase in spheroid size in the PLN-R14del hCSs was observed (Figure 3C). Interestingly, when modulating the ratio into +50% hiPSC-CMs, the PLN-R14del hCSs size significantly increased after 14 and 21 days of spheroid culturing (Supplementary Figure 4). This suggests that over time cardiomyocyte-specific cell interaction mediates the increase in size rather than the proliferation of existing hiPSC-derived fibroblasts in the hCSs, which is dramatically enhanced in the PLN-R14del patient lines.
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