308 Chapter 12 Figure 2. Comparison of morphology and cell numbers between hCSs from wild type (CTR lines) and PLN-R14del patients (R14del lines). (A) Cardiac spheroid morphology at days 2, 7, 14, and 21. Scale bar: 400 μm. Each dot represents the average of 27-304 spheroids, while 3-7 biological differentiations per condition were included in this analysis. (B) Quantification of spheroid size at day 21. **** P < 0.0001 by unpaired T-test. CTR = size measurement per spheroid of the 3 control lines, R14del = size measurement per spheroid of the 3 PLN-R14del lines. (C) Quantification of spheroid nuclei number at day 21. **** P < 0.0001 by unpaired T-test. CTR = nuclei number per spheroid of the 3 control lines, R14del = nuclei number per spheroid of the 3 PLN-R14del lines. Each dot represents one individual spheroid, and 2-3 biological, while 3-8 technical replicates per condition were included in this analysis. (D) Flow cytometry analysis of alpha-actinin positive cells before the generation of spheroids and after 21 days post-generation. (E) Quantification of alpha-actinin after 21 days post spheroid generation. **** P < 0.001 by Student’s unpaired T-test. Each dot represents the average of 5-25 flow cytometry measurements of 25.000 cells. 2-3 biological differentiations per condition were included in this analysis. (F) mRNA expression of proliferation marker ki67. Each dot represents one batch of hCSs (n=21 vs. n=18). 3 biological replicates per condition were included in this analysis. * P< 0.05 as calculated by an unpaired t-test. Abbreviations; hiPSC; human induced pluripotent stem cell-derived cardiomyocytes, CMs; Cardiomyocytes, CTR; Control hCSs (1CiCTR, 273iCTR, C31iCTR), R14del; PLN-R14del hCSs (D4iR14del, 6BiR14del, 10BiCTR). All data are represented as mean ± SD.
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