Human Induced Pluripotent Stem Cell-Derived Non-Cardiomyocytes Modulate Cardiac Electrophysiological Maturation Through Connexin 43-Mediated Cell-Cell Interactions

Figures

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  FIG. 1.. Characterization of sorted populations from cardiac differentiations reveals CM and cardiac fibroblast-like phenotypes. (A) Schematic of experimental design. Cardiac differentiations from hiPSCs were sorted on day 10 of differentiation into CM and non-CM populations. (B–E) Relative expression (FC) of cardiac fibroblast (B), CM (C), and ventricular CM (D) and atrial CM (E) genes in unsorted, non-CM, and CM subpopulations on day 10, as analyzed by qRT-PCR. Error bars represent range of fold change, calculated from standard deviation of ΔΔCt. Data are from eight sorts representing two clones each from three unrelated individuals. P values for non-CM versus CM samples were calculated from ΔΔCt values by a Student's t-test (*P ≤ 0.01; **P ≤ 0.001; ***P ≤ 0.0001). (F) Percentage of live cells in unsorted and CM populations positive for cTnI, cTnT, CD90, or CD31 by flow cytometry. Data are from 14 sorts representing 1–3 clones each from 4 unrelated individuals, analyzed between days 20 and 26. (G) Percentage of live cells in unsorted and non-CM populations positive for cTnI, cTnT, CD90, or CD31. Data are from nine sorts representing one to two clones each from three unrelated…

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  FIG. 2.. dFBs demonstrate the influence of a CD90-positive cell type upon hiPSC-CM electrophysiology. (A) Analysis of CD90 expression in dFBs by flow cytometry. (B) Optical tracings of negative change in fluorescence over fluorescence (-ΔF/F) permit analysis of AP properties of CMs, including APD50 (AP duration at 50% repolarization, horizontal red dotted line), APD90 (horizontal blue dotted line), amplitude (black line), and maximum upstroke velocity (Vmax, not shown). (C–G) Analysis of AP amplitude (C), Vmax (D), APD50 (E), APD90 (F), and APD90/APD50 (G) for 1:9 (1 part dFB to 9 parts CM) and 1:1 co-cultures. Data were collected from two different hiPSC-CM clones on day 40 of differentiation. 1:9: n = 22 cells; 7:3: n = 22 cells. Heavier dashed lines within each violin indicate medians and lighter dashed lines indicate interquartile range. P values were calculated by either Student's t-test (AP amplitude, APD50, APD90) or Mann-Whitney U test (Vmax, APD90/APD50). AP, action potential; APD50, action potential duration at 50% repolarization; APD90, Action potential duration at 90% repolarization; dFBs, dermal fibroblasts; hiPSC-CM, human induced pluripotent stem cell-derived cardiomyocyte.

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  FIG. 3.. Varied electrophysiological properties of hiPSC-CMs due to differing ratios of non-CMs versus CMs in co-cultures. (A–F) Analysis of optical AP amplitude (A), maximum upstroke velocities (Vmax) (B), APD50 (AP duration at 50% repolarization) (C), APD90 (D), APD90/APD50 (E), and interval between APs (F) for co-cultures with 1 part non-CMs to 9 parts CMs 1:9, 3:7 co-cultures, 1:1 co-cultures, and 7:3 co-cultures. Data were collected from eight independent differentiations representing seven clones from three unrelated individuals between days 39 and 40 of differentiation; 1:9: n = 152 cells; 3:7: n = 149 cells; 1:1: n = 160 cells; and 7:3: n = 138 cells. Heavier dashed lines within each violin indicate medians and lighter dashed lines indicate interquartile range. Data were initially analyzed by a Kruskal-Wallis test (n.s. for APD90 and P < 0.0001 for all other parameters) and pairwise P values were calculated by Mann-Whitney U test for AP amplitude, Vmax, APD50, APD90/APD50, and interval (*P ≤ 0.0083; **P ≤ 0.001; ***P ≤ 0.0001). (G) Representative inverted fluorescence traces (-ΔF) of optical APs for 7:3, 1:1, 3:7, and 1:9 co-culture conditions from the same experiment. Trace bars…

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  FIG. 4.. Non-CMs do not have a notable influence on CM electrophysiology through paracrine effects. (A-E) Analysis of optical AP amplitude (A), maximum upstroke velocities (Vmax) (B), APD50 (AP duration at 50% repolarization) (C), APD90 (D), and APD90/APD50 (E) for CMs cultured with conditioned media from CMs, non-CMs, co-culture with 1 part non-CM to 9 parts CM (1:9), or 7:3 co-culture. Data were collected from six independent differentiations representing five clones from two unrelated individuals between days 38 and 40 of differentiation. CM media: n = 121 cells; 1:9 media: n = 118 cells; and 7:3 media: n = 121 cells; non-CM media: n = 119 cells. Heavier dashed lines within each violin indicate medians and lighter dashed lines indicate interquartile range. Data were initially analyzed by a Kruskal-Wallis test (P < 0.01 for Vmax, P < 0.001 for APD90/APD50, and n.s. for all others) and pairwise P values were calculated by Mann-Whitney U tests for Vmax and APD90/APD50 (*P ≤ 0.0083; **P ≤ 0.001).

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  FIG. 5.. Both hiPSC-CMs and non-CMs, but not dFBs, exhibit Cx43 expression at cell-cell interfaces. (A, B) Immunofluorescence of cardiac troponin T (cTnT, green) and Cx43 (red) in hiPSC-CMs and non-CMs on day 41 of differentiation, showing Cx43 expression between cTnT-positive CMs (A) and between cTnT-negative non-CMs (B). Scale bars represent 50 μM. (C) Immunofluorescence of Cx43 expression at the interface of a cTnT-positive CM and a non-CM on day 41. Scale bars represent 10 μM. A-C are representative of four independent differentiations of three different clones representing two unrelated individuals between days 38 and 41. White arrows indicate examples of Cx43 staining at cell-cell boundaries. (D) Immunofluorescence of Cx43 in day 38 non-CMs (left) and dFBs (right, representative of cells from three different individuals) derived from the same individual. Scale bars represent 50 μM. Cx43, connexin 43.

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  FIG. 6.. Non-CMs mediate CM electrophysiology in a Cx43-dependent manner. (A) Immunofluorescence of Cx43 (green) in non-CMs transduced with either control or GJA1-targeted shRNAs, as indicated by RFP expression. Most stained cells did not express cTnT (white). Images are representative of two independent experiments. White arrow indicates Cx43 staining at cell-cell boundaries. Scale bars represent 50 μM. (B) Relative expression (FC) of GJA1 for knockdown and control samples, as analyzed by qRT-PCR. Error bars represent range of fold change, calculated from standard deviation of ΔΔCt. Data are from three differentiations representing clones from two unrelated individuals. P value was calculated from ΔΔCt values by a Student's t-test. (C–E) Analysis of optical AP amplitude (C), maximum upstroke velocities (Vmax) (D), and APD90/APD50 (E) for CMs and 1:1 co-cultures of CMs and control or GJA1 knockdown non-CMs. Data were collected in three independent experiments involving two clones in total (from distinct individuals) for both non-CMs and CMs. CMs: n = 120 cells (60 plated together and 60 non-CMs plated on top); control shRNA: n = 116 cells (60 plated together and 56 non-CMs plated on top);…