In vitro generation of human embryonic stem cell-derived heart organoids possessing physiological ion currents

Figures

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  Fig. 1. In vitro recapitulation of developing hearts. (A) Experimental scheme to generate hESC-derived heart organoids (HOs) in vitro. The hEBs derived from hESCs were cultured on the gelated LN/ET complex with HO medium containing FGF4. (B) Morphogenetic shift during HO formation. The hEBs were cultured with FGF4 only (batch n = 6, hEBs n = 36, day 15 HOs n = 36), BBFL(batch n = 2, hEBs n = 12, day 15 HOs n = 11), BBBFL (batch n = 8, hEBs n = 80, day 15 HOs n = 74), and Y (batch n = 2, hEBs n = 12, day 15 HOs n = 10) conditions on the LN/ET complex. The batch indicates independent differentiation. Top: The sizes (areas) of EBs and HOs were measured from microscopic images at day 0 (for EBs) and day 15 (for HOs) using Fiji (ImageJ). For the statistical analysis, GraphPad Prism 11.0 was used. A nested t-test was applied to the size change from EBs to day 15 heart organoids for each culture condition. Area data were presented as a superplot with individual data points (each EB and each heart organoid; small transparent dots) and the batch mean (large solid dot). ∗∗P = 0.0019, ∗P = 0.0323, ∗∗∗∗P < 0.0001. Bottom: A nested one-way ANOVA was used to compare the sizes across culture…

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  Fig. 2. Evaluation of hESC-derived HOs. (A) Immunofluorescence staining for MLC2V (red) and MLC2A (green) in the hESC-derived HOs. Top: a representative hESC-HO (n = 6). Scale bar: 25 μm. Bottom: mouse embryonic heart from E10.5. Scale bar: 50 μm. (B) Immunostaining to detect Cx43 (red)/Cx40 (green), (left panels), and Cx43 (red)/Cx45 (green) (right panels). Top: hESC-HOs (n = 6 for Cx43/Cx40, n = 4 for Cx43/Cx45). Bottom: mouse embryonic hearts from E10.5. Scale bar: 50 μm. (C) Immunostaining to detect one of potassium channel proteins, Kir2.1 in a representative HO (top, n = 10), E10.5 mouse embryonic heart (bottom left) and adult mouse heart (right, bottom). Scale bar: 150 μm. (D) hERG immunostaining in a representative HO (top and right, bottom, n = 10), E10.5 mouse embryonic heart (left, bottom). Scale bar: 75 μm. (E) Gene expression levels of MLC2V, MLC2A, Cx40, Cx43, Cx45, Kir2.1 and hERG in human fetal hearts (HE5W, HE6W, HE7W, HE9W, and HE13W for LA, LV, RA, and RV). The scRNAseq dataset was obtained from NCBI GEO (Accession: GSE106118) [11]. The log-transformed normalized values of the target genes were presented as a dot plot (mean ± SEM). log1p represents log (x + 1), and…

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  Fig. 3. Expression of sodium and calcium channel proteins (Nav1.5 and Cav1.2) in hESC-derived HOs. (A) Nav1.5 immunostaining to detect sodium channel in hESC-derived HOs (left and center, n = 9), E10.5 mouse embryonic heart (top right), and mouse adult heart (bottom right). Scale bar: 90 μm. (B) Cav1.2 immunostaining to detect calcium channel in hESC-derived HOs (left and center, n = 11), E12.5, E13.5, E15.5, and E18.5 mouse embryonic heart (right). Scale bar: 75 μm for left, 50 μm for center and 200 μm for right panels. (C) Gene expression levels of Nav1.5 and Cav1.2 in human fetal hearts (HE5W, HE6W, HE7W, HE9W, and HE13W for LA, LV, RA, and RV). The scRNAseq dataset was obtained from NCBI GEO (Accession: GSE106118) [11]. The log-transformed normalized values of the target genes were presented as a dot plot (mean ± SEM). log1p represents log (x + 1), and CP10K represents counts per 10,000.

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  Fig. 4. Electrophysiological analyses of CMs isolated from hESC-derived HOs in the whole-cell patch-clamp. (A, left) Representative Na current traces were elicited by the depolarizing voltage protocol, as shown in the inset. (A, right) Current-voltage relationships of peak INa with maximal inward currents at −20 mV (n = 4). Normalized peak amplitudes of INa to the maximal value were plotted as a function of the membrane potential. (B) Inward currents (blue) were substantially reduced by replacing Na+ ions with NMDG (top, red) or inclusion of TTX at 20 μM (bottom, red), and the addition of CoCl at 4 mM abolished the residual currents (top and bottom, black).