A Human iPSC Double-Reporter System Enables Purification of Cardiac Lineage Subpopulations with Distinct Function and Drug Response Profiles

Abstract

The diversity of cardiac lineages contributes to the heterogeneity of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs). Here, we report the generation of a hiPSC TBX5^Clover2 and NKX2-5^TagRFP double reporter to delineate cardiac lineages and isolate lineage-specific subpopulations. Molecular analyses reveal that four different subpopulations can be isolated based on the differential expression of TBX5 and NKX2-5, TBX5+NKX2-5+, TBX5+NKX2-5-, TBX5-NKX2-5+, and TBX5-NKX2-5-, mimicking the first heart field, epicardial, second heart field, and endothelial lineages, respectively. Genetic and functional characterization indicates that each subpopulation differentiates into specific cardiac cells. We further identify CORIN as a cell-surface marker for isolating the TBX5+NKX2-5+ subpopulation and demonstrate the use of lineage-specific CMs for precise drug testing. We anticipate that this tool will facilitate the investigation of cardiac lineage specification and isolation of specific cardiac subpopulations for drug screening, tissue engineering, and disease modeling.

Keywords: CORIN; NKX2-5; TBX5; cardiomyocyte subtypes; endothelial cell lineage; epicardial lineage; hiPSC double reporter; human first and second heart field; precise drug testing; purification.

Figure 1.. Isolation of cardiac subpopulations from TBX5^Clover2/NKX2-5^TagRFP hiPSC-derived cells.

(A) Representative images show G+R+ (yellow color), G+R− (asterisk), and G−R+ (arrow) subpopulations. Scale bars, 100 μm. (B) Fluorescence-activated cell sorting (FACS) plots show separation of subpopulations. Numbers in the quadrants represent the respective percentage of subpopulations. (C-D) Percentage of subpopulations from 5 to 16 independent experiments. (E) Gene expression in individual subpopulation isolated on day 10 (n=3). (F) Gene expression in G+R−, G−R+, and G+R+ derivatives on day 35 (n=3). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (mean±SEM). See also Figure S1, Tables S1, S2, and S4.

Figure 2.. Functional characterization of individual subpopulation.

(A) Optical imaging of action potentials (APs) using ASAP2 shows “ventricular-like” (top), “atrial-like” (middle), and “nodal-like” (bottom) hiPSC-CMs between days 35 and 40. (B) Percentage of subtypes in G+R+ and non-sorted hiPSC-CMs. Numbers indicate the recorded cell number. (C) Representative whole cell patch-clamp recordings of G+R+ and non-sorted hiPSC-CMs. (D) Percentage of subtypes in G+R+ and non-sorted hiPSC-CMs by patch-clamp. Numbers indicate the recorded cell number. (E) Maximum diastolic potential (MDP). (F) Expression of ion channel genes (n=3). (G-H) Western blots and quantification of the expression of Kir2.1 (n=3). (I-J) Quantification of contraction velocity and contraction deformation distance (G+R+, n=14; non-sorted, n=33). (K) Seahorse extracellular-flux assays measuring oxygen consumption rate under palmitate condition. (L) Analyses of Seahorse extracellular-flux assay results (n=8). (M) Optical imaging of G+R− and G−R+ hiPSC-CM APs using ASAP2. (N) Percentage of subtypes in G+R− and G−R+ hiPSC-CMs. Numbers indicate the recorded cell number. (O-Q) Quantification of beating rate, AP amplitude, and AP duration (APD) in G+R−, G−R+, G+R+, and non-sorted hiPSC-CMs (G+R+, n=92; G+R−, n=58; G−R+, 68; Non-sorted, n=99). (R) Gene expression of endothelial cell (EC) markers in G−R− cells on day 35 (n=3). (S-T) G−R− cells formed “vasculature-like” structure (S) in vitro and (T) in vivo. Scale bars, 30 μm. *p<0.01, **p<0.01, ***p<0.001, ****p<0.0001 (mean±SEM). See also Figures S2 and S3 and Table S4.

Figure 3.. Transcriptomic analyses of subpopulations.

(A) Principal component analysis (PCA) plots with the computation of the closest neighboring subpopulations. (B) Venn diagram shows the overlap between differentially expressed genes in subpopulations. (C-F) Heatmaps show hierarchical clustering of differentially expressed genes that are enriched in (C) G+R+, (D) G−R+, (E) G+R−, and (F) G−R− with at least 2-fold higher than at least two other subpopulations. Representative upregulated genes are listed in the box. (G-J) GO analyses of upregulated genes in (G) G+R+, (H) G−R+, (I) G+R−, and (J) G−R−. The significance is shown as –Log10FDR. See also Figure S4 and Table S3.

Figure 4.. Application of TBX5^Clover2/NKX2-5^TagRFP hiPSC reporter.

(A) Expression of CORIN in isolated subpopulations, hiPSC-derived endothelial cells (hiPSC-ECs), hiPSC-derived fibroblasts (hiPSC-Fs), and primary cardiac fibroblasts (CFs) (n=3). (B) Flow cytometry of CORIN and MLC-2v in day 30 G+R+ CMs. (C) Expression of CORIN in human fetal tissue. (D-E) Dynamic expression of CORIN during cardiac differentiation in (D) the reporter and (E) unmodified hiPSC-CMs from three independent differentiation. (F) Representative flow cytometry plot of day 10 live cells shows CORIN+ (blue box) and CORIN− (black box) cells. (G) Expression of CORIN, TBX5, and NKX2-5 (n=3). (H-J) Comparable AP properties including beating rate, AP amplitude, and APD between day 30 CORIN+ and G+R+ CMs (CORIN+, n=38; G+R+, n=55). (K) Optical imaging of APs in G+R− hiPSC-CMs with and without treatment of ivabradine or carbamylcholine chloride (CCH). (L-M) The effect of ivabradine and CCH on (L) APD and (M) beating rate (G+R− baseline, n=36; G+R− 1 μM ivabradine, n=21; G+R− CCH, n=19; G+R+ baseline, n=31; G+R+ 1 μM ivabradine, n=20; G+R+ 9 μM ivabradine, n=16; G+R+ CCH, n=22). #, stop beating. (N) Optical imaging of APs in G−R+ hiPSC-CMs with and without treatment of vernakalant. (O-P) The effect of vernakalant on (O) APD and (P) beating rate (G−R+ baseline, n=45; G−R+ vernakalant, n=47; G+R+ baseline, n=25; G+R+ vernakalant, n=34). (Q) Vernakalant induces arrhythmias in G−R+ CMs. Numbers indicate the cell number with and without arrhythmias. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (mean±SEM). See also Table S4.