Isolation and characterization of human embryonic stem cell-derived heart field-specific cardiomyocytes unravels new insights into their transcriptional and electrophysiological profiles

Abstract

Aims: We prospectively isolate and characterize first and second heart field- and nodal-like cardiomyocytes using a double reporter line from human embryonic stem cells. Our double reporter line utilizes two important transcription factors in cardiac development, TBX5 and NKX2-5. TBX5 expression marks first heart field progenitors and cardiomyocytes while NKX2-5 is expressed in nearly all myocytes of the developing heart (excluding nodal cells). We address the shortcomings of prior work in the generation of heart field-specific cardiomyocytes from induced pluripotent stem cells and provide a comprehensive early developmental transcriptomic as well as electrophysiological analyses of these three populations.

Methods and results: Transcriptional, immunocytochemical, and functional studies support the cellular identities of isolated populations based on the expression pattern of NKX2-5 and TBX5. Importantly, bulk and single-cell RNA sequencing analyses provide evidence of unique molecular signatures of isolated first and second heart field cardiomyocytes, as well as nodal-like cells. Extensive electrophysiological analyses reveal dominant atrial action potential phenotypes in first and second heart fields in alignment with our findings in single-cell RNA sequencing. Lastly, we identify two novel surface markers, POPDC2 and CORIN, that enable purification of cardiomyocytes and first heart field cardiomyocytes, respectively.

Conclusions: We describe a high-yield approach for isolation and characterization of human embryonic stem cell-derived heart field-specific and nodal-like cardiomyocytes. Obtaining enriched populations of these different cardiomyocyte subtypes increases the resolution of gene expression profiling during early cardiogenesis, arrhythmia modelling, and drug screening. This paves the way for the development of effective stem cell therapy to treat diseases that affect specific regions of the heart- or chamber-specific congenital heart defects.

Keywords: Cardiac differentiation; Corin; Electrophysiology; Heart field-specific cardiomyocytes; hESCs.

Graphical abstract

Figure 1

Generation and transcriptional profiling of heart field specific progenitors. (A) Schematic representation of the cardiomyocyte differentiation strategy used. (B) Flow cytometric analysis and fluorescent imaging of TdTomato (TdT) expression in differentiating hESCs at Days 0, 7, and 10. Scale bar = 200 µm. (C) Immunocytochemistry for cardiac progenitor TBX5, cardiac markers TNNT2 and ACTN2 on Days 10 and 60 of differentiation. Scale bar = 20 µm. (D) Heatmap and (E) bar plot of select genes from cardiac contraction, second heart field, pluripotency, and cell type specific gene groups in undifferentiated Day 0 hESCs and Day 7 TBX5⁺ and TBX5⁻ cells (n = 3 biological replicates per group), TPM = transcripts per million. (F) Flow analysis of intracellular TNNT2⁺ within TBX5⁺ or TBX5⁻ populations. (G) Immunocytochemistry of TNNT2 and ACTN2 in sorted TBX5⁺ and TBX5⁻ cells. Scale bar = 100 µm. Statistical analysis is performed by using one-way ANOVA, *P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = not significant. Error bars = SEM.

Figure 2

Transcriptional profiling of FHF- and SHF-like CMs at different cardiac differentiation stages. (A) Schematics and flow cytometric plots showing the cell sorting strategy for TBX5⁺ and TBX5⁻ populations at Days 7 (CPCs) and 10 (CMs) of differentiation. (B) Heatmap comparing cardiac contraction and second heart field related genes in TBX5⁺ and TBX5⁻ CPCs (Day 7) and CMs (Day 10). Violin plots of TNNT2 and ISL1 (right) of Days 7 and 10 populations (n = 3 biological replicates per group, Student’s t-test, * P < 0.05, ** P < 0.01, TPM = transcripts per million). (C) GO analysis of pathways enriched in Day 7 TBX5⁻ CPCs (top) and Day 10 TBX5⁻ NKX2-5⁺ CMs (bottom). (D) GO analysis of pathways enriched in Day 7 TBX5⁺ CPCs (top) and Day 10 TBX5⁺ NKX2-5⁺ CMs (bottom). (E) Optimization of FHF and SHF cardiomyocyte populations via small-molecule modulation of Wnt signalling and cell seeding densities. Representative flow cytometric analysis at Day 10 of cardiac differentiation showing the expression of TBX5 and NKX2-5. Column represents varying seeding densities and row shows varying concentrations of CHIR99021 (CHIR). Inset tables show results from all combinations of seeding densities and CHIR concentrations. Yellow, green, and red highlighted boxes represent the maximum percentage of TBX5⁺NKX2-5⁺, TBX5⁻NKX2-5⁺, and TBX5⁺NKX2-5⁻ cells obtained, respectively (pool of three biological replicates).

Figure 3

Characterization of hESC-derived FHF-, SHF-, and nodal-like cardiomyocytes. Fluorescence microscopy and representative flow cytometry plot of (A) TBX5⁺NKX2-5⁺ (FHF), (B) TBX5⁻ NKX2-5⁺ (SHF), and (C) TBX5⁺NKX2-5⁻ (nodal) optimized differentiations at Day 10 (pool of three biological replicates). Scale bar = 100 μm. Quantitative expression analysis of Day 10 sorted (D) FHF-, (E) SHF-, and (F) nodal-like CMs (n = 3 biological replicates, three technical replicates each) (left) and isolated human foetal left and right ventricles (LV and RV, respectively) (right) (n = 1 biological replicates, three technical replicates each) for FHF (TBX5 and HAND1), SHF (ISL1 and TBX1), and nodal genes SHOX2, HCN4, and TBX18. Immunocytochemistry analysis of Day 10 sorted (G) FHF- and (H) SHF-like, and (I) nodal-like cardiomyocytes for specific markers of FHF (TBX5), SHF (ISL1), and nodal cells (SHOX2). Presence of cardiomyocytes is indicated by expression of TNNT2 and all nuclei were visualized with DAPI. Scale bars = 20 µm. Statistical analysis is performed by using one-way ANOVA and Student’s t-test, *P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Error bar = SEM.

Figure 4

Single-cell RNA sequencing of FHF- and SHF-like cardiomyocytes. (A) Schematic of single cell capture using 10X Genomics. (B) UMAP showing cells labelled according to their input identity (FHF-like CMs: yellow, SHF-like CMs: green) (pool of three biological replicates, statistical analysis is performed by using Student’s t-test, *P < 0.05, **** P < 0.0001.). UMAP and boxplots showing gene expression quantification for the (C) cardiac contractile genes TNNT2, ACTN2, and MYH6, (D) atrial genes MYL7, DKK3, and NPPA, (E) ventricular genes MYL2, FHL2, and IRX4, and (F) nodal genes SHOX2, TBX18, and KCNJ3. (G) Heatmaps from bulk RNA sequencing of Day 20 FHF and SHF populations showing genes corresponding to (C–F) (n = 3 biological replicates per group).

Figure 5

Electrophysiological analyses of FHF- and SHF-like cardiomyocytes. Snapshots of optical mapping show spontaneous AP which propagates across monolayer of (A) FHF-like CMs and (B) islands of SHF-like CMs (pool of three biological replicates). The yellow arrow denotes direction of AP propagation. Numbers denote the area where APs were recorded. Red and pink represent depolarized and repolarized phases, respectively. (C) APD₉₀ histogram of APs recorded from FHF- (n = 6,660 pixels) and SHF-like CMs (n = 4,750 pixels). These histograms are superimposed on the right. (D) AP parameters of FHF- and SHF-like CMs (values are mean ± SD, statistical analysis is performed by using Student’s t-test, ** P<0.01, *** P<0.001. APA was calculated from patch-clamp studies; all others were calculated from optical mapping experiments). Each pixel size = 10 x 10 µm. Beating rates of (E) FHF- and (F) SHF-like cardiomyocytes following application of β–adrenergic agonist (isoproterenol, Iso, 1 μM) and antagonist (propranolol, Prop, 10 μM) (n = 10 biological replicates).

Figure 6

Isolation and enrichment of FHF-like CMs using the surface marker CORIN. (A) Heatmap showing co-expression of cardiac contraction and surface marker genes in TBX5⁺ and TBX5⁻ populations at different time points (three biological replicates per group). (B) CORIN and POPDC2 expression assessed using the (top) Kaessmann online database (one-way ANOVA, P-value (heart vs. other tissues, <0.0001), biological replicates: brain (n = 52), cerebellum (n = 58), heart (n = 44), kidney (n = 36), liver (n = 49), ovary (n = 18), testis (n = 39) for organ specificity, (middle) single-cell RNA sequencing analysis of human foetal heart (10-week gestation) for CM specificity (one biological replicate), and (bottom) Day 20 FHF- and SHF-like CMs for heart field specificity (pool of three biological replicates). (C) qPCR analysis of CORIN expression in (left) FHF and SHF-like CMs (three biological replicates, three technical replicates each) and (right) human foetal LV and RV (18-week gestation, one biological replicate, three technical replicates each). (D) Flow cytometric analysis of CORIN⁺ cells for TNNT2 expression (pool of 3 biological replicates). (E) Flow cytometric analysis of TBX5 and NKX2-5 in CORIN⁺ and CORIN⁻ cells (pool of three biological replicates) using FHF (top) and SHF (bottom) protocols. (F) Quantitative PCR analysis of FHF (TBX5) and SHF (ISL1) in CORIN⁺ and CORIN⁻ Day 10 sorted cells (three biological replicates, three technical replicates each). (G) Immunocytochemistry of Day 10 sorted CORIN⁺ CMs for markers of FHF (TBX5), SHF (ISL1), and nodal cells (SHOX2). Cardiomyocytes are shown by TNNT2 staining and all nuclei were shown by DAPI. Scale bar = 20 μm. (H) Snapshots of optical mapping show spontaneous AP propagation across monolayer of CORIN⁺ CMs. The yellow arrow denotes direction of AP propagation. Numbers denote the area from which the APs were recorded. Red and pink represent depolarized and repolarized phases, respectively (pool of three biological replicates). Statistical analysis is performed by using Student’s t-test, *P < 0.05, **** P < 0.0001, ns = not significant. Error bar = SEM.