Figure 4. Time course single cell RNA-sequencing of SANCM.

(A) Timeline of hiPSC differentiation to SANCM representing sample collection time points. (B) UMAP representation of single cell transcriptomes collected at different time points throughout differentiation from hiPSC to SANCM. Arrow indicates course of differentiation. (C) UMAP feature plots and violin plots showing TNNT2 and ACTN2 gene expression at different stages of SANCM differentiation. (D–H) Violin plots of pluripotency genes (D), mesodermal genes (E), posterior cardiac progenitor genes (F), proepicardial genes (G), and SAN-associated transcription factor genes (H). hiPSCs, human induced pluripotent stem cells; CPC, cardiac progenitor cells; CMs, cardiomyocytes; UMAP, uniform manifold approximation and projection; SANCM, sinoatrial node-like cardiomyocyte. Also see Figure 4—figure supplement 1 and Figure 4—figure supplement 2.

Figure 4—figure supplement 1. Time course single cell RNA-sequencing of sinoatrial node-like cardiomyocyte (SANCM).

(A) Heatmap showing top 10 differentially expressed genes of all clusters identified before filtering low-quality cells. Two clusters with low-quality/ERCC-spike-in DNA-enriched transcriptomes (spike_in_1 and spike_in_2) were excluded from the analysis. (B) Uniform manifold approximation and projection (UMAP) showing the original identifier of each collection time point. (C) Violin plots depicting expression of endoderm-associated genes in cluster D4_2. (D) UMAP depicting annotation of cluster 7 (sinus venosus-like cluster, Figure 3A) on entire time series dataset. Majority of cluster 7 (Figure 3A) cluster together with cells collected on day 10 (cluster D10_1).

Figure 4—figure supplement 2. Time course RT-qPCR of sinoatrial node (SAN)-subpopulation markers.

(A–C) Time course RT-qPCR of SAN progenitor-enriched genes (A), SAN-head-enriched genes (B) and SAN-tail (KCNIP2) or SAN-TZ-enriched genes (C). N=4 independent differentiations; corrected to GEOMEAN of reference genes RPLP0 and GUSB.