Figure 1.

hPSC-Derived HVPs by Modulation of Wnt Signaling via Small Molecules

(A) Schematic protocol for defined differentiation of hPSCs to ventricular progenitors. (B) Brachyury expression was detected on day 1. (C) MESP1 expression was detected on day 3 with flow cytometry. (D and E) 14 days after initiation of hPSC differentiation using the protocol shown above, cells were analyzed for MLC2v (D) and cTnT (E) expression by flow cytometry. (F) Protein expression was assessed by western blot at different time points during differentiation. (G) RNA-seq analysis of the rapid and synchronous HVP generation process. Gene expression profiles projected onto the first two principal components. Cells differentiated from different days are designated by colors. (H) Cluster analysis of gene expression profiles at different time points during HVP differentiation identifies stage-specific signature genes. These genes were clustered hierarchically on the basis of similarity of their expression profiles. Expression pattern of each gene is displayed as a horizontal strip. The intensity of the red and blue color is proportional to the relative gene induction (red) or repression (blue). ES, embryonic stem cells stage; MES, mesoderm stage; CMES, cardiac mesoderm stage; CPC1 and CPC2, cardiac progenitor stages; CM1 and CM2, cardiomyocyte stages; Rnd1–4, unclassified groups. (I) Venn diagram displaying number of genes in each category, with 5 candidate cell surface marker genes (JAG1, FZD4, FGFR3, LIFR, and TNFSF9) satisfying all 4 categories. (J) Flow cytometry analysis of the 5 candidate genes showed that LIFR is highly expressed (>87%) on day 6 of differentiation. (K) Flow cytometry analysis revealed that >86% of D6 cells co-express LIFR and ISL1. (L) Schematic diagram illustrating leukaemia inhibitory factor (LIF) binding to heterodimers of LIF receptor and gp130. LIFR-gp130 heterodimers can also associate with other receptor subunits to bind CT-1.