Figure 3.
Functional Significance of LPHN2 during In Vitro Cardiac Differentiation
(A) Gross morphology of control induced pluripotent stem cells (iPSCs) and Lphn2-knockdown (KD) iPSCs transduced by control-shRNA and Lphn2-shRNA, respectively. No differences were observed in the colony formation or EB formation. Scale bar, 200 μm. EB, embryoid body.
(B) Effects of Lphn2 KD on cardiac differentiation. Transcriptional profiling of Lphn2, Nanog, Oct3/4, Flk-1, Mesp1, Nkx2.5, Myh6, and cTnT in control iPSC- and Lphn2-KD iPSC-derived cells by qPCR. ∗p < 0.05, ∗∗p < 0.01, N.S., not significant, one-way ANOVA and post hoc Bonferroni test; n = 3 independent replicates.
(C) Quantification of spontaneous beating foci in control and Lphn2-KD cells on day 12 of cardiac differentiation. ∗p < 0.05, Mann-Whitney U test; n = 4 independent replicates.
(D) Immunostaining for LPHN2 (green) expression in wild-type embryonic stem cell (WT-ESC) and Lphn2-knockout (KO) ESC-derived cells on day 7 of cardiac differentiation. Blue, nuclear counterstaining with DAPI. Scale bar, 50 μm.
(E) WT-ESCs express LPHN2 (green) and α-SA (red) with striations at day 14 after differentiation, indicating cardiomyocyte differentiation, but Lphn2-KO ESCs did not differentiate into cardiomyocytes. Blue, nuclear counterstaining with DAPI. Scale bar, 20 μm.
(F and G) qPCR analysis of gene expression of cardiac-specific Mesp1, Nkx2.5, and cTnT after stimulation with presumptive ligands. Treatment with α-latrotoxin (F) and FLRT3 (G) did not increase the expression of cardiac lineage genes in mESCs or Lphn2-KO ESCs after 7 days under cardiac differentiation conditions. Values are shown relative to the expression in WT-mESCs on day 0. N.S., not significant, one-way ANOVA and post hoc Bonferroni test; n = 3 independent replicates. UD, undifferentiated; Veh, vehicle.