PMC

Generation of pulmonary artery smooth muscle cell (PASMC)-like induced pluripotent stem cell (iPSC)-derived smooth muscle cells (SMCs). (A) Wild-type and BMPR2^+/− iPSCs differentiated into iPSC-SMCs from lateral plate mesoderm (LM), paraxial mesoderm (PM), and neuroectoderm (NE) express SMA (smooth muscle actin) and calponin (green), (DAPI; blue) (scale bars, 20 μm). (B) Gene expression patterns of all samples (human PASMCs, PM-SMCs, NE-SMCs, and LM-SMCs) were analyzed using Illumina HumanHT-12 v4 Expression BeadChip microarrays. Gene expression patterns of samples were sorted based on similarity by hierarchical clustering. For this analysis, 171 SMC-specific genes were selected based on the wikipathway database (WikiPathway WP2064 revision 47071). The two-dimensional principal component analysis for differential gene expression is plotted in B. The red squares inside black boxes represent PASMCs, dark blue squares represent LM-SMCs, green squares represent NE-SMCs, and light blue squares represent PM-SMCs. (C) LM-SMCs are not growth suppressed by BMP4 (50 ng/ml), unlike PM- and NE-SMCs. (D) The apoptotic response in wild-type LM-SMCs is reduced in the presence of exogenous BMP4 (50 ng/ml), as previously described for distal PASMCs. Data in C and D are presented as mean ± SEM of three biological replicates (*P < 0.05; ***P < 0.001; one-way ANOVA [C] and Student’s t test [D]). BMP = bone morphogenetic protein; BMPR = BMP receptor; FITC = fluorescein isothiocyanate.

Effect of BMPR2 (bone morphogenetic protein receptor 2) heterozygosity on proliferation, apoptosis, and inner mitochondrial membrane (IMM) polarization of induced pluripotent stem cell (iPSC)-derived smooth muscle cells (SMCs) and iPSC–endothelial cells (ECs). (A and B) C2 W9X^+/− iPSC-SMCs are (A) significantly less apoptotic and (B) more proliferative than wild-type C2 iPSC-SMCs under serum-free conditions, assessed using the Caspase-3/7 Glo assay (A) and by measuring double-stranded DNA content by Vybrant DyeCycle Ruby staining (B). (C and E) In contrast, there was no significant difference in (C) apoptosis and (E) proliferation between C2 and C2 W9X^+/− iPSC-ECs under serum-free conditions. (D and F) C2 W9X^+/− iPSC-ECs became significantly more (D) apoptotic and (F) proliferative relative to isogenic wild-type C2 iPSC-ECs after exposure to 10% FBS for 1 week. (G) Serum-free BMPR2^+/− iPSC-SMCs display a hypopolarized IMM compared with isogenic wild-type cells, assessed using tetramethylrhodamine ethyl ester (TMRE) staining and flow cytometry. (H and K) BMPR2^+/− iPSC-SMCs became hyperpolarized compared with isogenic wild-type cells (H) after 2 weeks of exposure to serum or (K) after 1 week of exposure to serum and TNFα (tumor necrosis factor α) (1 ng/ml). (L) After 1 week of exposure to serum + TNFα, TNFα was removed and all cells were cultured for 1 further week in serum only to see whether the polarization state would recover. The polarization state of BMPR2^+/− iPSC-SMCs did not normalize and was significantly higher than exposure to serum only for 2 weeks, and was also significantly higher than in isogenic wild types treated the same way. (I and J) BMPR2^+/− iPSC-ECs (I) do not have a hyperpolarized IMM relative to the isogenic wild-type under serum-free conditions but (J) become significantly hyperpolarized after exposure to serum for 1 week. Wild-type iPSC-ECs showed significantly higher TMRE fluorescence after TNFα treatment (1 ng/ml), suggesting that TNFα increases IMM hyperpolarization. Cotreatment of C2 W9X^+/− iPSC-ECs with TNFα (1 ng/ml) and BMP9 (1 ng/ml) for 1 week resulted in significantly reduced TMRE fluorescence, and hence IMM polarization, compared with the effect of TNFα alone. Data are presented as mean ± SEM of the results from three independent differentiations (A–F) and three technical replicates per differentiation (*P < 0.05; **P < 0.01; ***P < 0.001; Student’s t test [A–F], one-way ANOVA [G–I], or two-way ANOVA [J–L]). AU = arbitrary units; BMP9 = bone morphogenetic protein 9; C2 = control 2, patient cell line; C2 δExon 1 = C2 carrying deletion of exon 1 in the gene BMPR2; C2 W9X = C2 carrying a W9X mutation in the gene BMPR2; FBS = fetal bovine serum; n.s. = not significant.