Contributions of BMPR2 Mutations and Extrinsic Factors to Cellular Phenotypes of Pulmonary Arterial Hypertension Revealed by Induced Pluripotent Stem Cell …

FN Kiskin, CH Chang, CJZ Huang… - American Journal of …, 2018 - atsjournals.org
FN Kiskin, CH Chang, CJZ Huang, B Kwieder, C Cheung, BJ Dunmore, F Serrano, S Sinha
American Journal of Respiratory and Critical Care Medicine, 2018atsjournals.org
Methods Using clustered regularly interspaced short palindromic repeats–Cas9–mediated
homologous recombination in a wild-type iPSC line, two isogenic sublines carrying either a
known causal BMPR2 mutation (W9X; referred to as C2 W9X+/−) or a deletion of exon 1 (C2
ΔExon1) were generated. Serum-free, chemically defined iPSC differentiation protocols
were used to generate iPSC-derived SMCs (iPSC-SMCs) and iPSC-ECs. This was achieved
by differentiating iPSCs into iPSC-SMCs via a lateral plate mesoderm, paraxial mesoderm …
Methods
Using clustered regularly interspaced short palindromic repeats–Cas9–mediated homologous recombination in a wild-type iPSC line, two isogenic sublines carrying either a known causal BMPR2 mutation (W9X; referred to as C2 W9X+/−) or a deletion of exon 1 (C2 ΔExon1) were generated. Serum-free, chemically defined iPSC differentiation protocols were used to generate iPSC-derived SMCs (iPSC-SMCs) and iPSC-ECs. This was achieved by differentiating iPSCs into iPSC-SMCs via a lateral plate mesoderm, paraxial mesoderm, or neural ectoderm lineage followed by 12 days in TGF-β1 (transforming growth factor β 1) and PDGF-BB (platelet-derived growth factor BB)±BMP4 (bone morphogenetic protein 4)(Figure 1A)(3), and into ECs via FGF-2 (fibroblast growth factor 2)–induced, BMP4-induced, and LY294002-induced mesoderm followed by FGF-2 and VEGF (vascular endothelial growth factor)±BMP4. iPSC-SMCs were compared with adult distal and proximal pulmonary artery smooth muscle cells (PASMCs) by microarray analysis. Cells were used postdifferentiation and in chemically defined conditions, and exposed to additional factors such as serum, BMP4, and TNFα (tumor necrosis factor α). Key PAH-associated cellular phenotypes, including altered apoptosis (via caspase cleavage and annexin/propidium iodide staining), proliferation (via DNA content and cell counts), and IMM polarization (via tetramethylrhodamine ethyl ester staining), which are cellular changes common to both SMCs and ECs (4), were assessed.
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