Contributions of <i>BMPR2</i> Mutations and Extrinsic Factors to Cellular Phenotypes of Pulmonary Arterial Hypertension Revealed by Induced Pluripotent Stem Cell Modeling
Authors: Fedir N. Kiskin, C-Hong Chang, Christopher J. Z. Huang, Baraa Kwieder, Christine Cheung, Benjamin J. Dunmore, Felipe Serrano, Sanjay Sinha, Nicholas W. Morrell, Amer A. Rana
Journal: American Journal of Respiratory and Critical Care Medicine (2018)

Reduced BMPR2 (bone morphogenetic protein receptor 2) signaling is central to the pathobiology of pulmonary arterial hypertension (PAH).However, the reduced penetrance of BMPR2 mutations in families suggests that other factors are required to establish disease (1).To date, it has proved difficult to elucidate these factors because of a lack of appropriate models.Sa and colleagues (2) developed an induced pluripotent stem cell (iPSC)-derived endothelial cell (iPSC-EC) model of PAH that recapitulated some of the previously described phenotypes of patient-derived pulmonary artery endothelial cells (PAECs), as well as appropriate responsiveness to Elafin and FK506 (2).This demonstrated a potential utility of iPSCs in modeling PAECs in PAH.However, other phenotypes such as inner mitochondrial membrane (IMM) hyperpolarization could not be recapitulated.Therefore, there is a need to better understand the contribution of BMPR2 mutations to PAH-associated phenotypes and the requirement for other factors in this process.Two advantages of iPSCs in disease modeling are their amenability to genome editing and their differentiation into specific cell types under serum-free, chemically defined conditions.This allows the assessment of the effect of a BMPR2 mutation without the confounding effects of genetic differences between cell lines, and the determination of the effect of controlled exposure to extrinsic factors that may influence the acquisition of a diseased state.In addition, no iPSC-smooth muscle cell (SMC) model of PAH has yet been described.We have addressed these issues. MethodsUsing 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 1/2 ) or a deletion of exon 1 (C2 DExon1) 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-b1 (transforming growth factor b 1) and PDGF-BB (plateletderived growth factor BB) 6 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) 6 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 TNFa (tumor necrosis factor a). 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.