Abstract 698: Functional Analysis of Smad3 Deficiency in VSMCs Derived From Crispr/cas9-modified Human Induced Pluripotent Stem Cells

Introduction: Transcription factor Smad3 plays key roles in both TGF-β signaling transduction and vascular smooth muscle cells(VSMCs) differentiation. SMAD3 gene mutations cause aneurysms-osteoarthritis syndrome(AOS), an autosomal dominant genetic disease with high rate occurrence of thoracic aortic aneurysm(TAA) and early-onset osteoarthritis. The production of patient-derived hiPSCs and genomic-modified ihPSCs by using Crispr/Cas9 technique provide a promise for in vitro disease modeling of SMAD3 mutation in VSMCs as well as drug screening for potential therapeutic targets. Hypothesis: We hypothesis that introducing SMAD3 mutations into hIPSCs that mimic the identified genotypic alteration in AOS patients could recapture the phenotypic defects of differentiation, contractibility and extracellular matrix(ECM) of VSMCs derived from hIPSCs-differentiated neural crest stem cells(NCSCs), the embryonic origin of VSMCs for both aorta root and ascending aorta. Method: hIPSCs were generated from non-TAA patients’ peripheral blood mononuclear cells by overexpression of Yamanaka factors. The indel mutation targeting SMAD3 exon5 were induced by using Crispr/Cas9 technique, which form frameshift mutation close to one in identified AOS patients. Selected cell clones of genomic modified hIPSCs were differentiated into NCSCs-derived VSMCs. Comparing analysis of VSMCs function were proceed in both hIPSCs lines with or without SMAD3 mutation. Result: We generated a homozygote with SMAD3 frameshift mutation. It contains a 7-base pair deletion(c.633_639delGATGTCC), which is similar with a pathogenic Smad3 mutation (c.652 delA). This mutation leads to a premature stop codon and remove the Smad3 protein. The qRCR and WB result showed that the expression of ACTA2 decreased in the differentiated SMC with SMAD3 mutation. Also, the expression of elastin, collagen type III were significantly down-regulated in the SMAD3 mutation cell line while the MMP9 was up-regulated. Conclusion: SMAD3 mutation may cause defects of differentiation, contractibility and ECM synthesis of VSMCs derived from NCSC.