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Introduction Thoracic aortic aneurysms (TAAs) are life-threatening conditions currently primarily managed surgically. Our aim is to model Marfan syndrome using patient iPSCs-derived vascular smooth muscle cells (vSMCs) bearing pathogenic variants in FBN1 with the intention of studying phenotypic changes to enable early disease detection and therapeutic intervention. Previously, using Marfan iPSC-vSMCs we showed that pathogenic variants affected fibrillin deposition and increased matrix proteolytic activity compromising its integrity (Granata et al, Nat Gen 2016).Methods In this study we cultured Marfan vSMCs on substrates with either physiological or pathological stiffness. We used bulk transcriptomics to analyse two Marfan lines against two WT controls. We re-analysed single-cell transcriptomic dataset GSE186845 for phenotype switching signatures (Pedroza et al, ATVB, 2020) and spatial transcriptomic data of human outflow tract development (Lázár, E. et al, Nat Genetics, 2020).Results and Implications Disease-like phenotypic switching events mediated through Klf4 were detected in vitro, characterized by an upregulation of chondrogenic markers across two Marfan patient lines, compared to two WT lines. Consequently, we hypothesized that the aberrant FBN1 matrix deposited by Marfan SMCs would be stiffer due to it being a cartilage-like extracellular matrix (ECM). Indeed, even on soft hydrogels (12kPa), within Marfan SMCs YAP1 was localized to the nucleus whilst still being cytoplasmic in WT cells thus indicating increased stiffness-sensing. This finding is supported by upregulation in Marfan vSMCs of cartilaginous matrix protein transcripts (MATN3, DCN, COMP, LUM), elastin anchoring EFEMP1, enhanced stiffness-compensatory secretion of extracellular matrix modifiers (ADAMTSs and MMPs), and proteoglycans (MGP, OGN, HSPG) likely to soften the matrix. Marfan MgR mice aortas also showed increased nuclear YAP1 compared to WT mice. The chondrogenic markers which we identified were mapped onto the Marfan mouse Fbn1C1041G/+ aortic aneurysm single cell transcriptomic dataset (Pedroza et al, ATVB, 2020) and confirmed the upregulation of chondrogenic process in vivo as well. This was also validated by our own study which showed increased percentages of positive cell populations of chondrogenic and phenotype switching markers Foxc1 and KLF4 at aortic root of Marfan mice Fbn1 (C1039G/+) compared to WT mice. Aortic calcification has been associated with elastin breaks in mouse model Fbn1 (C1039G/+) (Wanga et al, Journal of Pathology, 2017) suggesting that in time the cartilage deposition may progress to ossification.We hypothesize that Marfan cells are unable to regulate this developmental aortic root/valve chondrogenic program as shown by the persistence of chondrogenic transcription factors such as Sox9, FOSB, FOXD1 and FOXC1 as well as relevant proteoglycans. Wnt/β-catenin signaling pathway inhibits Sox9-driven chondrogenic program in adult valve interstitial cells, with failure of this process leading to chondrogenic nodules in aortic valves (Fang et al, ATVB 2014). We previously reported that Wnt signaling is repressed in MFS-iPSC VSMCs and a drug screen identified that GSK3β inhibitors could ameliorate proteolysis across 4 Marfan patient lines (Daavapil et al, Stem Cell Reports 2023). We are currently testing the possibility to control the chondrogenic phenotype switch in vitro using GSK3β inhibitors.Studies are ongoing to define the links between aberrant extracellular FBN1 and modulation of Wnt signalling by quantifying of ECM collagen and proteoglycan deposition and turnover along with Wnt status. However, our current findings establish aberrant chondrogenic switching as a potential disease mechanism and therapeutic target for early intervention in Marfan syndrome.