Endothelial Cells/Pericytes Co-culture Model and Human Blood Vessel Organoids: Modeling Vasculopathy in Systemic Sclerosis

Background and Objectives: Systemic sclerosis (SSc) is a complex autoimmune disease characterized by vascular abnormalities and fibrosis. Induced pluripotent stem cells (iPSCs) offer a promising avenue for modeling disease pathogenesis and screening for potential therapeutics. This study aimed to establish a co-culture model of iPSC-derived endothelial cells (ECs) and pericytes and a three-dimensional (3D) iPSC-derived model of blood vessel organoids (BVOs) for the study of vasculopathy in SSc. Methods: iPSCs obtained from healthy donors and SSc patients were directed to differentiate into ECs and pericytes using a combination of mesoderm and vascular differentiation media. Immunofluorescence staining was employed to assess the expression of EC and pericyte markers. Coculturemodels of ECs and pericytes were exposed to serum from healthy donors and SSc patients with or without active digital ulcers to evaluate angiogenesis. Furthermore, 3D BVOs were generated from both healthy and SSc-iPSCs and treated with serum from healthy individuals and SSc patients with or without active digital ulcers to assess vasculopathy. Vascular morphology of the organoids was captured using LSM880 confocal microscopy, and parameters such as vessel density, average vessel length, total vessel length, and junctions were quantified using Angiotool to evaluate angiogenesis. RNA-seq analysis was conducted to identify phenotypic differences at a transcriptomic level between SSc BVOs treated with healthy serum and those with active digital ulcers. Results: iPSCs derived from both healthy donors and SSc patients were successfully differentiated into ECs and pericytes, with comparable efficiency between the two groups. Co-culture models derived from SSc-iPSCs demonstrated augmented angiogenesis under healthy serum conditions compared to healthy co-culture models, but diminished neovascularization when exposed to serum from SSc patients with active digital ulcers. Moreover, BVOs derived from SSc-iPSCs exhibited impaired angiogenesis when treated with serum from SSc patients with active digital ulcers. RNA-seq analysis revealed differentially expressed genes associated with inflammation, immune response, adhesion, migration, and angiogenesis in SSc BVOs treated with serum from SSc patients with active digital ulcers in comparison to SSc BVOs treated with serum from healthy donors. Conclusion: Our study demonstrates that SSc-iPSC-, but not healthy-iPSC-derived vascular models, i.e. the EC-pericyte co-culture and BVOs, reproduce the aberrant vascular phenotype in SSc patients when exposed to SSc serum from patients with severe, clinically manifest vascular pathology. This provides first experimental evidence that clinically manifest vascular damage in SSc can be induced by circulating serum factors only in genetically predisposed individuals. The aberrant gene expression profile identified offers insights into the mechanisms driving SSc-associated vasculopathy, thereby providing potential avenues for therapeutic intervention in systemic sclerosis.