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bioRxiv · 10.1101/2025.06.11.659155

An iPSC-based iMSC Model for Osteogenesis Imperfecta: A Platform for Disease Modeling and Drug Screening

Kumar, A.·Kumar, V.·Selina, A.·Madhuri, V.·Thamodaran, V.
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Abstract

IntroductionOsteogenesis Imperfecta (OI) is a rare genetic disorder of connective tissue, primarily caused by mutations in the COL1A1 or COL1A2 genes. Research is hampered by the limitations of primary patient-derived cells, which are obtained through invasive methods and have finite proliferative capacity and high variability. While induced pluripotent stem cell (iPSC) models exist, direct differentiation to osteoblasts is often inefficient and may not fully replicate disease characteristics. Our study aimed to develop and characterize a robust iPSC-derived mesenchymal stem cell (iMSC) model of OI using a simple two-step differentiation protocol to serve as a platform for disease modeling and drug screening. MethodsBone marrow MSCs (BMMSCs) were isolated from two OI patients with heterozygous missense mutations, in COL1A1 (c.2299G >A) and COL1A2 (c.982G>A). The patient BMMSCs were reprogrammed into iPSCs using an integration-free Sendai virus. The resulting OI-iPSCs were characterized for pluripotency via immunofluorescence and RT-PCR, trilineage differentiation potential, and karyotype analysis. A two-step protocol was used to differentiate the OI-iPSCs into OI-iMSCs via mesodermal lineage. The OI-iMSCs were then characterized for morphology, immunophenotype by flow cytometry, and trilineage differentiation capacity. Functional osteogenic differentiation was assessed by Alizarin Red S staining and RT-PCR analysis of key osteogenic genes. Sanger sequencing confirmed the retention of patient-specific mutations in the OI-iMSCs. ResultsPatient-derived BMMSCs were successfully differentiated into adipocytes and chondrocytes but showed impaired osteogenic potential. The reprogramming process successfully generated stable OI-iPSC lines that expressed key pluripotency markers, demonstrated trilineage potential, and maintained a normal karyotype. Critically, while direct osteogenic differentiation of these iPSCs failed to recapitulate the primary cell phenotype, the two-step differentiation protocol successfully produced a homogeneous population of iMSCs. These OI-iMSCs displayed characteristics of MSC surface markers (CD73+, CD90+, CD105+) and recapitulated the disease phenotype seen in the original patient BMMSCs. Specifically, COL1A1-iMSCs exhibited cellular rolling and detachment, while COL1A2-iMSCs showed poor mineralization during osteogenic differentiation. Both OI-iMSC lines showed significantly decreased calcium deposition and downregulation of key osteogenic genes (RUNX2, ALP, COL1) compared to controls. ConclusionOur study successfully established an iMSC-based cellular model of OI that recapitulates patient-specific disease phenotypes, including impaired osteogenic differentiation. The two-stage differentiation of iPSCs to iMSCs proved more reliable than direct osteogenic differentiation for modeling the disease. iMSC model circumvents the limitations of primary cells by providing a scalable and homogeneous source of patient-specific cells. Our platform offers a valuable and robust tool for investigating OI pathophysiology and for high-throughput screening of potential therapeutic molecules, advancing efforts toward personalized therapies

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Kumar, A., Kumar, V., Selina, A., Madhuri, V., Thamodaran, V.. 2025-06-16. An iPSC-based iMSC Model for Osteogenesis Imperfecta: A Platform for Disease Modeling and Drug Screening. https://doi.org/10.1101/2025.06.11.659155

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