Application of Fibronectin-Coated Microparticles for Isolation of Human iPSC-Derived MSCs

Abstract

Abstract BACKGROUND : Bone marrow–derived MSCs (BMMSCs) are limited by availability, donor variability, and age-related decline, highlighting the need for alternative MSC sources. Induced pluripotent stem cells (iPSCs) offer a scalable solution. This study introduces a novel three-dimensional culture platform based on fibronectin (FN)-coated microparticles to efficiently derive and expand human iPSC-derived MSCs (iMSCs). METHODS : The system utilizes FN-coated non-porous (CytoDex) and porous (CytoPore) microparticles. FN coating efficacy on microparticle was confirmed using Rhodamine-labeled FN and confocal microscope. Base on various molecular cell biological experiment, the utilization of FN-coated CytoDex and CytoPore leverages an expanded surface area to improve iMSCs isolation. The characteristics and cell behaviors of iMSCs generated from FN-coated CytoDex and CytoPore was evaluated by immunophenotype analysis, cell proliferation, and senescence related assays. In vivo tissue regeneration was evaluated by microcomputed tomography and histopathological analyses. RESULTS : The porous microparticle CytoPore significantly enhanced FN coating efficiency, cell attachment, and proliferation compared to CytoDex. FN-coated CytoPore enabled the selective isolation of a high-purity MSC population from spontaneously differentiated iPSCs (SD-iPSCs) by day 7, confirmed by distinct morphology and mesodermal marker expression. The resulting iMSCs exhibited immunophenotypic characteristics comparable to adult MSCs, along with superior proliferative capacity, extended telomere length, and minimal senescence over 10 passages, in contrast to BMMSCs. Furthermore, iMSCs demonstrated effective in vivo tissue regeneration in an osteochondral defect model. CONCLUSION : This novel FN-coated microparticle-based 3D culture platform enables efficient, large-scale production of high-quality iMSCs and holds strong potential for clinical applications in stem cell therapeutics and regenerative medicine.