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SB431542 conditioned hASCs-derived extracellular vesicles enhance in vitro and in vivo osteogenesis via a miR20-a-5p-BAMBI-BMP/Wnt signaling axis

ABSTRACT: Critical-sized bone defects resulting from tumors, trauma, or surgery present a significant clinical challenge due to its poor self-regenerating capabilities and limited therapeutic options. Mesenchymal stem cells (MSCs) have shown promise in bone regeneration, possibly mediated through their secretion of bioactive factors rather than direct engraftment and differentiation. MSC-derived extracellular vesicles (EVs) deliver micro-RNAs (miRs), proteins and lipids that may modulate bone regeneration. EVs were isolated by differential ultracentrifugation from osteogenically induced human adipose tissue-derived MSCs (hASCs), treated with or without the small molecule SB431542, an inhibitor of TGFß-signaling (EV-hASCs[-/+]SB). EVs were characterized using Western Blot and Nanoparticle Tracking Analysis. hASCs were cultured with EV-hASCs[-/+]SBs to assess bone regeneration in vitro. Bone regeneration was assessed in vivo in calvarial defects treated with (EV-hASCs[-/+]SB). EV content was analyzed via miR-analysis and gain- and loss-of-function transfection assays were performed with identified miR. Enhanced osteogenesis is shown in vitro and in vivo upon treatment with EV-hASCs[+]SB. EV-hASCs[-/+]SB-enriched miR-20a-5p was identified as a modulating factor in osteogenesis, silencing BAMBI and enhancing early downstream phosphorylation of SMAD1/5 and late activation of ß-Catenin in ASCs undergoing osteogenic differentiation, targets of BMP- and Wnt-signaling pathways. EVs act as carriers for miR-mediated paracrine signaling, harnessing the therapeutic potential of MSCs, while circumventing the high immunogenicity and risk of neoplastic transformation associated with cell-based therapies.

ORGANISM(S): Homo sapiens

PROVIDER: GSE286905 | GEO | 2026/04/30

REPOSITORIES: GEO

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Project description:Human mesenchymal stem/stromal cells (hMSCs) hold significant potential for regenerative medicine due to their anti-inflammatory and pro-angiogenic secretome. In particular, 3D hMSC aggregates secrete EVs that have enhanced anti-inflammatory and immunomodulatory properties. However, safety and efficacy concerns associated with stem cell therapy have led to the discovery of cell-free approaches utilizing hMSC-derived extracellular vesicles (EVs). Despite their therapeutic promise, the clinical application of hMSC-EVs is limited by low production yield. This study investigates the scalable generation of hMSC-EVs from 3D aggregates in a novel Vertical-Wheel Bioreactor (VWBR) through shear stress-mediated biochemical cues, promoting cargo expression relevant to nerve regeneration and neuropathic treatment. Bone marrow-derived hMSCs were cultured as 3D aggregates in VWBRs and exposed to two different culture media—αMEM/FBS (serum-containing) and DMEM/F12/B27 (serum-free)—under three agitation speeds (25, 40, and 64 rpm). Metabolite and gene analyses were performed to assess EV biogenesis, focusing on ESCRT machinery markers. Results showed that hMSCs cultured in VWBR exhibited higher expression of EV biogenesis genes and glycolytic genes compared to static culture. αMEM/FBS (serum-containing) condition was more robust than DMEM/F12/B27 (serum-free) condition. EV yield (EV number per cell) increased by 3-10 fold (in serum-containing medium) in VWBR compared to static culture, with size of 120-180 nm and EV marker expression. microRNA-sequencing of the EVs shows the upregulation of miR-29a-3p, miR-451a, miR-224-5p, miR-16-5p, miR-133a-3p, and miR-143-3p, etc., reflecting a combination that enhances EV biogenesis, promotes metabolic reprogramming, and supports immunomodulatory behavior characteristics. Functional assays demonstrated that the generated EVs effectively modulated Schwann cell responses under neural inflammation, supporting their therapeutic potential. These findings highlight the role of VWBR-driven hydrodynamics in promoting EV production from 3D hMSC aggregates. This study advances the knowledge of dynamic aggregation and metabolic influence on 3D hMSC-EV production and fundamentally scale up the preconditioned techniques used to promote hMSC aggregation and the consequent EV secretion.

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SB431542 conditioned hASCs-derived extracellular vesicles enhance in vitro and in vivo osteogenesis via a miR20-a-5p-BAMBI-BMP/Wnt signaling axis

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