Multiplexed Molecular Imaging Strategy Integrated with RNA Sequencing in the Assessment of the Therapeutic Effect of Wharton’s Jelly Mesenchymal Stem Cell-Derived Extracellular Vesicles for Osteoporosis

Cheng-Hsiu Lu,1– 3 Yi-An Chen,4,5 Chien-Chih Ke,6– 8 Sain-Jhih Chiu,5 Fong-Shya Jeng,5 Chao-Cheng Chen,4 Ya-Ju Hsieh,6– 8 Bang-Hung Yang,4,9 Chi-Wei Chang,9 Feng-Sheng Wang,2,3,10,11 Ren-Shyan Liu1,4,9,12 1Industrial Ph.D. Program of Biomedical Science and Engineering, National Yang Ming Chiao Tung University, Taipei, Taiwan; 2Core Facility for Phenomics and Diagnostics, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan; 3Department of Medical Research, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan; 4Department of Biomedical Imaging and Radiological Sciences, National Yang Ming Chiao Tung University, Taipei, Taiwan; 5Molecular and Genetic Imaging Core/Taiwan Mouse Clinic, National Comprehensive Mouse Phenotyping and Drug Testing Center, Taipei, Taiwan; 6Department of Medical Imaging and Radiological Sciences, Kaohsiung Medical University, Kaohsiung, Taiwan; 7Drug Development and Value Creation Research Center, Kaohsiung Medical University, Kaohsiung, Taiwan; 8Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan; 9PET Center, Department of Nuclear Medicine, Taipei Veterans General Hospital, Taipei, Taiwan; 10Center for Mitochondrial Research and Medicine, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan; 11Graduate Institute of Clinical Medical Science, Chang Gung University College of Medicine, Kaohsiung, Taiwan; 12Department of Nuclear Medicine, Cheng Hsin Hospital, Taipei, TaiwanCorrespondence: Chien-Chih Ke; Ren-Shyan Liu Email ccke@kmu.edu.tw; rsliuvgh@gmail.comIntroduction: Osteoporosis is a result of an imbalance in bone remodeling. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have been considered as a potentially promising treatment for osteoporosis. However, the therapeutic effect, genetic alterations, and in vivo behavior of exogenous EVs for osteoporosis in mice models remain poorly understood.Methods: A multiplexed molecular imaging strategy was constructed by micro-positron emission tomography (μPET)/computed tomography (CT), μCT, and optical imaging modality which reflected the osteoblastic activity, microstructure, and in vivo behavior of EVs, respectively. RNA sequencing was used to analyze the cargo of EVs, and the bone tissues of ovariectomized (OVX) mice post EV treatment.Results: The result of [18F]NaF μPET showed an increase in osteoblastic activity in the distal femur of EV-treated mice, and the bone structural parameters derived from μCT were also improved. In terms of in vivo behavior of exogenous EVs, fluorescent dye-labeled EVs could target the distal femur of mice, whereas the uptakes of bone tissues were not significantly different between OVX mice and healthy mice. RNA sequencing demonstrated upregulation of ECM-related genes, which might associate with the PI3K/AKT signaling pathway, in line with the results of microRNA analysis showing that mir-21, mir-29, mir-221, and let-7a were enriched in Wharton’s jelly-MSC-EVs and correlated to the BMP and PI3K/AKT signaling pathways.Conclusion: The therapeutic effect of exogenous WJ-MSC-EVs in the treatment of osteoporosis was successfully assessed by a multiplexed molecular imaging strategy. The RNA sequencing demonstrated the possible molecular targets in the regulation of bone remodeling. The results highlight the novelty of diagnostic and therapeutic strategies of EV-based treatment for osteoporosis.Keywords: Wharton’s jelly mesenchymal stem cells, extracellular vesicles, osteoporosis, [18F]NaF, RNA sequencing