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Extracellular vesicles from human urine-derived stem cells prevent osteoporosis by transferring CTHRC1 and OPG.

ABSTRACT: Osteoporosis is a debilitating bone disease affecting millions of people. Here, we used human urine-derived stem cells (USCs), which were noninvasively harvested from unlimited and easily available urine, as a "factory" to obtain extracellular vesicles (USC-EVs) and demonstrated that the systemic injection of USC-EVs effectively alleviates bone loss and maintains bone strength in osteoporotic mice by enhancing osteoblastic bone formation and suppressing osteoclastic bone resorption. More importantly, the anti-osteoporotic properties of USC-EVs are not notably disrupted by the age, gender, or health condition (with or without osteoporosis) of the USC donor. Mechanistic studies determined that collagen triple-helix repeat containing 1 (CTHRC1) and osteoprotegerin (OPG) proteins are enriched in USC-EVs and required for USC-EV-induced pro-osteogenic and anti-osteoclastic effects. Our results suggest that autologous USC-EVs represent a promising novel therapeutic agent for osteoporosis by promoting osteogenesis and inhibiting osteoclastogenesis by transferring CTHRC1 and OPG.

SUBMITTER: Chen CY

PROVIDER: S-EPMC6594995 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Extracellular vesicles from human urine-derived stem cells prevent osteoporosis by transferring CTHRC1 and OPG.

Chen Chun-Yuan CY Rao Shan-Shan SS Tan Yi-Juan YJ Luo Ming-Jie MJ Hu Xiong-Ke XK Yin Hao H Huang Jie J Hu Yin Y Luo Zhong-Wei ZW Liu Zheng-Zhao ZZ Wang Zhen-Xing ZX Cao Jia J Liu Yi-Wei YW Li Hong-Ming HM Chen Yang Y Du Wei W Liu Jiang-Hua JH Zhang Yan Y Chen Tuan-Hui TH Liu Hao-Ming HM Wu Ben B Yue Tao T Wang Yi-Yi YY Xia Kun K Lei Peng-Fei PF Tang Si-Yuan SY Xie Hui H

Bone research 20190626

Osteoporosis is a debilitating bone disease affecting millions of people. Here, we used human urine-derived stem cells (USCs), which were noninvasively harvested from unlimited and easily available urine, as a "factory" to obtain extracellular vesicles (USC-EVs) and demonstrated that the systemic injection of USC-EVs effectively alleviates bone loss and maintains bone strength in osteoporotic mice by enhancing osteoblastic bone formation and suppressing osteoclastic bone resorption. More importa ...[more]

PMID: 31263627

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Project description:With limited therapeutic intervention in preventing the progression to end-stage renal disease, chronic kidney disease (CKD) remains a global health-care burden. Aristolochic acid (AA) induced nephropathy is a model of CKD characterised by inflammation, tubular injury, and interstitial fibrosis. Human liver stem cell-derived extracellular vesicles (HLSC-EVs) have been reported to exhibit therapeutic properties in various disease models including acute kidney injury. In the present study, we aimed to investigate the effects of HLSC-EVs on tubular regeneration and interstitial fibrosis in an AA-induced mouse model of CKD. NSG mice were injected with HLSC-EVs 3 days after administering AA on a weekly basis for 4 weeks. Mice injected with AA significantly lost weight over the 4-week period. Deterioration in kidney function was also observed. Histology was performed to evaluate tubular necrosis, interstitial fibrosis, as well as infiltration of inflammatory cells/fibroblasts. Kidneys were also subjected to gene array analyses to evaluate regulation of microRNAs (miRNAs) and pro-fibrotic genes. The effect of HLSC-EVs was also tested in vitro to assess pro-fibrotic gene regulation in fibroblasts cocultured with AA pretreated tubular epithelial cells. Histological analyses showed that treatment with HLSC-EVs significantly reduced tubular necrosis, interstitial fibrosis, infiltration of CD45 cells and fibroblasts, which were all elevated during AA induced injury. At a molecular level, HLSC-EVs significantly inhibited the upregulation of the pro-fibrotic genes α-Sma, Tgfb1, and Col1a1 in vivo and in vitro. Fibrosis gene array analyses revealed an upregulation of 35 pro-fibrotic genes in AA injured mice. Treatment with HLSC-EVs downregulated 14 pro-fibrotic genes in total, out of which, 5 were upregulated in mice injured with AA. Analyses of the total mouse miRnome identified several miRNAs involved in the regulation of fibrotic pathways, which were found to be modulated post-treatment with HLSC-EVs. These results indicate that HLSC-EVs play a regenerative role in CKD possibly through the regulation of genes and miRNAs that are activated during the progression of the disease.

Project description:Osteoporosis is one of the most common skeletal disorders caused by the imbalance between bone formation and resorption, resulting in quantitative loss of bone tissue. Since stem cell-derived extracellular vesicles (EVs) are growing attention as novel cell-free therapeutics that have advantages over parental stem cells, the therapeutic effects of EVs from adipose tissue-derived stem cells (ASC-EVs) on osteoporosis pathogenesis were investigated. ASC-EVs were isolated by a multi-filtration system based on the tangential flow filtration (TFF) system and characterized using transmission electron microscopy, dynamic light scattering, zeta potential, flow cytometry, cytokine arrays, and enzyme-linked immunosorbent assay. EVs are rich in growth factors and cytokines related to bone metabolism and mesenchymal stem cell (MSC) migration. In particular, osteoprotegerin (OPG), a natural inhibitor of receptor activator of nuclear factor-κB ligand (RANKL), was highly enriched in ASC-EVs. We found that the intravenous administration of ASC-EVs attenuated bone loss in osteoporosis mice. Also, ASC-EVs significantly inhibited osteoclast differentiation of macrophages and promoted the migration of bone marrow-derived MSCs (BM-MSCs). However, OPG-depleted ASC-EVs did not show anti-osteoclastogenesis effects, demonstrating that OPG is critical for the therapeutic effects of ASC-EVs. Additionally, small RNA sequencing data were analysed to identify miRNA candidates related to anti-osteoporosis effects. miR-21-5p in ASC-EVs inhibited osteoclast differentiation through Acvr2a down-regulation. Also, let-7b-5p in ASC-EVs significantly reduced the expression of genes related to osteoclastogenesis. Finally, ASC-EVs reached the bone tissue after they were injected intravenously, and they remained longer. OPG, miR-21-5p, and let-7b-5p in ASC-EVs inhibit osteoclast differentiation and reduce gene expression related to bone resorption, suggesting that ASC-EVs are highly promising as cell-free therapeutic agents for osteoporosis treatment.

Project description:BackgroundMesenchymal stromal cell (MSC) therapy holds great potential yet efficacy and safety concerns with cell therapy persist. The beneficial effects of MSCs are often attributed to their secretome that includes extracellular vesicles (EVs). EVs carry biologically active molecules, protected by a lipid bilayer. However, several barriers hinder large-scale MSC EV production. A serum-free culturing approach is preferred for producing clinical-grade MSC-derived EVs but this can affect both yield and purity. Consequently, new strategies have been explored, including genetically engineering MSCs to alter EV compositions to enhance potency, increase circulation time or mediate targeting. However, efficient transfection of MSCs is challenging. Typical sources of MSC include adipose tissue and bone marrow, which both require invasive extraction procedures. Here, we investigate the use of urine-derived stem cells (USCs) as a non-invasive and inexhaustible source of MSCs for EV production.MethodsWe isolated, expanded, and characterized urine-derived stem cells (USCs) harvested from eight healthy donors at three different time points during the day. We evaluated the number of clones per urination, proliferation capacity and conducted flow cytometry to establish expression of surface markers. EVs were produced in chemically defined media and characterized. PEI/DNA transfection was used to genetically engineer USCs using transposon technology.ResultsThere were no differences between time points for clone number, doubling time or viability. USCs showed immunophenotypic characteristics of MSCs, such as expression of CD73, CD90 and CD105, with no difference at the assessed time points, however, male donors had reduced CD73 + cells. Expanded USCs were incubated without growth factors or serum for 72 h without a loss in viability and EVs were isolated. USCs were transfected with high efficiency and after 10 days of selection, pure engineered cell cultures were established.ConclusionsIsolation and expansion of MSCs from urine is non-invasive, robust, and without apparent sex-related differences. The sampling time point did not affect any measured markers or USC isolation potential. USCs offer an attractive production platform for EVs, both native and engineered.

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Extracellular vesicles from human urine-derived stem cells prevent osteoporosis by transferring CTHRC1 and OPG.

Publications

Extracellular vesicles from human urine-derived stem cells prevent osteoporosis by transferring CTHRC1 and OPG.

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