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MSC exosomes alleviate osteoarthritis through restoration of matrix homeostasis
S52
Poster Abstract Presentations
oxidation state. Aggression of renal cells induced a decrease in the mitochondrial membrane polarization. The CellROX probe also showed an increase in cytoplasmic oxidative stress. We also developed a model of inflammation using THP1 cell line treated with SC. The SC induced an increase in TNFa secretion by THP1. Our main preliminary results indicated that MSCs improved the viability of renal cells, partially restored their mitochondrial membrane potential and decreased their cytoplasmic oxidative stress. In addition, they can exert an anti-inflammatory effect by inhibited the secretion of TNFa by THP1 and increased their secretion of the anti-inflammatory cytokine IL1RA. Finally, the pre-clinical model mimicking a polytrauma could be a formidable tool to evaluate the benefit of MSC therapy, away from shock. 176 MSC EXOSOMES ALLEVIATE OSTEOARTHRITIS THROUGH RESTORATION OF MATRIX HOMEOSTASIS K. TEO1, S. Zhang1, S. Chuah1, R. Lai4, S. Lim4,2 & W. Toh1,3 1 Faculty of Dentistry, National University of Singapore, Singapore, Singapore, Singapore, 2Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore, 3Tissue Engineering Program, Life Sciences Institute, National University of Singapore, Singapore, Singapore, 4Institute of Medical Biology, Agency for Science, Technology and Research, Singapore, Singapore Background & Aim: The efficacy of mesenchymal stromal cells (MSC) therapies is increasingly attributed to paracrine secretion, particularly exosomes. This study aims to elucidate the mechanisms of action of MSC exosomes in an immunocompetent rat model of temporomandibular joint osteoarthritis (TMJ-OA). Methods, Results & Conclusion: Exosomes were isolated from conditioned medium of human MSCs. OA of bilateral TMJs was induced in rats by injection of monosodium iodoacetate. Thereafter, weekly intra-articular injections of exosomes (100mg/50ml) or 50ml of vehicle (PBS) were given over 2, 4 and 8 weeks. Analyses were performed by head withdrawal threshold (HWT) measurement, micro-computed tomography, histology and immunohistochemistry, and Mankin scoring. Gene expression changes were measured by transcriptomic analysis of the condylar cartilage tissue. Cell-based assays were performed to determine the cellular processes and signalling pathways activated by MSC exosomes. Our results showed that exosome-mediated repair of the osteoarthritic TMJs was characterized by early suppression of pain and degeneration with reduced inflammation, followed by sustained proliferation and gradual improvements in matrix expression and subchondral bone architecture, leading to overall joint restoration and regeneration by the end of treatment. By 8 weeks, exosome-treated rats had reduced pain with HWT comparable to that of sham rats, and displayed cartilage and subchondral bone restoration including appropriate cartilage thickness, cellularity, matrix and subchondral bone architecture that closely resembled that of the naive rats. Using an in-vitro chondrocyte model of OA, MSC exosomes increased sulphated glycosaminoglycan synthesis and reduced nitric oxide production in interleukin-1b-treated chondrocytes through exosomal CD73-mediated adenosine activation of AKT and AMPK signalling. These effects were partially abrogated by theophylline, wortmannin or compound C, which inhibited adenosine receptor activation, AKT and AMPK phosphorylation, respectively. Together, our observations suggest that MSC exosomes promote TMJ repair and regeneration in OA through a well-orchestrated mechanism of action that involved multiple cellular processes to restore the matrix and overall joint homeostasis.
lymphocytes in human. In this study, the NK cells were isolated and cultured under different period of time, the ultrastructure of the highly purified activated NK cells were analyzed by transmission electron microscopy (TEM). The increase in the number of the NK cells were measured by flow cytometry. Methods, Results & Conclusion Methods: The peripheral blood mononuclear cells (MNCs) were isolated by Ficoll centrifugation followed by phosphate buffer saline (PBS) with 10% Acid Citrate Dextrose (ACD). The MNCs were cultured according to the protocol of Cellex Natural Killer Cell Kit (Japan). The NK cells at Day 0, and cultured at Day 3, 5, 7 and 14 were sampled and fixed with 2.5% glutaraldehyde for 24 hours at 4°C, and processed for routine TEM examination. Samples at Day 0 and Day 14 were quantified by flow cytometry. Results: The resting NK cell at Day 0 was about 3.5um with a round nucleus of about 1.7um in diameter. Electron-dense granules (EDGs) of varying sizes were observed (100-200nm) in figure 1. The NK cells started to proliferate after cytokine activation cultured at Day 3. The average size of the NK cells (6um) and nuclei (4um) increased in sizes when compared with Day 0. Membrane-bounded vesicles were observed at the proximity of Golgi area (Fig. 2). Electro-dense granules of similar sizes as at Day 0 were observed at Day 5.
Figure 1. The resting NK cell was characterized with small Golgi region (Gol) short granular endoplasmic reticulum (thick arrows), and a few mitochondria (Mit). Electron-dense granules (EDGs) of varying sizes were observed (100-200 nm). (Mag. £ 2200)
177 WILL NOT BE PRESENTED 178 WILL NOT BE PRESENTED 179 ULTRASTRUCTURE OF CYTOKINE INDUCED HUMAN NATURAL KILLER CELLS W. Ng1, D. Tam2, D. Liao1, W. Lee3 & F. Chan3 1 Genepath Technology Limited, Hong Kong SAR, China, 2Cryolife Company Limited, Hong Kong SAR, China, 3Electron Microscope Unit, The University of Hong Kong, Hong Kong SAR, China Background & Aim: Natural Killer (NK) cells account for 3.66-26.74% of lymphocytes in the human peripheral blood. They secrete exosomes which express both NK-cell markers and cytokines. It has been shown that Interkin2 (IL-2) stimulated the proliferation of natural killer cells, a sub-class of
Figure 2. NK cells after IL2 stimulation for 3 days increased in size. Small vesicles were found within membrane-bounded vacuoles (thin arrows). (Mag. x 5200)
Poster Abstract Presentations
oxidation state. Aggression of renal cells induced a decrease in the mitochondrial membrane polarization. The CellROX probe also showed an increase in cytoplasmic oxidative stress. We also developed a model of inflammation using THP1 cell line treated with SC. The SC induced an increase in TNFa secretion by THP1. Our main preliminary results indicated that MSCs improved the viability of renal cells, partially restored their mitochondrial membrane potential and decreased their cytoplasmic oxidative stress. In addition, they can exert an anti-inflammatory effect by inhibited the secretion of TNFa by THP1 and increased their secretion of the anti-inflammatory cytokine IL1RA. Finally, the pre-clinical model mimicking a polytrauma could be a formidable tool to evaluate the benefit of MSC therapy, away from shock. 176 MSC EXOSOMES ALLEVIATE OSTEOARTHRITIS THROUGH RESTORATION OF MATRIX HOMEOSTASIS K. TEO1, S. Zhang1, S. Chuah1, R. Lai4, S. Lim4,2 & W. Toh1,3 1 Faculty of Dentistry, National University of Singapore, Singapore, Singapore, Singapore, 2Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore, 3Tissue Engineering Program, Life Sciences Institute, National University of Singapore, Singapore, Singapore, 4Institute of Medical Biology, Agency for Science, Technology and Research, Singapore, Singapore Background & Aim: The efficacy of mesenchymal stromal cells (MSC) therapies is increasingly attributed to paracrine secretion, particularly exosomes. This study aims to elucidate the mechanisms of action of MSC exosomes in an immunocompetent rat model of temporomandibular joint osteoarthritis (TMJ-OA). Methods, Results & Conclusion: Exosomes were isolated from conditioned medium of human MSCs. OA of bilateral TMJs was induced in rats by injection of monosodium iodoacetate. Thereafter, weekly intra-articular injections of exosomes (100mg/50ml) or 50ml of vehicle (PBS) were given over 2, 4 and 8 weeks. Analyses were performed by head withdrawal threshold (HWT) measurement, micro-computed tomography, histology and immunohistochemistry, and Mankin scoring. Gene expression changes were measured by transcriptomic analysis of the condylar cartilage tissue. Cell-based assays were performed to determine the cellular processes and signalling pathways activated by MSC exosomes. Our results showed that exosome-mediated repair of the osteoarthritic TMJs was characterized by early suppression of pain and degeneration with reduced inflammation, followed by sustained proliferation and gradual improvements in matrix expression and subchondral bone architecture, leading to overall joint restoration and regeneration by the end of treatment. By 8 weeks, exosome-treated rats had reduced pain with HWT comparable to that of sham rats, and displayed cartilage and subchondral bone restoration including appropriate cartilage thickness, cellularity, matrix and subchondral bone architecture that closely resembled that of the naive rats. Using an in-vitro chondrocyte model of OA, MSC exosomes increased sulphated glycosaminoglycan synthesis and reduced nitric oxide production in interleukin-1b-treated chondrocytes through exosomal CD73-mediated adenosine activation of AKT and AMPK signalling. These effects were partially abrogated by theophylline, wortmannin or compound C, which inhibited adenosine receptor activation, AKT and AMPK phosphorylation, respectively. Together, our observations suggest that MSC exosomes promote TMJ repair and regeneration in OA through a well-orchestrated mechanism of action that involved multiple cellular processes to restore the matrix and overall joint homeostasis.
lymphocytes in human. In this study, the NK cells were isolated and cultured under different period of time, the ultrastructure of the highly purified activated NK cells were analyzed by transmission electron microscopy (TEM). The increase in the number of the NK cells were measured by flow cytometry. Methods, Results & Conclusion Methods: The peripheral blood mononuclear cells (MNCs) were isolated by Ficoll centrifugation followed by phosphate buffer saline (PBS) with 10% Acid Citrate Dextrose (ACD). The MNCs were cultured according to the protocol of Cellex Natural Killer Cell Kit (Japan). The NK cells at Day 0, and cultured at Day 3, 5, 7 and 14 were sampled and fixed with 2.5% glutaraldehyde for 24 hours at 4°C, and processed for routine TEM examination. Samples at Day 0 and Day 14 were quantified by flow cytometry. Results: The resting NK cell at Day 0 was about 3.5um with a round nucleus of about 1.7um in diameter. Electron-dense granules (EDGs) of varying sizes were observed (100-200nm) in figure 1. The NK cells started to proliferate after cytokine activation cultured at Day 3. The average size of the NK cells (6um) and nuclei (4um) increased in sizes when compared with Day 0. Membrane-bounded vesicles were observed at the proximity of Golgi area (Fig. 2). Electro-dense granules of similar sizes as at Day 0 were observed at Day 5.
Figure 1. The resting NK cell was characterized with small Golgi region (Gol) short granular endoplasmic reticulum (thick arrows), and a few mitochondria (Mit). Electron-dense granules (EDGs) of varying sizes were observed (100-200 nm). (Mag. £ 2200)
177 WILL NOT BE PRESENTED 178 WILL NOT BE PRESENTED 179 ULTRASTRUCTURE OF CYTOKINE INDUCED HUMAN NATURAL KILLER CELLS W. Ng1, D. Tam2, D. Liao1, W. Lee3 & F. Chan3 1 Genepath Technology Limited, Hong Kong SAR, China, 2Cryolife Company Limited, Hong Kong SAR, China, 3Electron Microscope Unit, The University of Hong Kong, Hong Kong SAR, China Background & Aim: Natural Killer (NK) cells account for 3.66-26.74% of lymphocytes in the human peripheral blood. They secrete exosomes which express both NK-cell markers and cytokines. It has been shown that Interkin2 (IL-2) stimulated the proliferation of natural killer cells, a sub-class of
Figure 2. NK cells after IL2 stimulation for 3 days increased in size. Small vesicles were found within membrane-bounded vacuoles (thin arrows). (Mag. x 5200)