Secretome derived from mesenchymal stem cells (MSCs) have profound effects on tissue regeneration, which could become the basis of future MSCs therapies. Hypoxia, as the physiologic environment of MSCs, has great pote...Secretome derived from mesenchymal stem cells (MSCs) have profound effects on tissue regeneration, which could become the basis of future MSCs therapies. Hypoxia, as the physiologic environment of MSCs, has great potential to enhance MSCs paracrine therapeutic effect. In our study, the paracrine effects of secretome derived from MSCs preconditioned in normoxia and hypoxia was compared through both in vitro functional assays and an in vivo rat osteochondral defect model. Specifically, the paracrine effect of total EVs were compared to that of soluble factors to characterize the predominant active components in the hypoxic secretome. We demonstrated that hypoxia conditioned medium, as well as the corresponding EVs, at a relatively low dosage, were efficient in promoting the repair of critical-sized osteochondral defects and mitigated the joint inflammation in a rat osteochondral defect model, relative to their normoxia counterpart. In vitro functional test shows enhancement through chondrocyte proliferation, migration, and matrix deposition, while inhibit IL-1β-induced chondrocytes senescence, inflammation, matrix degradation, and pro-inflammatory macrophage activity. Multiple functional proteins, as well as a change in EVs’ size profile, with enrichment of specific EV-miRNAs were detected with hypoxia preconditioning, implicating complex molecular pathways involved in hypoxia pre-conditioned MSCs secretome generated cartilage regeneration.展开更多
Bone marrow-derived mesenchymal stem cell(MSC)is one of the most actively studied cell types due to its regenerative potential and immunomodulatory properties.Conventional cell expansion methods using 2D tissue cultur...Bone marrow-derived mesenchymal stem cell(MSC)is one of the most actively studied cell types due to its regenerative potential and immunomodulatory properties.Conventional cell expansion methods using 2D tissue culture plates and 2.5D microcarriers in bioreactors can generate large cell numbers,but they compromise stem cell potency and lack mechanical preconditioning to prepare MSC for physiological loading expected in vivo.To overcome these challenges,in this work,we describe a 3D dynamic hydrogel using magneto-stimulation for direct MSC manufacturing to therapy.With our technology,we found that dynamic mechanical stimulation(DMS)enhanced matrix-integrinβ1 interactions which induced MSCs spreading and proliferation.In addition,DMS could modulate MSC biofunctions including directing MSC differentiation into specific lineages and boosting paracrine activities(e.g.,growth factor secretion)through YAP nuclear localization and FAK-ERK pathway.With our magnetic hydrogel,complex procedures from MSC manufacturing to final clinical use,can be integrated into one single platform,and we believe this‘all-in-one’technology could offer a paradigm shift to existing standards in MSC therapy.展开更多
Background There is a difficulty in evaluating the in heterogeneity among cartilage affected by osteoarthritis vivo functionality of individual chondrocytes, and there is much （OA）. In this study, in vitro cultured ...Background There is a difficulty in evaluating the in heterogeneity among cartilage affected by osteoarthritis vivo functionality of individual chondrocytes, and there is much （OA）. In this study, in vitro cultured chondrocytes harvested from varying stages of degeneration were studied as a projective model to further understand the pathogenesis of osteoarthritis. Methods Cartilage of varying degeneration of end-stage OA was harvested, while cell yield and matrix glycosaminoglycan （GAG） content were measured. Cell morphology, proliferation, and gene expression of collagen type 1,‘11, and X, aggrecan, matrix metalloproteinase 13 （MMP-13）, and ADAMTS5 of the acquired chondrocytes were measured during subsequent in vitro culture. Results Both the number of cells and the GAG content increased with increasing severity of OA. Cell spreading area increased and gradually showed spindle-like morphology during in vitro culture. Gene expression of collagen type II, collagen type X as well as GAG decreased with severity of cartilage degeneration, while expression of collagen type I increased. Expression of MMP-13 increased with severity of cartilage degeneration, while expression of ADAMTS-5 remained stable. Expression of collagen type II, X, GAG, and MMP-13 substantially decreased with in vitro culture. Expression of collagen type I increased with in vitro cultures, while expression of ADAMTS 5 remained stable. Conclusions Expression of functional genes such as collagen type Ⅱ and GAG decreased during severe degeneration of OA cartilage and in vitro dedifferentiation. Gene expression of collagen I and MMP-13 increased with severity of cartilage degeneration.展开更多
基金supported by National Medical Research Council of Singapore(MOH-000371-00)the National Research Foundation,Prime Minister’s Office,Singapore under its Campus for Research Excellence and Technological Enterprise(CREATE)program,through Singapore-MIT Alliance for Research and Technology(SMART):Critical Analytics for Manufacturing Personalized-Medicine(CAMP)Inter-Disciplinary Research Group.YY was supported by NUS Research Scholarship.
文摘Secretome derived from mesenchymal stem cells (MSCs) have profound effects on tissue regeneration, which could become the basis of future MSCs therapies. Hypoxia, as the physiologic environment of MSCs, has great potential to enhance MSCs paracrine therapeutic effect. In our study, the paracrine effects of secretome derived from MSCs preconditioned in normoxia and hypoxia was compared through both in vitro functional assays and an in vivo rat osteochondral defect model. Specifically, the paracrine effect of total EVs were compared to that of soluble factors to characterize the predominant active components in the hypoxic secretome. We demonstrated that hypoxia conditioned medium, as well as the corresponding EVs, at a relatively low dosage, were efficient in promoting the repair of critical-sized osteochondral defects and mitigated the joint inflammation in a rat osteochondral defect model, relative to their normoxia counterpart. In vitro functional test shows enhancement through chondrocyte proliferation, migration, and matrix deposition, while inhibit IL-1β-induced chondrocytes senescence, inflammation, matrix degradation, and pro-inflammatory macrophage activity. Multiple functional proteins, as well as a change in EVs’ size profile, with enrichment of specific EV-miRNAs were detected with hypoxia preconditioning, implicating complex molecular pathways involved in hypoxia pre-conditioned MSCs secretome generated cartilage regeneration.
基金supported by NUS Presidential Young Professorship,MOE Tier 1 grantsupported by the NUS Research Scholarship.
文摘Bone marrow-derived mesenchymal stem cell(MSC)is one of the most actively studied cell types due to its regenerative potential and immunomodulatory properties.Conventional cell expansion methods using 2D tissue culture plates and 2.5D microcarriers in bioreactors can generate large cell numbers,but they compromise stem cell potency and lack mechanical preconditioning to prepare MSC for physiological loading expected in vivo.To overcome these challenges,in this work,we describe a 3D dynamic hydrogel using magneto-stimulation for direct MSC manufacturing to therapy.With our technology,we found that dynamic mechanical stimulation(DMS)enhanced matrix-integrinβ1 interactions which induced MSCs spreading and proliferation.In addition,DMS could modulate MSC biofunctions including directing MSC differentiation into specific lineages and boosting paracrine activities(e.g.,growth factor secretion)through YAP nuclear localization and FAK-ERK pathway.With our magnetic hydrogel,complex procedures from MSC manufacturing to final clinical use,can be integrated into one single platform,and we believe this‘all-in-one’technology could offer a paradigm shift to existing standards in MSC therapy.
基金This work is supported by National Basic Research Program of China （973 Program） （No. 2011CB707500, 2012CB619102） and National Natural Science Foundation of China （No. 30772198, 30970881, 31011140348）.
文摘Background There is a difficulty in evaluating the in heterogeneity among cartilage affected by osteoarthritis vivo functionality of individual chondrocytes, and there is much （OA）. In this study, in vitro cultured chondrocytes harvested from varying stages of degeneration were studied as a projective model to further understand the pathogenesis of osteoarthritis. Methods Cartilage of varying degeneration of end-stage OA was harvested, while cell yield and matrix glycosaminoglycan （GAG） content were measured. Cell morphology, proliferation, and gene expression of collagen type 1,‘11, and X, aggrecan, matrix metalloproteinase 13 （MMP-13）, and ADAMTS5 of the acquired chondrocytes were measured during subsequent in vitro culture. Results Both the number of cells and the GAG content increased with increasing severity of OA. Cell spreading area increased and gradually showed spindle-like morphology during in vitro culture. Gene expression of collagen type II, collagen type X as well as GAG decreased with severity of cartilage degeneration, while expression of collagen type I increased. Expression of MMP-13 increased with severity of cartilage degeneration, while expression of ADAMTS-5 remained stable. Expression of collagen type II, X, GAG, and MMP-13 substantially decreased with in vitro culture. Expression of collagen type I increased with in vitro cultures, while expression of ADAMTS 5 remained stable. Conclusions Expression of functional genes such as collagen type Ⅱ and GAG decreased during severe degeneration of OA cartilage and in vitro dedifferentiation. Gene expression of collagen I and MMP-13 increased with severity of cartilage degeneration.
