Harvest of functional mesenchymal stem cells derived from in vivo osteo-organoids

Bone marrow-derived mesenchymal stem cells(BM-MSCs)play a crucial role in stem cell therapy and are extensively used in regenerative medicine research.However,current methods for harvesting BM-MSCs present challenges,including a low yield of primary cells,long time of in vitro expansion,and diminished differentiation capability after passaging.Meanwhile mesenchymal stem cells(MSCs)recovered from cell banks also face issues like toxic effects of cryopreservation media.In this study,we provide a detailed protocol for the isolation and evaluation of MSCs derived from in vivo osteo-organoids,presenting an alternative to autologous MSCs.We used recombinant human bone morphogenetic protein 2-loaded gelatin sponge scaffolds to construct in vivo osteo-organoids,which were stable sources of MSCs with large quantity,high purity,and strong stemness.Compared with protocols using bone marrow,our protocol can obtain large numbers of high-purity MSCs in a shorter time(6 days vs.12 days for obtaining passage 1 MSCs)while maintaining higher stemness.Notably,we found that the in vivo osteo-organoid-derived MSCs exhibited stronger anti-replicative senescence capacity during passage and amplification,compared to BM-MSCs.The use of osteo-organoid-derived MSCs addresses the conflict between the limitations of autologous cells and the risks associated with allogeneic sources in stem cell transplantation.Consequently,our protocol emerges as a superior alternative for both stem cell research and tissue engineering.

Amphiphilic cytokine traps remodel marrow adipose tissue for hematopoietic microenvironment amelioration

Hematopoietic stem cell transplantation(HSCT)is extensively employed in the treatment of hematological malignancies but is markedly constrained by the paucity of hematopoietic stem/progenitor cells(HSPCs).Recent studies have found that marrow adipose tissue(MAT)acts on hematopoiesis through complicated mechanisms.Therefore,the osteo-organoids fabricated in vivo using biomaterials loaded with recombinant human bone morphogenetic protein 2(rhBMP-2)have been used as models of MAT for our research.To obtain sufficient amounts of therapeutic HSPCs and healthy MAT,we have developed amphiphilic chitosan(AC)-gelatin as carriers of rhBMP-2 to the regulate type conversion of adipose tissue and trap hematopoietic growth factors.Unlike medicine interventions or cell therapies,the traps based on AC not only attenuate the occupancy of adipocytes within the hematopoietic microenvironment while preserving stem cell factor concentrations,but also improve marrow metabolism by promoting MAT browning.In conclusion,this approach increases the proportion of HSPCs in osteo-organoids,and optimizes the composition and metabolic status of MAT.These findings furnish an experimental basis for regulating hematopoiesis in vivo through materials that promote the development of autologous HSPCs.Additionally,this approach presents a theoretical model of rapid adipogenesis for the study of adipose-related pathologies and potential pharmacological targets.