CLONING AND SEQUENCING OF MATURE FRAGMENT OF HUMAN BMP4 GENE

Objective To study the cloning and sequencing of mature fragment of human bone morphogenetic protein 4 gene. Methods The template DNA was obtained from the human osteosarcoma cell line U2OS. By using RT PCR method, the cDNA coding for the mature fragment of BMP 4 was amplified, cloned into the vector pUC19, and sequenced by Sanger Dideoxy mediated Chain Termination method. Results The mature fragment of BMP4 cDNA was obtained by RT PCR and determined by sequencing. Through the computer search on Genebank, the analysis showed that the homology of nucleotides and amino acids between cDNA of rhBMP4 mature fragment of this study and the published sequence was 99%. Sequence analysis showed that there were two differences, one was at base 1154(201): G→C, which had no influence on the corresponding amino acids(Val). Another was at base1222(269):C→T, the mutation at the base 1222 had the change of Ala to Val. Conclusion The mature fragment of BMP4 gene has been cloned. The results will be of great significance in treatment of skeletal injuries and diseases.

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.