جداسازي سلولهاي بنيادي مزانشيمي CD34 مثبت از مغز استخوان موش

Isolation of CD34+ mesenchymal stem cells from mouse bone marrow

Background and Objectives The isolation and purification of mesenchymal stem cells (MSCs) from mouse via plastic adherent cultures are arduous due to the unwanted growth of hematopoietic cells and non-MSCs. In this study, homogenous populations of CD34+ MSCs from mouse bone marrow were purified through positive immunoselection. Materials and Methods In this experimental study, C57BL/6 mice were sacrificed. Their bone marrow cells were aspirated and incubated with anti-CD34 conjugated magnetic beads. After immunoselection, a sample of the cells was prepared for flow cytometry in order to examine the expression of CD34 antigen and the remains were cultivated. 24 hours later, non-adherent cells, mostly consisting of CD34+ hematopoietic stem cells were removed and the plastic adherent cultivated cells were investigated for surface markers (CD44, Sca-1, Vcam-1, CD34, CD11b and CD45). The plastic adherent cultivated cells were differentiated into the osteoblastic and adipogenic lineages to investigate the mesenchymal nature. Furthermore, the expression of some surface markers were investigated through flow cytometry. Results Purified populations of fibroblast-like CD34+ cells were achieved in the first passage (one week after initiative cultivation). The CD34, CD44, Sca-1 and Vcam-1 markers were expressed but CD 11b and CD45 were absent. Osteocyte (osteocalsin, osteopontin, parathyroid hormone receptor) and adipocyte (lipo protein lipase and adipsin) differentiating genes were expressed in these cells. Conclusions This study indicates that this protocol can result in efficient isolation of homogenous populations of MSCs from C57BL/6 mouse bone marrow. We showed that plastic adherent murine bone marrow derived CD34+ cells with capability of differentiating into skeletal lineages in vitro are MSCs.