The Effect Of Bone Marrow Microenvironment On The Property Of Mesenchymal Stem Cells

Mesenchymal stem cells(MSCs)are important stem cells except the hematopoieticstem cells (HSCs) in bone marrow.MSCs are characterized by their intrinsicself-renewal capacity and multilineage differentiation potentials.They can differentiateinto a variety of cells such as adipocytes, osteoblasts and endothelial cells. Therefore,MSCs are of great importance for the reparation of damaged tissue. Furthermore, MSCshave hematopoietic-supporting and immune regulatory capacities. MSCs are easy toculture and proliferate in vitro and they can be isolated from other tissue like muscle.Because of these characteristics, MSCs have been used in clinic trials and showed goodtherapeutic effect. However, many key questions such as immune regulatory mechanismand the effectof the bone marrow microenvironment on the property of MSCs are stillunclear. So,establishing a convenient and efficient method for isolating and culturingmurine MSCs in vitro will lay a good foundation for the follow-up studies.In our study, a new method was developed to isolate and culture murine MSCsfrom bone marrow plus bone fragment. By comparing with flushing-out bone marrowand collagenase-digested bone fragment, MSCs colonies from bone marrow plus bonefragment came out earliest, and the total cell yields of MSCs was best. MSCs showedspindle adherent growth and flow cytometry data showed the cultured cells werepositive for Sca-1, CD44and CD29, and negative for pan-leukocyte surface markerCD45and endothelial cell marker CD31. The isolated and cultured MSCs coulddifferentiate into osteoblast at the osteogenic diffierentiation condition, or adipocyte atadipogenic differentiation condition. All these showed that the cells had typicalcharacteristics of MSCs.Because MSCs have many kind of superior features, especially the reparationability of damaged tissues, the study about MSC functions become a hotpot. It is worthnoting that MSCs are important stem cells in bone marrow, although they play an important role to other cells in bone marrow microenvironment such ashematopoietic-supporting, their characteristics will be affected by the bone marrowmicroenvironment. When the body becomes age, the mobilization and migration ofMSCs in the process of wound healing is slower than that in the young body. So thisstudy would focus on the effect of bone marrow from young mice (BMyoung) and bonemarrow from old mice (BMold) on the property of MSCs.Working in the co-culture of bone fragment and bone marrow (BMyoungand BMoldrespectively), we have found that BMoldinhibit MSC colony formation. Flow cytometrydata showed that the proportion of B220+cells in BMoldwas significantly lower thanthat in BMyoung, while the proportion of CD11b+,CD3+,Gr-1+and F4/80+cells are on thecontrary.CD11b+, B220+, Ter119+cells from BMoldwere separated by MACS andcultured with bone fragment, however, these three subgroups didn’t suppress MSCcolony formation, instead, they promoted MSC colony formation. Then we comparedthe effect of BMoldand BMyoungon the proliferation and migration of MSCs. In theproliferation assay, BMoldand BMyoungexhibited similar effect on cell proliferation ofMSCs. While wound healing and transwell assays showed the migration of MSCs wassignificantly lower when cultured with BMold. Consistently, QPCR data revealed thatMSCs cultured with BMoldshowed lower expression of SDF-1mRNA.Western blotresults showed that both BMyoungand BMoldinduced the phosphorylation of p38/MAPK,JNK/SAPK and ERK/MAPKinMSCs.But only the phosphorylation of JNK/SAPKshowed the difference between BMyoungand BMoldstimulated MSCs. The BMoldgroupexpressed lower p-JNK/SAPK than the one in BMyoung.In conclusion, the new method of bone marrow plus bone fragment is convenientand efficient for isolating and culturing murine MSCs. BMoldmicroenvironment caninhibit the migration of MSCs from the stem cell niche, but which cells or extracellularmatrix or cell factors in the bone marrow play a role in the mechanism of migrationneeds further study.