The Characteristics Of Fetal Non-adherent Bone Marrow Stromal Cells

Bone marrow mesenchymal stem cells (BMSCs) is another multipotential stem cells in the bone marrow besides hematopoietic stem cells. Because they have the potential of differentiated into osteoblasts,adipocytes,chondrocytes,nerve cells et al, and have the characteristic of low immunogenicity, they absorb more investigators attentions. The traditional culture method is adherent culture method and can get the most adherent BMSCs.In the 9th decade of last century Clarke discovered some non-adherent cells can become adherent and can form into colony units in the long-term culture system, and the appearance and the phenotypes of the cells are similar with the MSCs, so they think there maybe more primitive stromal stem cells consist with the MSCs in the bone marrow. After then, more and more researches find the exist of non-adherent stem cells, Scutt A find the PGE2,forskolin and Dibutyryl Adenosine Cyclophosphate can induce the non-adherent stem cells translated into mesenchymal stem cells, and the non-adherent stromal stem cells makes an important effects in the reconstruction of hematopoiesis. The NA-MSCs derived from the mouse with normal gene were imported into the mouse without the VDR gene and have a lethal dose irradiation by Zhang ZL, and they found that haemogram of the mouse that were imported the NA-MSCs were recovered quickly and the VDR gene were found in different tissues. T his can indicate that NA-MSCs have a strong ability of tissue repair. Recently Fricke S find that the transplantation of NA-MSCs is more useful than the transplantation of bone marrow only in the aspects of recovery of hematopoiesis use the model of transgenic mice, and they also made a study of the fundamental properties of NA-MSCs, they think the NA-MSCs of mouse maybe a mixed cell colonies that can express many stromal cells and can promote the rebuilt of the hematopoiesis. Their ability ?of form into CFU-F is week than the MSCs, but they also can form into CFU-F, the analysis of gene expression display that NA-MSCs include the gene expressions of stromal cell,mesenchymal stem cell,endotheliocyte and monocyte ,but the expression of osteoid,lymphocyte,eyrthrocytic and hematopoetic stem cell are low. The pathology results from the mouse with lethal dose irradiation that transplanted NA-MSCs shows that the ability of promote hematopoiesis and tissue repair are stonger than MSCs.On the background of the upper studies, we mainly study the culture method and the fundamental characters of the non-adherent fetal bone marrow stromal stem cells, and study the cytokines that they express, and make a comparison with the MSCs.Objective: To explore the isolation and foundational characteristic of non-adherent bone marrow stromal cells (NA-BMSCs) . Method: To culture fetal bone marrow derived non-adherent bone marrow stromal cells(NA-BMSCs), the cells were isolated by the method of adherent separation repeatly ("pull-offs"),the cells were incubated in another culture bottle every 24 hours for 4 times . The phenotype at the 1,3,5 passages (P1,3,5) of the NA-BMSCs was determined with flow cytometry. MTT assay was employed to determine the proliferation of NA-BMSCs. The hematopoietic colony assay, osteogenic and adipogenic differentiation assay were employed to determine their ability to differentiation in vitro.Result: After four times'"pull-off",the resulting cells were continue to culture for 7 days and those cells were the primary NA-MSCs(P0). The morphologic f the cells is uneven,they highly expressed the integrin and the surface markers of endothelial cell with a middle level of CD106. Interestingly,they also express the surface marker of hematopoietic cells CD34 .those cells had multi-differentiation potency in vitro and they can differentiated into osteoblasts, adipocyte and hematopoietic cells under dif- ferent inducing medium, but the capability of lipid differentiation is weak . Conclusion: The NA-MSCs can be obtained by the method of"pull-offs", the NA- MSCs may be the common progenitor cells of hematopoietic stem cells and mesen- chymal stem cells.Objective: To investigate the hematopoietic related cytokines profiles expressed in non-adherent bone marrow stromal cells(NA-BMSCs). Method: To detect the expression of TPO,EPO,G-CSF,GM-CSF,IL-3,IL-6,IL-11,SCF,FLT-3 ligand, mRNA isolated from P1 of NA-BMSCs with standard methods and real time PCR was used to analyzed the expression of those cytokines. The results were compared to those of mesenchymal stem cells that cultured in routine way.Result: The NA-BMSCs express G-CSF,GM-CSF,IL-6,IL-11,SCF,FLT-3ligand and TPO as those expressed in MSCs,but don't express EPO or IL-3,and the expression of SCF and TPO in NA-BMSCs is apparently high than the expression in MSCs.Conclusion: The expression of cytokines in NA-MSCs is similar to those in the MSCs ,but expression of stem cell related factors such as SCF and TPO is higher in NA-BMSCs, which mean that NA-MSCs might be the more primitive cells than those of the MSCs.