Effects Of FN-TPO Gene Modified Mesenchymal Stem Cells On EX Vivo Amplification And Reconstruction Of Hematopoietic Cells

【Background】Hematopoietic stem/progenitor cell (HSPC) transplantation is one of the most effective treatments to cure many diseases that seriously threatened human beings'life and health. Both insufficiency of HSPC and dysfunction of hematopoietic inductive microenvironment (HIM) are important reasons for the failure of HSPC transplantation. Mesenchymal stem cells (MSCs) are another group of stem cells that located in bone marrow beside HSPC. MSCs are the main source of bone marrow stromal cells which constitute the bone marrow HIM. MSCs can support hematopoietic cells to expand in vitro efficiently. And if co-transplant with HSPC it can support hematopoiesis reconstitution by rescuring the HIM. Thrombopoietin is an efficient early hematopoietic cell promoting cytokine. Fibronectin (FN) is one of the most important component which mediate interactions between HSPC and HIM. Both TPO and FN are mainly secreted by MSCs resourced stromal cells in bone marrow. Previously researchers have demonstrated that biological characters of MSCs could be improved by gene modification. The hypothesis that gene modification of MSCs can improve hematopoietic cells amplification and hematopoiesis reconstitution capacity is getting more and more attention.【Objective】Establish in vitro retrovirus vector mediated FN-TPO gene modification system for bone morrow MSCs and investigate the effect of FN-TPO gene modified MSCs on hematopoietic cells amplification and hematopoietic system reconstruction.【Methods】Constructed retroviral vector that containing FN-TPO fusion gene and modified bone marrow MSCs in vitro by the vector. The transcription of FN-TPO gene was assayed by RT-PCR. The proliferation of bone marrow MSCs were detected by trypan blue exclusion assay. The ability of bone marrow MSCs to adhere hemopoietic cells were assayed by MTT and the concentrations of TPO in the bone marrow MSCs culture supernatant were assayed by ELISA. Cord blood CD34+ cells that selected by immunomagnetic beads were seeded on FN-TPO gene modified MSCs formed layers and several essential growth factors (SCF,FLT3-L and IL-11) were added too. After co-culturing in vitro for 7 days, the number of CD34+ cells and their colony forming capacities were assayed by flow cytometry and semisolid culture assay. The SCID mice treated by sublethal radiation were transplanted with the FN-TPO gene modified MSCs and cord blood mononuclear cells. General state of health, peripheral blood RT on different time, hnman CD45+ cell percentage and the survival rate of the SCID mice were assayed to determine the hematopoiesis reconstitution capacity of the FN-TPO gene modified MSCs.【Results】Retroviral vector that containing FN-TPO fusion gene was successfully constructed and bone morrow MSCs were gene modified. FN-TPO gene expression was detected in modified MSCs by RT-PCR. The viability of MSCs had no significant difference between pre-gene-modified and post-gene-modified [(6.92±0.77)×10~4/ml Vs (7.18±0.89)×10~4/ml,P>0.05]. The ability of gene modified bone marrow MSCs to adhere hematopoietic cells were reinforced (0.188±0.018 Vs 0.167±0.017, P<0.01). The concentration of TPO in the bone morrow MSCs culture supernatant was raised from 5.58±0.37 to 7.46±0.59 (P﹤0.01) and showed no significant decline in short-time (7.46±0.59 ng/ml Vs 7.15±0.19 ng/ml,P>0.05), but was influenced by the state of cell growth. 2.0×10~4 of cord blood CD34+ hematopoietic stem/progenitor cells were co-cultured with gene modified MSCs in vitro for 7 days, the absolute number of nuclear cells, the percentage of CD34+ cells and the numbers of BFU-E, CFU-GM, CFU-GEMM were (29.9±2.7)×10~4, (33.3±2.8)%, 109.3±4.1, 163.7±7.1, 13.3±1.5, respectively, were significantly higher than that co-cultured with non-modified MSCs (P<0.01). The hematopoietic systems of SCID mice were effectively reconstructed by co-transplantation with the gene modified MSCs and cord blood MNCs. Survival rate of these mice were increased. 【Conclusion】1. In vitro FN-TPO fusion gene modification system for bone morrow MSCs is successfully established and the target gene is stably and effectively expressed in bone marrow MSCs.2. The abilities of FN-TPO gene modified bone marrow MSCs to adhere hemopoietic cells and to secrete TPO are reinforced.3. FN-TPO gene modified bone marrow MSCs can effectively amplify cord blood CD34+ hematopoietic stem/progenitor cells.4. The hematopoietic systems of SCID mice after treated with sublethal radiation were effectively reconstituted by co-transplanted of FN-TPO gene modified MSCs with cord blood MNCs.