Studies On Culture Of Clone-derived Human Mesenchymal Stem Cells And Their Hematopoietic Support Effect

Research background: Bone marrow mesenchymal stem cells (MSCs) are a third kind of adult stem cells besides hematopoietic stem cells and endothelial progenitors in bone marrow which endowing multi-differentiation potential and hematopoietic support effect, and also possessing vast application potential in tissue engineering and gene therapy as well. It is thought at present that MSCs is a complex of heterogeneous cells which are readily proliferated in vitro. But duo to the lack of exclusive phenotype, their isolation and purification proved to be difficult. Uptodate lots of their biological characteristics remained unknown. No exclusive phenotype has been found and still now no feasible way has been gained to culture them in vitro. We are still uncertain their differentiation principles and ultimate differentiation potential. Recently focus has been drawn on roles of accelerating hematopoiesis reconstitution and reducing GVHD of the receiptor when cotransplantated MSCs and HSCs. Clone culture of MSCs may be a feasible way to further study their biological characteristics. This experiment aims at exploring ex vivo culture method of MSCs and assaying their in vivo and in vitro hematopoiesis support effects. Methods: 1. Culture and expansion of clone-derived MSCs To get mononuclear cells by density gradient centrifugation and to culture MSCs according to their ahesion characteristics to plastic. MSCs are detached with 0.5% trypsin-EDTA when confluent and a clone circle is used to get clone-derived MSCs. Culture conditions are as follows: DMEM-LG+10%FBS+10ng/mlEGF+10ng/mlPDGF-BB, at 37℃, 5%CO2 and saturation wetness with the media changed every 3 days. Biological characteristics assays of MSCs including(1)Phenotypes assay of MSCs. (2) Growth curves assay of MSCs. (3) CPU-forming ability .(4)Refrigeration and revivification of MSCs.(5) Atomic force microscope detection. (6) Commited induced differentiation of MSCs ex vivo. (7) GFP gene transfection.2. Hematopoiesis support effect of MSCs on cord blood hematopoietic stem cells.Culture methods of MSCs are as mentioned above. Disposal of MSCs: nearly onfluent MSCs were undergone irradiation for15Gy of 60CO.To prepare human umbilical cord blood mononuclear cells by densitygradient centrifugation and purify CD34+ cells by miniMACS according tothe instructions of the manual.In vitro expansion of umbilical cord blood CD34+ cells. CD34+ cells at thedensity of 3×104 are plated in 24-well plates. Three groups are set for eachcondition, each group contain 3 wells. (1)MSCs +cytokine group, (2)Cytokine group, (3)MSCs group. Comparison between P5 and P15 MSCs onthe effect of hematopoietic support was performed.CPU forming capability of CD34+ cells: expanded cord blood mononuclearcells as mentioned above are subcultured in plates containing CPU-MIXmedium, count CFU-GM, CFU-E, CFU-GEMM, BFU-E at day7,day!4, andday21 respectively.3. Studies on in vivo hematopoietic support effect of clone-derived humanMSCs.Mononuclear cells preparation of umbilical cord blood and assay of CD34+ cells:four groups are set: 1. MNCs group: each mouse is administer a dose of 5 X 106 cord blood mononuclear cells; 2. MNCs+1 XMSCs group: each mouse is adminishtered 5 × 106 of mononuclear cells and 3 × 105 MSC; 3. MNCs +10 ×MSCs group: each mouse is administered 5×106 of mononuclear cells and 3 × 106 of MSCs; 4. Control group: each mouse is irradiated only. Cells are infused from tail vein.Count bone marrow and peripheral blood mononuclear cells and assay their CPU-forming capability 2 weeks, 4weeks, 6weeks post-transplantation. Mice post-transplantation are killed by dislocation of cervical vertabra , get the femurs, and prepare the mononuclear cells, adjust cell density to IX 106/ml. Add 2X105 mice MNCs in culture plates containing CPU-MIX media, count colonys containing more than 50 cells after 14 days. Implantation testimony assay of HSCs: Prepare bone marrow mononuclea...