Study On Hematopietic Differentiation Potential Of Human Mesenchymal Stem Cells Derived From Bone Marrow

Hematopoietic stem cells (HSC) and mesenchymal stem cells (MSC) are two maily stem cells in bone marrow. MSC is a kind of stem cells capable of multilineage differentiated potential, which are able to differentiate into neural cells, osteoblasts, adipocytes, chondrocytes, myocytes and to be culture-expanded maintaining with its genetics in vitro. Hematopoietic stem cells transplantation (HSCT) as important biological or cellular therapy, is limited by lack of donors, difficult to be culture-expanded in vitro, contamination with tumor cells, immunological rejective reaction after transplantation. MSC and HSC are original of mesoderm, it is possible that MSC differentiate into HSC under some special conditions. The study about MSC differentiation is limited to some small animals such as mice and dogs. Whether hMSC can be differentiation into hematopoietic stem cells or progenitor cells? Whether this cells have the hematopoietic differentiation potential and can achieve the hematopoiesis reconstitution after transplantation? It is sure that exploration and resolution of these problems will be beneficial for the application of hMSC in clinical setting, and provide an useful tool for the research on regulating mechanism of hematopoietic differentiation. To above mentioned, Firstly, hMSC derived from adult bone were isolated, culture-expanded efficiently in vitro and its biological features are detected. Secondly, Fetal liver stromal cell conditioned medium (FLSC-CM), and some hematopoietic factors were well prepared and adapted to induce hMSC toward its differentiate into hematopoietic lineages respectively. Thirdly, we transfect hMSC with green fluorescent protein (GFP), then get the cell line hMSC/EGFP that express GFP stablely. Last, we establish the human-node mice xenotransplantation model that donor cells are hMSC and hMSC/EGFP, identify the differentiation potential of culture-expanded hMSC derived from adult bone marrow in vivo, anal the ability to reconstitute human hematopoiesis in the sublethally irradiated node mice after hMSC transplantation through GFP and FACS. It provide experimental basis about the original of stem cell transplantation donor and the study of stem cells transversal development. Part One Isolation, characterization and ex vivo expansion of human BM MSC in a large-scale. BM cells were obtained from iliac crest aspirates from healthy donors after informed consent. BM was layered over 1.073 g/ml Percoll solution and centrifuged at 900g for 30 min. Mononuclear cells collecting at the interface were recovered, resuspended in Dulbecco's Modified Eagles Medium-Low Glucose (DMEM-LG) supplemented with 10% fetal bovine serum (FBS) and 2mM L-glutamine, and incubated at 37℃in 5%CO2 in air. Then the morphology, cell growth curve, cell cycle, phenotype , and multi-differentiation potentiality were detected in vitro. All the results were repeated three times, and statistical comparison were performed using the t test and sing-factor variance analysis. Results: TheMSC from adult bone marrow was homogenously spindle shaped fiberoblast appearance. They had a high proliferation capacity ex vivo, which made expansion ex vivo in a large-scale a reality. (3.46~3.60)×107 MSC can be harvested when 10ml heparinized bone marrow after the third passage(P3). The total amount of MSC we got had not significant difference among the different donors(P=0.896). The expanded time of different donors P0 passage were different(P=0.000), the shortest was 9d, the longest was 16d. After serial cultivation, MSC proliferated quicker, but the speed were different among the differet donors(P=0.000). Analysis of cell surface molecules and cell cycle was performed using flow cytometry and the following procedures. No known hematopoietic markers were found on each MSC passage, such as CD34,CD45. In addition, Cells from passage 5 were positive for CD29,CD44,CD90,CD105,CD106, G0/G1 accounted for 90.03%, S+G2+M9.97%. With the induction reagents in vitro, MSCs can be differentiated into osteoblast cells, adipocytes. Part two To investigate the hematopoietic differentiation potential of hMSC in vitro. 12.5-14.5dpe(days post coitus) of KM mice were used for the preparation of fetal liver stromal cells conditioning medium(FLSC-CM) Culture-expanded hMSC were cultured with FLSC-CM and the combination of IL-6 and SCF. After 7-14days cultivation, the hematopoietic differentiated cells type of hMSC were characterized by morphology, CFU-GM assay, and immunofluorescence methods. There were some suspension cells derived from hMSC cultured with FLSC-CM after 5d, then the amount of suspension cells increasing after 8d, the suspension cells with round shape and clear nucleus distributed in the supernatant assembling or scattering. There wereonly small amount suspension cells in the cytokines group and control group. Immunofluorescence analysis of the suspension cells demonstrated the presence of the positive CD34 and CD45 cells which were labeled by both PE and FITC, and appear red and green respectively. The suspension cells remained viable with the morphology similar to monocytes or small lymphcytes, and had the ability to form hematopoietic progenitor cells colonies in methylcellulose culture system with recombined human granulocyte/ macrophage-colony stimulating factor (rhGM-CSF). Part three The study of transfection hMSC with green fluorescent protein and their biological features. The supernatant of package cell line PG13/EGFP which constantly express recombinant EGFP-RV transfected the hMSC. The resistant clone were screened by puromycin. The alternation of morphology of hMSC/EGFP was observed, as well as its cell growth curve, phenotype and multipotentiality. Expanded hMSC were successfully transfected by recombinant EGFP-Rv with nucleoprotamine or polybrene, and we got the resistant clone hMSC/EGFP after being screened by puromycin. Under fluorescence microscope the bright green fluorescence were seen among the hMSC/EGFP, the purity is 100% through FCM. The morphology, cell growth curve, and phenotype were similar to that of culture-expanded MSC, the hMSC/EGFP could be induced into adipocyte, osteoblast and maintained its multipotentiality. Part four The Study on hematopoietic differentiation potential after culture-expanded hMSC transplantation in vivo. Aside from control group (n=15), all female the nude mice were sublethally irradiated (350cGy) by 60Coγray, four hours before transplantation. The irradiated and non-irradiatedmice were randomly divided into several groups, hMSC or hMSC/EGFP were injected into a lateral tail vein. Controls consisted of mice receiving PBS. 8 weeks after injection, the mice which were still alive were sacrificed. During the course of transplantation, the survival rate and general state of health of the mice were observed. The weight, white blood cell count and classification of the mice were detected per week. 8 weeks after transplantation bone marrow slides and spleen, bone marrow histopathologic examination were detected. Colony assay, FACS, immunohistochernistry and immuocyto-chemistry technique, fluorescense microscope, and PCR were measured to identify the human hematopoietic engraftment . Expect of 3 nude mice dead of accident, the rest of TBI+/hMSC group and TBI+/hMSC/EGFP group were alive longer than 8 weeks, and their general conditions, leukocyte counts, leukocyte differential count, bone marrow figure reverted to the normal. There were no significant difference between the two groups. The cells with CD34 positive or CD45 positive were detected in bone marrow, peripheral blood, spleen through FACS and immunohistochemistry and immnocytochemistry stain, the percentage of the doner-derived cells CD34+ in bone marrow of recipients were higher than those in spleen and peripheral blood, and the cells CD45+ were in peripheral blood were higher than those in spleen and bone marrow. There were no significant difference between the TBI+/hMSC group and TBI+/hMSC/EGFP group. We got the specific amplificated human Alu gene products from spleen, bone marrow and peripheral blood cells of the TBI+/hMSC group and TBI+/hMSC/EGFP group in PCR analysis. The cells from bone marrow of the two groups formed the hematopoietic progenitor cell colony in CFU-GM. The cells with green...