| Objectives and backgroundEmbryonic and adult tissues contain a population of stem cells that have marked self-renewal and differentiation potential in the developmental process. The stem cell isolation and culture provide an ideal model for further exploration of embryogensis, tissue differentiation and gene regulation, and a novel approach to a spectrum of diseases. Previous studies have identified several adult stem cells with various origin and potential in bone marrow, such as hematopoietic stem cell (HSC), mesenchymal stem cell (MSC), hemangioblast and endothelial progenitor cell. Up to date, bone marrow derived-stem cell (BMSC) is an adult stem cell population of which phenotype and functional properties have been extensively characterized. Plasticity has been found in BMSC in a number of animal transplantation models, which can differentiate into hepatocyte, myocardic cell, muscle cell, neuron, pancreatic islet cells, as well as epithelium cells in kidney, lung and gastrointestine. Several mechanisms are involved in plasticity, that is. trans-differentiation, dedifferentiation and cell fusion, and so on. It is maybe that this common ancestor differentiates into stem cells for each type of tissue depending upon specific environmental conditions, function as a stem cell for many different tissues. If pluripotent stem cells can be isolated and expanded in culture, then a cell line was established, that will provide a technical platform for the further study on stem cell proliferation and differentiation regulation, and promising seed cells for cell therapy and tissue engineering.Several impediments are layed up before hematopoietic stem cell transplantation (HSCT), such as donor scarce, prolonged hematopoietic recovery, and graft verus host disease (GVHD). While some biological features of BMSC may be helpful to solve theproblems. First, researchers have isolated pluripotent stem cells from human, rat and mouse bone marrow, which have a capacity of differentiating into the tissues of three germ layers at the single cell level. And when the cells were transplanted into NOD/SCID mice through tail vein, the differentiated hematopoietic cells were detectable in bone marrow, peripheral blood and spleen, which indicated that BMSC maybe a novel resource of HSC. Second, BMSC do not express major histocompatibility class II (MHCII) antigens, express histocompatibility class I (MHC I) at a low level, if not. Then it is not substantially immunogenic, thus can not be rejected and ameliorate or even prevent GVHD reactions in allogeneic HSCT (allo-HSCT) settings. Third, BMSC is a special population identified within bone marrow MSC cultures, some characteristics of which is similar to MSC. Studies have demonstrated that co-transplantation of MSC and HSC enhanced engraftment and hematopoiesis recovery, suppressed rejection and ameliorated GVHD. While to date, the studies of BMSC are still at the preliminary stage, and its role in hematopoiesis recontitution and immunity modulation after HSCT is few reported, which deserves further explored.In this project, we have engaged into isolating murine marrow-derived multipotent stem cell (mMMSC), and establishing a cell line. Its cross-lineage and cross-germ layer multipotency and biological characteristics were studied, providing an ideal target cell for further study in vivo and in vitro. Meanwhile, mMMSC transplantation animal model was established, the feasibility of mMMSC as a novel source of HSC was ascertained, and its facilitating hematopoiesis recovery role was established.The establishment and characterization of a murine marrow-derived multipotent stem cell line providing a technical platform for the further study on immunologic modulation, and lay a solid foundation for cell therapy with human MMSC. Part One Isolation, culture and characterization of mMMSCMethods Bone marrow (BM) was collected from femur and tibia of male C57BL/6 under sterile condition. BM mononuclear cells (MNCs) were obtained by Ficoll-Paque density gradient centrifugation(p=1.077g/ml), then plated l><105/cm2 cells in the plates in 2ml medium supplemented with 60% DMEM-LG, 40%MCDB-201, 2% FBS, lOng/mlrhEGF, lOng/ml rhPBGF-BB, 1 X 103U/ml mLIF, in the wells of 6-well plate coated with lOOng/ml FN, incubating at 37°C with 5%CO2 in a fully humidified atmosphere. Once adherent cells were more than 75% confluency, all colonies weretrypsinized and combined. The CD45 Terll9 cells were sorted with magnetic cell sorting system (MACS) and replated in FN-coated wells of 96-well plates at a concentration of 10 cells/well. After colonies grows out, we expand them at densities between 0.5 and 1.5><10 /cm . Morphology, cell growth curve, cell cycle, phenotype, and karyotype were evaluated in each passage. In addition, transcription factor Oct-4 and Rex-1 were detected with RT-PCR. Its multilineage potency into adipocytes, osteoblasts, endothelium cells, hepatocytes, neural cells, and hematopoietic cell was also investigated. The pathogens such as bacteria, fungi, chlamydia and mycoplasm in culture system were detected. Cytogenetics stability was measured by chromosome G banding. Tumour formation was observed in nude mice that received mMMSC intravenously. Satistical comparision were tested with sing-factor variance analysis. The reported P value was 2-sided (a = 0.05). All calculations were performed using the software SPSS 11.5. Results The cultured mMMSCs were homogenously short-spindle or multi-angular shaped fiberoblast appearance. Its biological properties remained stable in 60 cell doublings, and cell-doubling time was 17.73—18.72 hours in log phase. Flow-cytometric analysis of cell cycle phase revealed that 42.74% and 89.60% of cells were at G0/G1 phase, 57.26% and 10.40% of cells were at S+G2+M phase which confluency was 30%-40% or more than 90%, respectively. The phenotype of cultured mMMSC was CD 117, CD45, and MHC-II negative; mMMSC express higher levels of CD29> CD44 and Sca-1, and low levels of CD13, CD90^ Flk-1 and MHC- I . RT-PCR showed that two transcription factors important in maintaining undifferentiated embryonic stem cell (ES), Oct-4 and Rex-1, were expressed in mMMSC. With the inductive reagents in vitro, mMMSC can be differentiated into adipocytes, osteoblast cells, endothelium cells, hepatocytes and neutral cells, but not hematopoietic cells. Cytogenetics remained stable in mMMSC during cell doubling. Tumor formation was not observed after 3 months since inoculated mMMSC subcutaneously in nude mice. The detection of bacteria, fungi, chlamydia and mycoplasm was negative in the supernatant of culture medium. mMMSCs through serial population doublings and from frozen stocks were homogenous in biology properties. It demonstrated that mMMSC had extensive proliferation and clonal multilineage differentiation potential. It remained biological characterizatics stable for more than 60 cell doubling, and free of tumorgenesis and pathogen pollution.Part two Hematopoiesis reconstitution with mMMSC in vivoMethods 1. Male C57BL/6 mice bone marrow suspension was prepared as donorcells. 2. Female C57BL/6 mice were recipients received lethally total body irradiated with 8 Gy ( 25cGy/min). 3. The recipients were defined into 4 groups randomly: A Control group (n=5) was injected with 0.2ml PBS/per mouse in 4-6 hours after irradiation( injection timepoint was same as follows); B BM group (n=5) was injected with 0.2ml of 6* 106 donor cells/per mouse; C group (n=5) was injected with 0.2ml of 2*106 mMMSC/per mouse; D mMMSC+BM group (n= 10) was injected with 0.2ml of 2xl06mMMSC+6xl06 BM /per mouse. 4. (1) At 3rd day after transplantation, 20f.il peripheral blood was collected from the retro-orbital plexus for blood routine evaluated. From then on, the peripheral blood was extracted once every week, until 42th day after transplantation. (2) Mice were sacrificed at 42th day after transplantation or dying mouse at any time, whose femurs, livers and spleens were fixed in 10% formaldehyde, sectioned, and stained with hematoxylin and eosin for histological studies. (3) At 42th day post-transplantation, bone marrow was evaluated for R band chromosome analysis, and sex-determining region of Y chromosome(sry) gene PCR in bone marrow, peripheral blood and spleen. (4) Mice were sacrificed at 42th day post-transplantation in mMMSC group, CD45cells sorted with MACS from bone marrow and peripheral blood were evaluated with R bind chromosome analysis, and detected by FISH using murine chromosome Y painting probe. 5.Statistics analysis: Comparisions were made using single-factor variance analysis. The reported P value was 2-sided (a = 0.05). All calculations v/ere performed using SPSS11.5. software. Results 1. After lethally irradiation, all mice in control group died between 14 and 17th days after transplantation. Before death, eytopenia was observed in peripheral blood. Hyperemia and congestion in medullary sinus was found on pathological slide of femur after HE staining, indicating all mice died of bone marrow failure. 2 WBC and HB were recovered to (4.03±0.61)x109/L and (127.33±24.00)g/L at 21th day after transplantation in mMMSC group, respectively. While at 28th day , PLT was recovered to (812.33±67.38)xl09/L. The count of WBC on 14th and 21th day in mMMSC group were lower than that of BM and mMMSC+BM groups(.P<0.05 ). The count of PLT in mMMSC group on 21th day was lower than that of BM and mMMSC groups; while, there was no significant difference on 28th ,35th and 42th day. 3. The recovery of WBC and PLT in mMMSC+BM group was quicker than that ofBM group (P<0.05), while on 28th ,35th and 42th day, there was no significant difference in WBC and PLT recovery (/>>0.05 ) . 4. There was no significant difference in HB after transplantation between groups (P>0.05) .5. Highly purified CD45+cells were sorted with MACS from bone marrow and peripheral blood in mMMSC group. FISH using the murine chromosome Y painting probe ascertained the being of CD45+Y+ cells, which positive rates were 31.18+1.83% and 24.65 + 2.77%, respectively. That confirmed mMMSC can differentiate into hematopoietic cells in vivo. Part three The engraftment and distribution of mMMSC/EGFP in vivoMethods 1. The supernatant of package cell line PT67/EGFP which constantly express pLNCG Cl transfected the mMMSCs. 2. The morphology of mMMSC/EGFP was observed, as well as cell growth curve, phenotype and multipotency. 3. The female C57BL/6 mice as recipients were defined into three groups, (l)control group(n=5) received 0.2ml PBS in 4-6 hours after 3.75Gy irradition(transplantation timepoint was same as follows); (2)mMMSC/EGFP - 3.75group(n=10) received 0.2ml of 2*106mMMSC/EGFP/per mouse after 3.75Gy irradition; (3)mMMSC/EGF-8.0 group (n=10) received 0.2ml of 2*106 mMMSC/EGFP/per mouse after 8Gy irradiation. 4. The engraftment and distribution of mMMSC/EGFP were detected at variable time points (24h> 7d. 14d, 28d^ 42d^ 56d post-transplantation) :(1 )The expression of EGFP in frozen slide were performed by laser confocal microscope; (2) DNA was extracted from bone marrow, peripheral blood , liver, spleen, lung, kidney, heart, brain and skin. Murine Y chromosone specific gene SRY was amplified with PCR. 5. mMMSC/GFP derived cells in BM and peripheral blood were detected with FACS on 42th day after transplantation. 6. Statistical analysis: Comparisions were made using single-factor variance analysis. The reported P value was 2-sided(a=0.05). All calculations were performed using SPSS 11.5 software. Results 1. mMMSCs were successfully transfected with an EGFP retroviral gene, and novel stain mMMSC/EGFP was established. 2. The morphology, cell growth curve, and phenotype were similar with that of cultured mMMSCs, and its multipotency was maintained. 3. mMMSC/EGFP transplanted mice was measured with PCR: (1) mMMSC/EGFP-3.75 group: SRY gene was detectable in liver, lung, kidney, and peripheral blood of recipient after 24 hours post-transplantation; and in bone marrow ,spleen and small intestine after 7 days; in skin after 28 days. But it were negative in cerebrum, myocardium and skeletal muscle. (2)... |
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