| Mesenchymal stem cells (MSCs) are highly attractive candidates for regenerated medicine approaches because they exhibit multilineage differentiation capacity. MSCs can be induced to differentiate into bone, adipose and cartilage if these cells are cultured under specific permissive conditions. The main source of MSCs has traditionally been the bone marrow (BM). The major disadvantages of BM as the source of MSCs, however, are the invasive collection procedure, high degree of viral exposure, and significant decrease in cell number, proliferation, and differentiation capacity with progressing age. This has spurred an ongoing search for alternative sources of MSCs for the clinical application. Recent researches showed that MSCs could be isolated from human placenta. Placenta-derived mesenchymal stem cells (PMSCs) also exhibit multilineage differentiation potentials as BM-derived MSCs.MSCs are the progenitor of most cell components in the hematopoietic microenviroment. Through direct interaction with hematopoietic cells, secreting extracellular matrix and factors, MSCs maintain the integrity of hematopoietic microenvironment and regulate hematopoiesis accurately. Recently, MSCs have been studied for their potential application to support hematopoiesis both in vitro and in vivo. In this study, PMSCs were isolated from human placenta successfully, whose effects on supporting hematopoiesis were investigated both in vitro and in vivo.This study was divided into two parts. In the first part, human PMSCs were isolated and identified firstly, followed by the investigation of the role of human PMSCs in supporting the growth of mononuclear cells (MNCs) from umbilical cord blood (UCB) in vitro. The placenta tissues were digested by collagenase IV and cultured with low-glucose DMEM supplemented with b-FGF. The adherent cells were collected and passaged. The phenotypes of the cultured cells were detected by flow cytometry. The osteogenic differentiation was induced withβ-glycerol phosphate disodium salt-pentahydrate, vitamin C and dexamethasone whereas the adipogenic differentiation was induced with 3-isobutyl-1-methylxanthine (IBMX), insulin, indomethacin and dexamthasone. The osteogenic differentiation was detected with alkaline phosphate assay and von kossa staining. The adipogenic differentiation was observed with Oil red O staining. Umbilical cord blood-derived MNCs (UCB-MNCs) isolated by Ficoll density centrifugation were cocultured with PMSCs as feeder layer. The growth characteristics were analyzed by cell count and flow cytometry. The results showed that: 1. PMSCs were isolated and expanded from human placenta successfully, which were adherent fibroblast-like cells. FACS analyses showed that the phenotypes of PMSCs were CD29~+, CD44~+, CD105~+, CD106~+, CD166~+, CD34~-, CD45~-, HLA-DR~-. 2. These cells were highly positive for alkaline phosphate staining and also showed mineralization detected with von kossa staining after 3 weeks culture with the induction of osteogenic differentiation. Futhermore, these cells were highly positive for Oil red O staining after 2 weeks culture with the induction of adipogenic differentiation. 3. The total cell number and CD45~+ cells of the PMSCs+UCB-MNCs coculture group at the different culture time points were significantly higher than that of UCB-MNCs alone group. On day 7, the amount of CD45~+, CD14~+and CD19~+ cells of the coculture group was also significantly higher than that of the control group separately. These results demonstrated that human PMSCs could support the growth of UCB-MNCs in vitro as feeder layer effectively.In the second part, a murine model (BALB/c→C57BL/6 ) for allogenic bone marrow transplantation was established by intra-bone marrow injection (IBMI) and intravenous injection (IV) firstly, followed by the analyses of the kinetics of hemopoietic reconstitution after IBMI or IV in the recipient mice. Secondly, the hematopoietic reconstitution of implanted SCID mice was evaluated after intra-bone marrow cavity injection of human UCB-MNCs and PMSCs. C57BL/6 recipient mice conditioned with lethal dose irradiation were transplanted with bone marrow MNCs from BALB/c mice by IBMI or IV. Sixty recipient mice were divided into three groups at random: IBMI group one (IBMI group), IBMI group two (IBM2 group), IV group. There were twenty recipient mice in every group. The total bone marrow cells per tibia of every recipient mouse were counted respectively at the 1st, 3rd, 6th and 9th day after transplantation. Donor engraftment level (total donor-derived cell number and donor-derived myeloid cell number) was analyzed by flow cytometry. SCID recipient mice conditioned with sublethal dose irradiation were transplanted with human UCB-MNCs and PMSCs by IBMI or IV. Nine recipient mice were divided into three groups at random: cotransplantation group A (PMSCs+UCB-MNCs by IBMI), single transplantation group B (UCB-MNCs by IBMI), cotransplantation groupC (PMSCs by IBMI, UCB-MNCs by IV). There were three recipient mice in every group. On day 14, the bone marrow cells of every recipient mouse were flushed out from the injected tibias and contralateral tibias, respectively. The percentage of human CD34~+ and CD45~+ hematopoietic cells was analyzed by flow cytometry. The results showed that: 1. On day 6 after allogeneic transplantation, the total bone marrow cells, total donor-derived cells and donor-derived myeloid cells in the injected tibias of IBM1 group and IBM2 group were all significantly higher than those of the IV group. 2. On day 14 after transplantation in SCID mice, the percentages of human CD34~+ and CD45~+ hematopoietic cells in the tibias of group B were both significantly lower than them in the injected tibias and contralateral tibias of group A. These results indicated that IBMI could further improve early hematopoietic reconstitution after allogenic bone marrow transplantation than IV did. Furthermore, human PMSCs could enhance the early engraftment of UCB-MNCs in SCID mice.In summary, PMSCs from human placenta tissues were isolated and expanded successfully. The result indicated that human PMSCs could support the growth of UCB-MNCs in vitro as feeder layer effectively. Moreover, with the cotransplantation of human PMSCs and UCB-MNCs into the SCID mice by IBMI, the result showed thathuman PMSCs could improve the early hematopoietic reconstitution of UCB-MNCsefficiently. These results may provide useful information to clarify the molecularmechanisms of the effects of PMSCs on supporting hematopoiesis both in vitro and invivo. |
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