| Mesenchymal stem cells (MSCs) are defined as a group of multi-potent stem cells that can functionally differentiate into at least bone, fat and cartilage in vitro and in vivo. As has been reported, stem cells and their precursor present in the hematopoietic development locus for AGM region in murine embryo. As the main component for AGM region, DA (dorsal aorta) region is critical in hematopoietic stem cell (HSC) development. Mesenchymal play an important role for the HSC generation. The elucidation of stem cell function in stromal environment has become a pressing issue in embryonic hematopoietic development.To identify more differentiation potential stem cells than MSC in murine embryonic DA region, the adherent cells from DA region in mouse 11.5dpc embryos were isolated, and then transfected by pSV3neo-SV40. The clones with G418 resistance were selected, and then characterized their reproductive activity, phenotype and multilineage differentiation potentials. Futhermore, the G418 resistance adherent cells were irradiated as a feeder layer to support the cells from mouse 10.5dpc embryonic DA region. Selected the clones which were different from G418 resisitance adherent cells, their reproductive activity, phenotype and multilineage differentiation potentials were investigated. The content we investigated and tested were included: morphological features, reproductive activity, phenotypes, multilineage differentiation potentials, the expression of special genes analyzed by RT-PCR, immunofluorescent staining, phagocytosis ability, angiogenic ability on the matrigel, their ability to generate Weibel-Palade body analyzed by electron microscopy, colony forming capacity, and their potential to differentiate into blood cells with bone marrow adherent cells co-cultured.The result showed that two kinds of single colony adherent cells which were named mDAF3 and mDAF18 were obtained and displayed the typical fibroblastoid morphology, the growth kinectics of single colony derived adherent cells was measured, the mean cumulative time of population doublings was 24 hours. The immunophenotype of the mDAF3 and mDAF18 was determined by flow cytometry. The cultured expanded adherent cells were stained positive for CD29, CD44, CD105, Sca-1, CD31 and CD34. mDAF3 and mDAF18 were capable of differentiating into adipocytes, osteocytes and chondrocytes. Adipogenic differentiation of the adherent cells was indicated by accumulation of oil-red-O staining lipid-rich vesicles. Under osteogenic condition for 4 weeks, they also could form aggregates or nodules displaying alkaline phosphatase activity. Finally, formation of type II collagen in the extracellular matrix of induced cells could be detected by Alcian blue staining after cultured in chondrogenic medium. In agreement with the results of differentiation assays, RT-PCR also demonstrated that the adherent cells displayed specific adipogenic and osteogenic inductive cultures, respectively. The morphologic, immunophenotypic, and differentiation assays described strongly indicated that the adherent cells were MSC resembling cells.Moreover, the other two kinds of colony cells which were named mDAV6 and mDAV8 were selected on mDAF3 as a feeder layer culture system. These adherent cells also displayed fibroblast-like morphology, and the mean cumulative time of population doublings was 22～24 hours. The immunophenotype of the adherent cells was expressed MSC phenotype, and also displayed the hemotopoietic and endothelial markers, i.e. CD31, CD34, CD45, CD11b, Flk-1 and CD144. Functionally, they could be induced into adipocytes, osteocytes, and chondrocytes. RT-PCR also indicated that the adherent cells displayed specific adipogenic and osteogenic markers. They can be formed tube-like and network structures in Matrigel, also can generate Weibel-Palade body which was specific marker for endothelial cells. The hemotopoietic markers changed after cells co-cultured with bone marrow adherent cells to test the hematopoietic differentiating ability for mDAV6 and mDAV8.In conclusion, the single colony MSC like adherent cells was obtained from murine embryonic DA region, this observation provide evidence for MSC residing in the murine embryonic DA region, and we established DA region derived MSC cell model to dissect hematopoietic and immunoregulatory features. Furthermore, the multipotential stem cells from mouse embryonic DA region were isolated and displayed multipotential differentiation capacity, which indicate that there may be MSC precursor cells in the embryonic DA region. The intrinsic discrepancy of mesenchymal precursors in embryonic DA region may contribute to the research on the modulation activity of DA microenvironment for embryonic hematopoiesis... |
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