| Mesenchymal stem cells (MSCs) originating from mesoblast, resides mainly in adultmammalian bone marrow, and coexists with hematopoietic stem cells (HSCs). Functionally MSCs not only exerts the crucial role of supporting hemopoiesis by participating in the formation of hematopoietic microenvironment, but many events showed they can be cultured and proliferated easily in vitro, autoplastic transplantation and have differentiation multipotentials into multiple cell lineages of the three germ layers, such as osteoblast, chondrocyte, adipocyte, tendon cell, muscle cell and neuron. MSCs with undifferentiated or after genetic manipulation would be one of the ideal seed cells source for cell/gene therapy and tissue engineering in future. The aim of this research is to illuminate the biological property and multipotentials of the mouse bone marrow MSCs and establishes expansion and culture condition for mouse MSCs, so as a model to apply MSCs to diseases cell or gene therapy in future. Therefore, the isolation, culture, proliferation and growth characteristics and in vitro differentiation of adult Kunming mouse marrow-derived MSCs were assayed and researched, systematically. It found an experimental base for further investigation and using of mesenchymal stem cells. The results are as follows:1. MSCs can be isolated from mouse bone marrow by cells adherence-dependent growth characters to tissue culture plates, successfully. And the MSCs can be sub-cultured and expanded stably. In our test, the mouse MSCs were successfully isolated from adult Kunming femur and tibia bone marrow and sub-cultured to the 8th passage and they have been expanded more than 5000 doublings. In morphology, the typical characteristics of the MSCs were different from other cells, such as the mouse MSCs is polygonal, triangle, or long fusiform in shape, the nucleus-plasma ratio was high and there were a few organelles in the cells, and have a strong ability of proliferation, similar to human and other animals MSCs, which were pure and well-organized cell groups, and steadily passage more than 8 passages in vitro.2. The Horse serum (HoS) or dexamethasone (DEX), 3-isobutyl-1-methylxanthine (IBMX), insulin (In), indomethacin could induce mouse MSCs to differentiate into the adipocytes. After induced with induction medium, lipid droplets appearance and the cells displayed a perinuclear accumulation of lipid vacuoles gradually, and their shape from long fusiform cells to round or polygonal, and Oil Red O stain positive. That established a model for MSCs differentiate into the adipocytes. The potential of adipocytes differentiation was an important criterion for distinguishing the bone marrow-derived mesenchymal stem cells from hematopoietic stem cells.3. Under the induction of dexamethasone (DEX), ascorbate-phosphate (Vita min C), and β-glycerophosphate (β-Gly), the mouse MSCs gradually changed their shape from polygonal, triangle, or long fusiform cells, to cubiform, then elliptic, and even rotundity. That meant the MSCs differentiated into osteoblasts, which were Von Kossa stain positive, and DEX, Vc, β-Gly were important factor for inducing MSCs differentiation into osteoblasts. MSCs would be one of the ideal seed cells source for bone tissue engineering in future.4. The mouse brain neurocytes which isolated in this research have the typical neuron characteristics and were used as positive control of immunocyte-fluorescence identification after MSCs induced differentiation into neurons successfully. This was a simple method for mouse brain neurocytes isolation and culture and satisfied to requirement of general experiment.5. The mouse MSCs can be induced to differentiate into neuron phenotype by the β-mercaptoethanol and all-trans-Retinoic acid. After the induced treatment the form of the cells were changed into neurocyte-likes and expressed the neuron molecule markers, such as NSE (neuron specific enoiase) and NF-200 (neurofilament-200), and keep invariable after subsequent culture in neural medium. But it was necessary for appraising their function to further study.In conclusion, we obtained mouse bone marrow mesenchymal stem cells successfully, which were capable of multipotential differentiation in vitro. According to the morphology and differentiation potentials, the cells derived from the adult Kunming mouse marrow should be identified as the MSCs. Although it is no doubt that the MSCs show an enormous potential applied prospect, but many works should be done before identified all biological properties of MSCs and applied in clinical practice.
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