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Non adherent bone marrow mesenchymal stem cells differentiation into neural cells in vitro

Author: WangYan
Tutor: ZuoXiaoMing;MiaoDengShun
School: Nanjing Medical University
Course: Pediatrics
Keywords: non-adherent BM-MSCs CFU-F pluripotent EGF proliferation Non-adherent bone marrow stem cells differentiation neuron-like cells non-adherent mesenchymal stem cells ischemic brain neuron-like cells
CLC: R329
Type: Master's thesis
Year: 2008
Downloads: 74
Quote: 1
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Abstract


PartⅠIsolation and identification of Non-adherent Bone-Marrow Mesenchymal Stem Cells and the regulation of EGF on the CFU-F forming efficiencyObjective:To determine whether non-adherent bone marrow mesenchymal stem cells（NA-BM-MSC）exist in non-adherent bone marrow cells （NA-BMC）from mouse,and whether the forming effiency of colony-forming units-fibroblast（CFU-F）could be regulated by Epidermal Growth Factor（EGF）.Methods:Bone marrow cells of mouse were isolated and cultured with or without EGF in vitro,non-adherent bone marrow cells（NA-BMC）were repeatedly-transferred into new dishes every day for 5 times,every dish was stained with methylene blue after 12 days cell culture for CFU-F.For the detection of differentiation,NA-BMC were collected at 4th day of the culture without EGF,3 conditioned medium were used to induce the NA-BMC into adipocyte,chondrocyte and osteoblast cells.Oil red staining,immunohistochemical staining of typeⅡcollagen and alizarin red staining were developed respectively to detect the differentiation of NA-BMCs.Results:In the process of repeatedly-transfer and culture,not only total bone marrow cells proliferated to CFU-F,NA-BMCs became the adherent cells and proliferated to CFU-F continuously.With appropriate condition they differentiated into cells which were typical for adipocyte,chondrocyte and osteoblast in vitro.Compared with the control,the clone number from the group with EGF increased significantly after every transfer and the adherent cells increased 3 times.Conclusion:Non-adherent bone marrow mesenchymal stem cells（NA-BM-MSC）exist in non-adherent bone marrow cells（NA-BMC）of mouse;NA-BMC could form CFU-F and have the pluripotent ability of differentiating into adipocyte,chondrocyte and osteoblast cells,which is typical for NA-BM-MSC.EGF can improve the proliferation of NA-BM-MSC significantly.PartⅡDifferentiation of Non-adherent Bone Marrow Mesenchymal Stem Cells into Neuron-like Cells in vitroObjective To determine whether NA-BM-MSC can differentiate into neuron-like cells' dissertation">neuron-like cells in vitro.Methods Bone marrow cells（BMC）of mouse were isolated and cultured.NA-BMC were collected at 4th day of the culture and cultured in new dish for another 4 days,and the adherent cells from total bone marrow cells were cultured with new medium.When become confluent,the cells were digested for passaging and purifying.The 4thpassage cells were induced at the density of 1X103/cm2 with conditioned medium with EGF and fibroblast growth factor-2（b-FGF）for 14days. Immunocytochemistry staining and toluidine blue staining were developed respectively to detect the expression of the neuron-specific proteins,such as NeuN and NF-200 and Nissl body.Results In mice,not only tocal BMC but also NA-BM-MSC can be induced into NeuN,NF-200 and Nissl body positive cells.Conclusion Our results indicate MSCs derived from NA-BMCs can be induced to differentiate into neuron-like cells in vitro. neuron-like cellsPartⅢDifferentiation of Non-adherent Bone Marrow Mesenchymal Stem Cells into Neural Cells in the isehemic brainObjective To determine whether non-adherent bone marrow mesenchymal stem cells（NA-BM-MSC）in mouse can differentiate into neuron-like cells in vivo,especicially in ischemic brain.Methods Bone marrow cells fromβ-gal transgenic mice constitutively expressingβ-galactosidase were isolated and cultured,NA-BMC were collected and transferred into a new dish at 4thday,When become confluent,the cells were digested for passaging and purifying.The 4thpassage cells were used to be transplanted into the mice model of middle cerebral artery occlusion（MCAO）,at 9th weeks after transplantation,LacZ staining and single or double immunohistochemistry staining of anti-β-Gal、anti-NeuN and anti-GFAP were performed to dectect the survival,distribution and the differentiation of the donor cells in the ischemic brain.Results LacZ staining show us NA-BM-MSCs can survive in ischemic brain and expressing the markerβ-galactosidase,numbers of the NeuN-positive and GFAP-positive cells inβ-Gal-positive cells in ischemic brain were detected with immunohistochemistry staining.Conclusion Our results indicate that NA-BM-MSCs in adult mouse bone marrow can differentiate into neuron-like cells in ischemic brain,which suggests that they can be candidates for transplantation therapy in neural diseases.

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