ABSTRACT
This study established superparamagnetic iron oxide (SPIO)-labeled nerve growth factor-β (NGF-β) gene-modified spinal cord-derived neural stem cells (NSCs).The E14 rat embryonic spinal cord-derived NSCs were isolated and cultured.The cells of the third passage were transfected with plasmid pcDNA3-hNGFβ by using FuGENE HD transfection reagent.The expression of NGF-β was measured by immunocytochemistry and Western blotting.The positive clones were selected,allowed to proliferate and then labeled with SPIO,which was mediated by FuGENE HD transfection reagent.Prussian blue staining and transmission electron microscopy (TEM) were used to identify the SPIO particles in the cells.The distinctive markers for stem cells (nestin),neuron (β-Ⅲ-tubulin),oligodendrocyte (CNPase) and astrocyte (GFAP) were employed to evaluate the differentiation ability of the labeled cells.The immunocytochemistry and western blotting showed that NGF-β was expressed in spinal cord-derived NSCs.Prussian blue staining indicated that numerous blue-stained particles appeared in the cytoplasma of the labeled cells.TEM showed that SPIO particles were found in vacuolar structures of different sizes and the cytoplasma.The immunocytochemistry demonstrated that the labeled cells were nestin-positive.After differentiation,the cells expressed β-Ⅲ-tubulin,CNPase and GFAE It was concluded that the SPIO-labeled NGF-β gene-modified spinal cord-derived NSC were successfully established,which are multipotent and capable of self-renewal.
ABSTRACT
Objective To induce directional differentiation from spinal cord-derived neural stem cells(SNSCs)into neurocytes in the simulated microenvironment of development of embryonic spinal cord of rat after isolation and culture of SNSCs and to investigate the proliferation and differentiation of SNSCs in vitro.Methods SNSCs were isolated mechanically from rat embryonic(E10 to 11d)spinal cord under microscope.SNSCs were cultured and maintained in serum-free medium.Directional differentiation from SNSCs to neurocytes was induced by adding N4 supplement.The morphologic features of the differentiated cells were noted.Cultured and differentiated cells were identified by immunochemistry stain and t he mean differentiating percentages of the positive cells were calculated.Resul ts SNSCs isolated by the mechanical method under microscope were vigorous and pr oliferative,and could differentiate into neurons,astrocytes and oligodendrocyt es.Conclusion The mechanical method under microscope of isolating SNSCs is simp le and efficient to obtain a high percentage of neurons.N4 supplement can induc e SNSCs to differentiate into neurocytes.