The Separation, Culture And Identification Of Neural Stem Cells In Sprague-Dawley

Parkinson’s disease (PD) is a degenerative disease of the nervous system which is related to age. Past studies suggest that adult mammalian’s central nervous system cells don’t have the ability of regeneration.When the factors of injury, disease and natural apoptosis are decreasing in adult mammalian’s nerve cells of brain or spinal cord,can not be renewed and replaced by other cells,thus appearing neurological degenerative diseases.Neural stem cells (NSCs) have the potential of multi-differentiation,which produce progeny of neural stem cells, neurons,astrocytes and oligodendrocytes by symmetric division and asymmetric division.If we can induce neural stem cells directional differentiation and transplant them into body,may provide a new approach for the treatment of Parkinson’s disease which is a representative for degenerative disease of the nervous system.ObjectiveIsolating neural stem cells from midbrain in pregnant 14d embryonic Sprague-Dawley(SD) rats,culturing, inducing differentiation and identification.Methods1.Isolating neural stem cells:Decapitating pregnant 14d embryonic SD rats, soaking 15min in 75% ethanol,removing the whole brain with sterile conditions.Put it into dish with PBS, remove cerebellum, brainstem, meninges, blood vessels and others, dissect embryonic ventral midbrain tissue with sterile conditions,clip fragments of 1mm3. Wash them by D-Hanks.Pipet by straw with flame polishing and prepare single cell suspension by 200 mesh screen filter.Centrifuge (800r/min,centrifuged radius 10cm)5minx3times,remove supernatant,adjust cell density into 2×105/ml.Neural stem cell complete medium DMEM/F12(Hyclone)、 1%N2(Gibeo)、20ng/mlEGF(R&D Systems、20 ng/mlalkaline fibroblast growth factor(R8LD Systems)], culture in 37℃,5%CO2.2.Culturing neural stem cells:culture by medium(DMEM/F12, alkaline fibroblast growth factor,EGF、N2)without serum.Observe cells growth every day, change 2～3d with half the amount,passage 5-7d.When passage,centrifuge and collect neurospheres, mechanical pipet into single cell.Passage with 1×105/ml, culture in 37℃,5%CO2.By Comparison of separation,observe cell morphology with Inverted microscope, determine growth curve of 3rd generation cell.3. Identificate neural stem cells:centrifuge and collect neurospheres, resuspend by neural stem cells differentiation medium VDMEM/F12、1%FBS (Gibco)、1%N2, inoculate coverslip in plates which prepositioned poly-lysine coated, culture in 37℃,5%CO21 h, identificate by Nestin(Abeam) immunocytochemistry.4. Induced differentiation neural stem cells:centrifuge and collect neurospheres, resuspend by neural stem cells differentiation medium VDMEM/F12、1%FBS (Gibco)、1%N2, inoculate coverslip in plates which prepositioned poly-lysine coated,culture in 37℃,5%CO27day. Observe differentiation of cells every day, after 7days, respectively identificate differentiation of cells by CD11c、GFAP and NeuN immunocytochemistry.Results1. Isolation and culture of neural stem cells:After 24 h, Most cells are adherent, exist by single or in pairs; After 48 h,observe dumbbell-shaped cell division phase; After 72h, dozens of cell aggregate into cell clusters, suspended growth; continue culture, rapid increase in cells, cell mass are increasing and form neurospheres; after 6～7 d neurospheres grow to about 200μm to passages.2. Identificate neural stem cells:3rd neurospheres culture with neural stem cells differentiation medium,after 1h cells are adherent.Immunocytochemistry with neural stem cell-specific markers Nestin,resulting in positive.3. Induced differentiation neural stem cells:3rd neurospheres culture with neural stem cells differentiation medium,after 1h cells are adherent. After 72h, a large number of cells from the neurospheres grow apophysis. After 7d, Immunocytochemistry, resulting in neurons positive by NeuN immunoreactivity,astrocytes positive by GFAP immunoreactivity,oligodendrocytes positive by CD11c immunoreactivity.ConclusionIn conclusion:Cells by isolated and cultured through neural stem cells specific markers Nestin Immunocytochemistry,resulting in positive.With strong proliferation and differentiation potential, prove there exist neural stem cells in SD embryonic rat brain,may provide a new approach for the treatment of Parkinson’s disease which is a representative for degenerative disease of the nervous system.