Isolation And Characterization Of Mouse Neural Stem Cells

Objectives:It had been long considered that the nervous system of adult animals and human was an organ lacking of regenerative capacity,and the brain could not be repaired once injured.This traditional concept has been overthrow by the discovery of neural stem/progenitor cells(NSC/NPCs).Since the concept of neural stem cell was first proposed by Reynolds in 1992,NSC/NPCs with potential self-renewal and differentiation capacity have been successfully isolated from adult brains of several mammal species including human being.Mouse neural stem cells have been widely studied,but their exact phenotype and functional natures remain unclear.The purpose of this paper,by utilizing various cellular and molecular methods,is to systematically dissect the cellular composition of NSCs in C57BL/6 mice,in order to uncover new subsets of mouse NSC/NPCs.The results from the present study will greatly enhance our understanding of NSC biology,and will also have important implications in the development of stem cell-based therapeutic strategies for neurodegenerative and other brain damage-related diseases.Methods:(1)Isolation of mouse nervous cells:mouse was sacrificed by cervical dislocation,and brain recovered.Isolated the hippocampal dentate gyrus tissues,were minced into 1-2mm3 small pieces and digested for 5min with trypsin.Single cell suspension was prepared by passing tissues through a 40?m cell strainer.(2)FACS analysis and sorting of cells:freshly prepared cells were stained with a panel of fluorescent-labeled anti-surface marker antibodies.The stained cells were then analyzed or sorted by flow cytometry analyzer or sorter.(3)In vitro proliferation and differentiation of the target cell populations:sorted target cells were seeded into 24 well-plates with a concentration of 1-2 × 105 cells/well and cultured in growth or differentiation medium.(4)Immunofluorescence assays:cells were fixed,permeated and blocked,and then incubated sequentially with primary and secondary antibodies.After adding into anti-fluorescence quenching sealing fluid,cells were observed and photographed under the fluorescence microscope.(5)Analysis of cell cycle:freshly isolated nerve cells were first stained with anti-surface marker protein fluorescent antibodies,then fixed and permeated with Fixation/Permeabilization,and finally stained with 7-aminoactinomycin D(7-AAD).The cell cycle distribution was analyzed by flow cytometry.(6)Statistical methods:Data were analyzed using SPSS 19.0 statistical software and the results were showed as mean ± standard deviation.Results:1.Identification of putative mouse NSC/NPCs.Based on current reports,we used a panel of anti-surface marker antibodies to label isolated mouse hippocampus dentate gyrus region cells,and analyzed by FACS.Two subsets were identified:CD133+GFAP+CD117+Sca-1+ cells and CD133+GFAP+CD117+Sca-1-cells,which accounted for 0.114±0.030%and 2.541 ±0.360%of brain hippocampus dentate gyrus region cells,respectively.2.Differentiation of CD133+GFAP+CD117+Sca-1+/-cells in vitro.Differentiation potential is a gold standard to evaluate stem cell's nature of a population.To determine whether the two identified cell populations of mouse hippocampus dentate gyrus region are endowed with stemness,sorted CD133+GFAP+CD117+Sca-1+ cells and CD133+GFAP+CD117+Sca-1-cells were seeded into 24 well-plate(1-2×105 cell/well),and cultured for 2-3 weeks in the presence of specific differentiation medium.As reflected by cell morphology changes,the population of CD133?GFAP+CD117+Sca-1+ cells started to differentiate 15days after culture,and almost all cells became differentiated on 20 days.Immunofluorescence staining further demonstrated that CD133+GFAP+CD117+Sca-1+ cells were able to differentiate into neurons,astrocytes and oligodendrocytes.In contrast,CD 133+GFAP+CD117+Sca-1-cells cultured in the same conditions could not form any cell type of above-mentioned.These results clearly indicated that,CD133+GFAP+CD117+Sca-1+ cells represent the native neural stem cell population resident in mouse brain.3.Proliferation of CD133+GFAP+CD117+Sca-1+ cells in vitro.Self-renewal is another signature function of stem cells.To determine the proliferation ability of CD133+GFAP+CD117+Sca-1+ cells,sorted cells were seeded into 24 well-plate(1-2×105cell/well)and cultured with growth medium.Remarkably,extensive formation of neurospheres in various shapes and sizes were detected after 20 days incubation,suggesting that CD133+GFAP+CD177+Sca-1+ cells possess potential capacity of proliferation and self-renewal.4.Cell cycle distribution of CD133+GFAP+CD117+Sca-1+ cells.Given the strong proliferation capacity of CD133+GFAP+CD117+Sca-1+ cells in vitro,we further analyzed their cell cycle distribution.The proportions of cells in G0/G1,S,G2/M phase were 97.6±1.239%,1.115±0.239%and 1.233±0.170%,respectively,within the whole hippocampus dentate gyrus cell compartment.These proportions had been changed in the CD 133+GFAP+CD117+Sca-1+ population to 73.9±3.522%?3.36±0.098%? 33.7± 1.053%,respectively.These results indicated that,the CD 133+GFAP+CD 117+Sca-1+ population contained more cells progressing into normal cell cycle(dividing cells)compared to the whole cells in mouse hippocampus dentate gyrus,providing further evidence that this cell population has self-renewal capacity and stem cell properties.5.Age-dependent change in abundance of mouse brain CD133+GFAP+CD117+Sca-1+ cells.To understand the change pattern of mouse NSCs with age,we examined the contents of CD 133+GFAP+CD 117+Sca-1+ cells in young,middle and aged mice,and CD133+GFAP+CD117+Sca-1-cell population was also tested as a control.Flow cytometry analysis showed that the two cell populations displayed a similar trend in that cell abundance was not apparently changed between young and middle age mice,but significantly decreased in aged mice.The percentage of CD133+GFAP+CD117+Sca-1+ cells among total hippocampus dentate gyrus cells were 0.114±0.030%,0.144±0.016%and 0.050±0.004%,respectively,in young,middle and aged mice(young or middle vs aged,P<0.01);while these percentages for the CD 133+GFAP+CD 117+Sca-1-population were 2.541 ±0.360%,2.329±0.071%and 0.443±0.026%,respectively,(young or middle vs aged,P<0.01).Conclusion:This study has identified a previously unreported mouse neural stem cell population.These cells,resident in the mouse hippocampus dentate gyrus area and displaying a CD133+GFAP+CD117+Sca-1+ phenotype,have proliferation and self-renewal capacity,and can differentiate into neurons,astrocytes and oligodendrocytes in vitro.The cell population was significantly reduced in terms of abundance in aged mouse.These findings will offer novel insights into neural stem cell biology,and may serve as a platform for creating stem cell-based therapies for neurodegenerative diseases.