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Neurotrophin-3 gene transduction of mouse neural stem cells promotes proliferation and neuronal differentiation in organotypic hippocampal slice cultures.

Lu HX, Hao ZM, Jiao Q, Xie WL, Zhang JF, Lu YF, Cai M, Wang YY, Yang ZQ, Parker T, Liu Y - Med. Sci. Monit. (2011)

Bottom Line: The effect of NT-3 over-expression on cell proliferation and differentiation in NSCs was observed by immunohistochemistry, cell culture and organotypic hippocampal slice cultures.<br /> The characteristics of self-renewal and multiple differentiation of NSCs were well-preserved.Furthermore, cells in the NSC-NT-3 group survived in a significantly higher percentage and undertook neuronal differentiation preferably in organotypic hippocampal slice cultures.Our results suggest that the transduction of NT-3 into NSCs could effectively promote NSCs survival, proliferation, and neuronal differentiation in vitro without change of the stemness of NSCs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neurobiology, Environment and Genes Related to Diseases Key Laboratory of Education Ministry, Xi'an Jiaotong University College of Medicine, Xi'an, PR China.

ABSTRACT

Background: The transplantation of neural stem cells (NSCs) has been accepted as a promising therapeutic strategy for central nervous system disorders. However, the beneficial effect of NSC transplantation upon functional recovery is limited due to the unfavorable microenvironment (niche) at the site of trauma or degenerative disease in the brain. Combination of transplantation of NSCs with neurotrophins may overcome the hurdles of impaired cell survival and neuronal differentiation.

Material/methods: In the current study, the neurotrophin-3 (NT-3) gene was transduced into cultured mouse embryonic cortical NSCs via an AAV vector (NSC-NT-3). The effect of NT-3 over-expression on cell proliferation and differentiation in NSCs was observed by immunohistochemistry, cell culture and organotypic hippocampal slice cultures.

Results: The characteristics of self-renewal and multiple differentiation of NSCs were well-preserved. Cells in the NSC-NT-3 group proliferated faster and differentiated into more β-tubulin III-positive neurons compared to the control group in vitro. Furthermore, cells in the NSC-NT-3 group survived in a significantly higher percentage and undertook neuronal differentiation preferably in organotypic hippocampal slice cultures.

Conclusions: Our results suggest that the transduction of NT-3 into NSCs could effectively promote NSCs survival, proliferation, and neuronal differentiation in vitro without change of the stemness of NSCs. This work also offers evidence to better understand the safety and efficiency of combined treatment with NT-3 and NSCs for the central nervous system disorders.

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NT-3 genetic modification promotes cell viability, proliferation and neuronal differentiation in vitro NSCs-NT-3 exhibited a high rate of proliferation (A) and produced significantly larger neurospheres (B, 7 DIV) than the control group. *** p<0.001. The majority of NSCs-NT-3 remained nestin-positive after 7 days in differentiation medium (C). Some of them were nestin/β-tubullin III double-positive and nestin/GFAP double-positive. Nearly two-thirds of NSCs-NT-3 (67.7%) were immunoreactive with β-tubullin III, which is significantly higher than controls (31.2% and 28.4%, respectively; p<0.05). Nevertheless, no significant difference was observed between NSCs-NT-3 and controls in the proportion of GFAP-positive cells (47.6% vs. 43.5% and 40.3%; p>0.05).
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f4-medscimonit-17-11-br305: NT-3 genetic modification promotes cell viability, proliferation and neuronal differentiation in vitro NSCs-NT-3 exhibited a high rate of proliferation (A) and produced significantly larger neurospheres (B, 7 DIV) than the control group. *** p<0.001. The majority of NSCs-NT-3 remained nestin-positive after 7 days in differentiation medium (C). Some of them were nestin/β-tubullin III double-positive and nestin/GFAP double-positive. Nearly two-thirds of NSCs-NT-3 (67.7%) were immunoreactive with β-tubullin III, which is significantly higher than controls (31.2% and 28.4%, respectively; p<0.05). Nevertheless, no significant difference was observed between NSCs-NT-3 and controls in the proportion of GFAP-positive cells (47.6% vs. 43.5% and 40.3%; p>0.05).

Mentions: During the assessment of cell survival and proliferation, NSCs were kept in culture for 8 days without medium replacement. Dark-colored neurospheres which contained more dead cells surrounded by abundant cell debris were observed in both blank controls and the AAV-GFP-transduced groups at 5 DIV. However, in the AAV-NT-3-transduced group, neurospheres were lighter colored and surrounded by little debris, even at 10 DIV. The trypan blue staining during the cell counting further confirmed that NSC-NT-3 contained more viable cells compared with the controls. The growth curve of the NSCs (passage 4), as well as the neurosphere diameter, was measured in the different groups throughout the 8-d growth period to evaluate cell proliferation. NSC-NT-3 proliferated at a higher rate compared with the NSC-GFP (AAV-GFP-transduced) and blank control cultures at 4 DIV (Figure 4A, n=3, p<0.05). At 6 DIV, more and larger-sized spherical cell clusters were visible in NSC-NT-3 than controls, likely due to the greater increase in cell numbers (Figure 4B, n=30, p<0.001).


Neurotrophin-3 gene transduction of mouse neural stem cells promotes proliferation and neuronal differentiation in organotypic hippocampal slice cultures.

Lu HX, Hao ZM, Jiao Q, Xie WL, Zhang JF, Lu YF, Cai M, Wang YY, Yang ZQ, Parker T, Liu Y - Med. Sci. Monit. (2011)

NT-3 genetic modification promotes cell viability, proliferation and neuronal differentiation in vitro NSCs-NT-3 exhibited a high rate of proliferation (A) and produced significantly larger neurospheres (B, 7 DIV) than the control group. *** p<0.001. The majority of NSCs-NT-3 remained nestin-positive after 7 days in differentiation medium (C). Some of them were nestin/β-tubullin III double-positive and nestin/GFAP double-positive. Nearly two-thirds of NSCs-NT-3 (67.7%) were immunoreactive with β-tubullin III, which is significantly higher than controls (31.2% and 28.4%, respectively; p<0.05). Nevertheless, no significant difference was observed between NSCs-NT-3 and controls in the proportion of GFAP-positive cells (47.6% vs. 43.5% and 40.3%; p>0.05).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3539508&req=5

f4-medscimonit-17-11-br305: NT-3 genetic modification promotes cell viability, proliferation and neuronal differentiation in vitro NSCs-NT-3 exhibited a high rate of proliferation (A) and produced significantly larger neurospheres (B, 7 DIV) than the control group. *** p<0.001. The majority of NSCs-NT-3 remained nestin-positive after 7 days in differentiation medium (C). Some of them were nestin/β-tubullin III double-positive and nestin/GFAP double-positive. Nearly two-thirds of NSCs-NT-3 (67.7%) were immunoreactive with β-tubullin III, which is significantly higher than controls (31.2% and 28.4%, respectively; p<0.05). Nevertheless, no significant difference was observed between NSCs-NT-3 and controls in the proportion of GFAP-positive cells (47.6% vs. 43.5% and 40.3%; p>0.05).
Mentions: During the assessment of cell survival and proliferation, NSCs were kept in culture for 8 days without medium replacement. Dark-colored neurospheres which contained more dead cells surrounded by abundant cell debris were observed in both blank controls and the AAV-GFP-transduced groups at 5 DIV. However, in the AAV-NT-3-transduced group, neurospheres were lighter colored and surrounded by little debris, even at 10 DIV. The trypan blue staining during the cell counting further confirmed that NSC-NT-3 contained more viable cells compared with the controls. The growth curve of the NSCs (passage 4), as well as the neurosphere diameter, was measured in the different groups throughout the 8-d growth period to evaluate cell proliferation. NSC-NT-3 proliferated at a higher rate compared with the NSC-GFP (AAV-GFP-transduced) and blank control cultures at 4 DIV (Figure 4A, n=3, p<0.05). At 6 DIV, more and larger-sized spherical cell clusters were visible in NSC-NT-3 than controls, likely due to the greater increase in cell numbers (Figure 4B, n=30, p<0.001).

Bottom Line: The effect of NT-3 over-expression on cell proliferation and differentiation in NSCs was observed by immunohistochemistry, cell culture and organotypic hippocampal slice cultures.<br /> The characteristics of self-renewal and multiple differentiation of NSCs were well-preserved.Furthermore, cells in the NSC-NT-3 group survived in a significantly higher percentage and undertook neuronal differentiation preferably in organotypic hippocampal slice cultures.Our results suggest that the transduction of NT-3 into NSCs could effectively promote NSCs survival, proliferation, and neuronal differentiation in vitro without change of the stemness of NSCs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neurobiology, Environment and Genes Related to Diseases Key Laboratory of Education Ministry, Xi'an Jiaotong University College of Medicine, Xi'an, PR China.

ABSTRACT

Background: The transplantation of neural stem cells (NSCs) has been accepted as a promising therapeutic strategy for central nervous system disorders. However, the beneficial effect of NSC transplantation upon functional recovery is limited due to the unfavorable microenvironment (niche) at the site of trauma or degenerative disease in the brain. Combination of transplantation of NSCs with neurotrophins may overcome the hurdles of impaired cell survival and neuronal differentiation.

Material/methods: In the current study, the neurotrophin-3 (NT-3) gene was transduced into cultured mouse embryonic cortical NSCs via an AAV vector (NSC-NT-3). The effect of NT-3 over-expression on cell proliferation and differentiation in NSCs was observed by immunohistochemistry, cell culture and organotypic hippocampal slice cultures.

Results: The characteristics of self-renewal and multiple differentiation of NSCs were well-preserved. Cells in the NSC-NT-3 group proliferated faster and differentiated into more β-tubulin III-positive neurons compared to the control group in vitro. Furthermore, cells in the NSC-NT-3 group survived in a significantly higher percentage and undertook neuronal differentiation preferably in organotypic hippocampal slice cultures.

Conclusions: Our results suggest that the transduction of NT-3 into NSCs could effectively promote NSCs survival, proliferation, and neuronal differentiation in vitro without change of the stemness of NSCs. This work also offers evidence to better understand the safety and efficiency of combined treatment with NT-3 and NSCs for the central nervous system disorders.

Show MeSH
Related in: MedlinePlus