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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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Related in: MedlinePlus

Expression of NT-3 Secreted NT-3 in the medium of AAV-NT-3, AAV-GFP and blank control groups was detected by ELISA at 3, 7, 14 and 28 DPI, respectively. AAV-NT-3-transduced NSCs produced a higher level of NT-3 compared with control groups. No significant difference was found between AAV-GFP and blank control. DPI: day post infection. *** p<0.001.
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f2-medscimonit-17-11-br305: Expression of NT-3 Secreted NT-3 in the medium of AAV-NT-3, AAV-GFP and blank control groups was detected by ELISA at 3, 7, 14 and 28 DPI, respectively. AAV-NT-3-transduced NSCs produced a higher level of NT-3 compared with control groups. No significant difference was found between AAV-GFP and blank control. DPI: day post infection. *** p<0.001.

Mentions: NSCs isolated from E14.5 mouse embryo cortex were cultured in growth medium. A large number of neurospheres emerged after 5–7 d in vitro (DIV; Figure 1A) and immunocytochemistry confirmed that most of these cells were nestin-positive NSCs (Figure 1B). Neurospheres (passage 3) were trypsinized into single cells 12 h prior to transduction. During the first 24 h following transduction, cell debris was observed in both AAV-NT-3 and AAV-GFP-transduced groups. At 72 h following transduction, results of FACS demonstrated that 15.3% and 16.1% of NSCs expressed GFP in AAV-NT-3- and AAV-GFP-transduced groups, respectively, indicating successful transduction (Figure 1C,D). The concentration of NT-3 protein, determined by ELISA, in the medium of NSC-NT-3 was 37.6 pg/ml at 3 DPI. It elevated slightly at 7 DPI (40.7 pg/ml) and then remain at a relatively high level until 28 DPI (33.8 pg/ml); it was significantly higher than that in control groups. (Figure 2, n=3, 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)

Expression of NT-3 Secreted NT-3 in the medium of AAV-NT-3, AAV-GFP and blank control groups was detected by ELISA at 3, 7, 14 and 28 DPI, respectively. AAV-NT-3-transduced NSCs produced a higher level of NT-3 compared with control groups. No significant difference was found between AAV-GFP and blank control. DPI: day post infection. *** p<0.001.
© Copyright Policy
Related In: Results  -  Collection

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

f2-medscimonit-17-11-br305: Expression of NT-3 Secreted NT-3 in the medium of AAV-NT-3, AAV-GFP and blank control groups was detected by ELISA at 3, 7, 14 and 28 DPI, respectively. AAV-NT-3-transduced NSCs produced a higher level of NT-3 compared with control groups. No significant difference was found between AAV-GFP and blank control. DPI: day post infection. *** p<0.001.
Mentions: NSCs isolated from E14.5 mouse embryo cortex were cultured in growth medium. A large number of neurospheres emerged after 5–7 d in vitro (DIV; Figure 1A) and immunocytochemistry confirmed that most of these cells were nestin-positive NSCs (Figure 1B). Neurospheres (passage 3) were trypsinized into single cells 12 h prior to transduction. During the first 24 h following transduction, cell debris was observed in both AAV-NT-3 and AAV-GFP-transduced groups. At 72 h following transduction, results of FACS demonstrated that 15.3% and 16.1% of NSCs expressed GFP in AAV-NT-3- and AAV-GFP-transduced groups, respectively, indicating successful transduction (Figure 1C,D). The concentration of NT-3 protein, determined by ELISA, in the medium of NSC-NT-3 was 37.6 pg/ml at 3 DPI. It elevated slightly at 7 DPI (40.7 pg/ml) and then remain at a relatively high level until 28 DPI (33.8 pg/ml); it was significantly higher than that in control groups. (Figure 2, n=3, 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