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

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.<br />

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.

Figure 1

Successful transduction of NSCs by AAV Cells isolated from E14.5 mouse embryo cortex proliferated into neurospheres at 7 DIV (A) and most of these cells were nestin positive NSCs (B). Neurospheres were trypsinized into single cells 12 h prior to AAV transduction. At 3 DPI, up to (B) 16.1% of NSCs were transduced by AAV-GFP and (C) 15.3% of NSCs were transduced by AAV-NT-3. E: embryonic day. DIV: day in vitro. DPI: day post-infection. B Scale bar, 30 um.

Figure 2

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.

Figure 3

Multiple differentiation of NSCs following AAV transduction (A–D) NSCs differentiated into various cell types with various morphological appearances following 7 days in differentiation medium. Immunocytochemistry further confirmed that these cells were: GFAP-positive astrocytes (E) and β-tubullin III-positive neurons (F). E,F Scal bar, 100 um

Figure 4

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).

Figure 5

The survival and differentiation of NSCs-NT-3 in orgnaotypic hippocampal slice cultures Hippocampal slice cultures remained healthy after addition of CM-DiI labeled neurospheres (A). CM-DiI labeled neurospheres (insert in B) appeared to all over the slices and more NSCs-NT-3 survived after transplanted into organotypic hippocampal slices than controls (B). Some of them differentiated into β-tubulin positive neurons (C) and GFAP positive astrocytes (D). A Scal bar, 100 um; B Scal bar, 50 um; C and D Scal bar, 20 um.