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. 2016 Apr;11(4):591-6.
doi: 10.4103/1673-5374.180743.

Nerve growth factor promotes in vitro proliferation of neural stem cells from tree shrews

Liu-Lin Xiong  1 , Zhi-Wei Chen  2 , Ting-Hua Wang  3
Affiliations

Nerve growth factor promotes in vitro proliferation of neural stem cells from tree shrews

Liu-Lin Xiong et al. Neural Regen Res. 2016 Apr.

Abstract

Neural stem cells promote neuronal regeneration and repair of brain tissue after injury, but have limited resources and proliferative ability in vivo. We hypothesized that nerve growth factor would promote in vitro proliferation of neural stem cells derived from the tree shrews, a primate-like mammal that has been proposed as an alternative to primates in biomedical translational research. We cultured neural stem cells from the hippocampus of tree shrews at embryonic day 38, and added nerve growth factor (100 μg/L) to the culture medium. Neural stem cells from the hippocampus of tree shrews cultured without nerve growth factor were used as controls. After 3 days, fluorescence microscopy after DAPI and nestin staining revealed that the number of neurospheres and DAPI/nestin-positive cells was markedly greater in the nerve growth factor-treated cells than in control cells. These findings demonstrate that nerve growth factor promotes the proliferation of neural stem cells derived from tree shrews.

Keywords: cell number; cell proliferation; cell therapy; embryo; hippocampus; in vitro; nerve growth factor; nerve regeneration; neural regeneration; neural stem cells; neurosphere; tree shrews.

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Conflict of interest statement

Conflicts of interest: None declared.

Figures

Figure 1
Figure 1
Dynamic temporal changes of growth of cultured tree shrew NSCs under the light field of the immunofluorescent microscope. (A) Cultured NSCs at 12 hours were mainly single cells, small and transparent, and round or oval in shape. (B) Tens of cells aggregated to form neurospheres after 2 days in culture. (C) Neurospheres enlarged over time; the surviving neurospheres showed suspended growth after 7 days in culture. NSCs: Neural stem cells. Scale bar: 100 μm.
Figure 2
Figure 2
Identification of third-passage tree shrew NSCs after 3 days in culture (immunofluorescence staining). (A) Neurospheres were positive for the NSC marker nestin (red fluorescence). (B) DAPI nuclear staining (blue fluorescence). (C) Merged image for nestin and DAPI, showing the cultured NSCs. NSCs: Neural stem cells; DAPI: 4′,6-diamidino-2-phenylindole. Scale bar: 50 μm.
Figure 3
Figure 3
Morphological changes of NSCs at 3 days in culture under the light field of immunofluorescent microscope. (A) Control group. Single cell and small cell clumps were visible in cultured control NSCs. (B) NGF-NSC group. Aggregation of multiple neurospheres was observed, with strong refraction. NSCs: Neural stem cells; NGF: nerve growth factor. Scale bar: 100 μm.
Figure 4
Figure 4
Effects of NGF on proliferation in cultured tree shrew-derived NSCs at 3 days. (A) Number of neurospheres in 6-well plates using an immunofluorescent microscope at 200× magnification. (B) Cell area in 6-well plates using an immunofluorescent microscope at 200× magnification. **P < 0.01, vs. control group (mean ± SD, n = 5, Student's t-test). NGF: Nerve growth factor; NSC: neural stem cells.
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