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Journal of Neurorestoratology  2018, Vol. 6 Issue (1): 19-27    doi: 10.2147/JN.S148794
Proliferation and differentiation of human fetal brain neural stem cells in vitro
Erica L McGrath1, Junling Gao1, Ping Wu1,2
1Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, TX, USA
2Beijing Institute for Brain Disorders, Capital Medical University, Beijing, People’s Republic of China
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Primary human fetal brain neural stem cells (hNSCs) are a unique non-genetically modified model system to study molecular mechanisms underlying human neural development, to model human diseases, and to screen drugs or validate new treatments. They may also be used for cell transplantation to treat various neurological diseases. This protocol details our methods that can be used to expand hNSCs in culture as well as how to differentiate them into various neuronal lineages and astrocytes.

Key wordsneural stem cell      proliferation      differentiation      neuron      astrocyte     
Published: 26 June 2018
Corresponding Authors: Ping Wu   
About author:

*These authors contributed equally to this work

Cite this article:

Erica L McGrath, Junling Gao, Ping Wu. Proliferation and differentiation of human fetal brain neural stem cells in vitro. Journal of Neurorestoratology, 2018, 6: 19-27.

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Medium componentsVolume per 10 mL of medium
TPPS173 μL
200 mM L-Glut50 μL
10 mg/mL insulin25 μL
20 μg/mL EGF10 μL
20 μg/mL bFGF10 μL
10 μg/mL Lif10 μL
5 mg/mL heparin10 μL
Table 1Growth factors for new proliferation media
Trypsin solutionVolume for 10 million cellsVolume for 30 million cells
dPBS1 mL3 mL
10% glucose60 μL180 μL
2.5% Trypsin10 μL30 μL
DNase5 μL15 μL
Table 2Preparation of trypsin
Trypsin inhibitorVolume for 10 million cellsVolume for 30 million cells
CM1 mL3 mL
Trypsin inhibitor10 μL30 μL
Table 3Preparation of trypsin inhibitor
Medium componentsVolume for 1 vialVolume for 3 vials
DFHGPS0.7 mL2.1 mL
FBS 20%0.2 mL0.6 mL
DMSO 10%0.1 mL0.3 mL
Table 4Preparation of freezing media
Medium componentsVolume
TPPS17.3 μL
200 mM L-Glut5 μL
10 mg/mL insulin2.5 μL
20 μg/mL EGF1 μL
10 μg/mL Lif1 μL
1 mg/mL laminin1 μL
Table 5ELL priming media
Medium componentsVolume
TPPS17.3 μL
200 mM L-Glut5 μL
10 mg/mL insulin2.5 μL
20 μg/mL bFGF0.5 μL
5 mg/mL heparin0.5 μL
1 mg/mL laminin1 μL
Table 6FHL priming media
ComponentFinal concentrationAmount
N2 supplement1%1 mL
GlutaMax-I supplement2 mM1 mL
FBS1%1 mL
Table 7StemPro NSC SFM media components
Figure 1Representative bright field image of hNSC sphere in culture.
Figure 2Representative bright field and fluorescent images of hNSCs following various differentiation treatments.
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