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Journal of Neurorestoratology  2018, Vol. 6 Issue (1): 19-27    doi: 10.2147/JN.S148794
Methodology     
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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Abstract  

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.

URL:

http://jnr.tsinghuajournals.com/10.2147/JN.S148794     OR     http://jnr.tsinghuajournals.com/Y2018/V6/I1/19

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
DFGHPS1 mL
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
DFGHPS1 mL
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
DMEM1X97 mL
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.
1.   Nam H, Lee KH, Nam DH, Joo KM. Adult human neural stem cell therapeutics: current developmental status and prospect. World J Stem Cells. 2015;7(1):126-136.
2.   Reekmans K, Praet J, Daans J, et al. Current challenges for the advancement of neural stem cell biology and transplantation research. Stem Cell Rev. 2012;8(1):262-278.
3.   Lee Y, Dawson VL, Dawson TM. Animal models of Parkinson’s disease: vertebrate genetics. Cold Spring Harb Perspect Med. 2012;2(10). pii: a009324.
4.   Mertens J, Marchetto MC, Bardy C, Gage FH. Evaluating cell reprogramming, differentiation and conversion technologies in neuroscience. Nat Rev Neurosci. 2016;17(7):424-437.
5.   Thonhoff JR, Ojeda L, Wu P. Stem cell-derived motor neurons: applications and challenges in amyotrophic lateral sclerosis. Curr Stem Cell Res Ther. 2009;4(3):178-199.
6.   Barrows NJ, Campos RK, Powell ST, et al. A screen of FDA-approved drugs for inhibitors of Zika virus infection. Cell Host Microbe. 2016;20(2):259-270.
7.   McGrath EL, Rossi SL, Gao J, et al. Differential responses of human fetal brain neural stem cells to Zika virus infection. Stem Cell Reports. 2017;8(3):715-727.
8.   Svendsen CN, ter Borg MG, Armstrong RJ, et al. A new method for the rapid and long term growth of human neural precursor cells. J Neurosci Methods. 1998;85(2):141-152.
9.   Wu P, Tarasenko YI, Gu Y, Huang LY, Coggeshall RE, Yu Y. Region-specific generation of cholinergic neurons from fetal human neural stem cells grafted in adult rat. Nat Neurosci. 2002;5(12):1271-1278.
10.   Jakel RJ, Schneider BL, Svendsen CN. Using human neural stem cells to model neurological disease. Nat Rev Genet. 2004;5(2):136-144.
11.   López-García I, Ger? D, Szczesny B, et al. Development of a stretch-induced neurotrauma model for medium-throughput screening in vitro: identification of rifampicin as a neuroprotectant. Br J Pharmacol. Epub 2016 Oct 9.
12.   Mich JK, Signer RA, Nakada D, et al. Prospective identification of functionally distinct stem cells and neurosphere-initiating cells in adult mouse forebrain. Elife. 2014;3:e02669.
13.   Gao J, Grill RJ, Dunn TJ, et al. Human neural stem cell transplantation-mediated alteration of microglial/macrophage phenotypes after traumatic brain injury. Cell Transplant. 2016;25(10):1863-1877.
14.   Lyczek A, Arnold A, Zhang J, et al. Transplanted human glial-restricted progenitors can rescue the survival of dysmyelinated mice independent of the production of mature compact myelin. Exp Neurol. 2017;291:74-86.
15.   Windrem MS, Schanz SJ, Guo M, et al. Neonatal chimerization with human glial progenitor cells can both remyelinate and rescue otherwise lethally hypomyelinated shiverer mouse. Cell Stem Cell. 2008;2(6):553-565.
16.   Sandrock RW, Wheatley W, Levinthal C, et al. Isolation, characterization, and preclinical development of human glial-restricted progenitor cells for treatment of neurological disorders. Regen Med. 2010;5(3):381-394.
17.   Gao J, Prough DS, McAdoo DJ, et al. Transplantation of primed human fetal neural stem cells improves cognitive function in rats after traumatic brain injury. Exp Neurol. 2006;201(2):281-292.
18.   Tarasenko YI, Gao J, Nie L, et al. Human fetal neural stem cells grafted into contusion-injured rat spinal cords improve behavior. J Neurosci Res. 2007;85(1):47-57.
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