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Culture of Neuroepithelial Stem Cells

Thomas Hazel¹, Thomas Müller¹

¹National Institute of Neurological Disorders and Stroke, Bethesda, Maryland

Publication Name:

Current Protocols in Neuroscience

Unit Number:

Unit 3.1

DOI:

10.1002/0471142301.ns0301s00

Online Posting Date:

May, 2001

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Abstract

In vitro culture of multipotent neural precursors allows researchers to study mechanisms regulating such processes as proliferation and lineage commitment within the developing central nervous system (CNS). The protocols presented in this unit describe the isolation and maintenance of stem cells from both fetal and adult rodent neural tissue. A procedure for treating tissue culture dishes with poly-l-ornithine and fibronectin, which is necessary before the dishes are used for neuroepithelial stem cell culture, is also included.

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Unit Introduction
Basic Protocol: Fetal Rat Brain Neuroepithelial Stem Cell Culture
Alternate Protocol: Adult Rodent Brain Neuroepithelial Stem Cell Culture
Support Protocol: Preparation of Polyornithine/Fibronectin-Coated Tissue Culture Dishes
Reagents and Solutions
Commentary
Bibliography

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Materials

Basic Protocol: Fetal Rat Brain Neuroepithelial Stem Cell Culture

Materials

Pregnant rat
HBSS/HEPES (see recipe), room temperature and 37°C
N2 medium (see recipe)
0.2% trypan blue
10-cm tissue culture dishes coated with poly-l-ornithine and fibronectin (see Support Protocol)
Basic fibroblast growth factor (bFGF)
Cell lifters (Costar)

Additional reagents and equipment for tissue culture (see cpmb appendix 3F and appendix 1A in this manual)

NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.

NOTE: All tissue culture incubations should be performed in a humidified 37°C, 5% CO₂ incubator unless otherwise specified.

Alternate Protocol: Adult Rodent Brain Neuroepithelial Stem Cell Culture

Materials

Adult rat or mouse
L15 medium
Artificial cerebrospinal fluid (aCSF, rodent; appendix 2A) supplemented with 0.2 mg/ml kynurenic acid and aerated with 95% O₂/5% CO₂; or HBSS/HEPES (see recipe) supplemented with 10 mM glucose and 0.2 mg/ml kynurenic acid
Trypsin (tissue culture grade, ~10,000 BAEE U/mg, from bovine pancreas; Sigma)
Hyaluronidase (1500 U/mg; Type V, Sigma)
DMEM/F-12 (appendix 2A) medium supplemented with 0.2 mg/ml kynurenic acid, 0.05% DNase I (Worthington), and 0.7 mg/ml trypsin inhibitor from chicken egg white (Boehringer Mannheim)
HBSS/HEPES
10-cm tissue culture dishes coated with poly-l-ornithine and fibronectin (see Support Protocol)
N2 medium (see recipe)
B-27 supplement (Life Technologies; optional)
Basic fibroblast growth factor (bFGF)

Dissection instruments, including forceps and a sharpened tungsten needle, or a 25-G needle attached to 1-ml syringe
50- and 15-ml Falcon tubes
32° to 35°C shaking water bath

NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.

NOTE: All tissue culture incubations should be performed in a humidified 37°C, 5% CO₂ incubator unless otherwise specified.

Support Protocol: Preparation of Polyornithine/Fibronectin-Coated Tissue Culture Dishes

Materials

15 µg/ml poly-l-ornithine (unit 3.2)
PBS (appendix 2A), sterile
1 mg/liter bovine fibronectin in sterile PBS
10-cm tissue culture dishes

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Literature Cited

Literature Cited
	Davis, A.A. and Temple, S. 1994. A self-renewing multipotential stem cell in embryonic rat cerebral cortex. Nature 372:263-266.
	Gage, F.H., Ray, J., and Fisher, L.J. 1995. Isolation, characterization, and use of stem cells from the CNS. Annu. Rev. Neurosci. 18:159-192.
	Johe, K.K., Hazel, T.G., Muller, T., Dugich-Djordjevic, M.M., and McKay, R.D.G. 1996. Single factors direct the differentiation of stem cells from the fetal and adult central nervous system. Genes & Dev. 10:3129-3140.
	Kilpatrick, T.J. and Bartlett, P.F. 1993. Cloning and growth of multipotential neural precursors: Requirements for proliferation and differentiation. Neuron 10:255-265.
	Paxinos, G. and Watson, C. 1982. The Rat Brain in Sterotaxic Coordinates. Academic Press, San Diego.
	Vescovi, A.L., Reynolds, B.A., Fraser, D.D., and Weiss, S. 1993. bFGF regulates the proliferative fate of unipotent (neuronal) and bipotent (neuronal/astroglial) EGF-generated CNS progenitor cells. Neuron 11:951-966.
Key Reference
	Johe et al., 1996. See above.
Describes the differentiative properties of cells isolated using these protocols.