Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Summary

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

Abstract

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development ^1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation ^6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype ^10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21.

Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

Protocol

1. Preparation of solutions and materials for dissection and maintenance of neural stem cell culture

1. Prepare 100ml dissection medium (KNOCKOUT DMEM/F12, Invitogen) ahead of time and refrigerate.
2. Prepare100 ml culture medium (Stem Pro NSC SFM, Invitrogen) and keep at 37 °C in a water bath.
3. Prepare cell-freezing medium (KNOCKOUT DMEM/F12+10%FBS+ 5%DMSO) for long term cryopreservation of cells.
4. If desired, prepare 4% paraformaldehyde (PFA) for tissue fixation.
5. Sterile, autoclaved forceps and scalpel blades with handles are used for dissection.
6. Set aside a pipette gun, 10 ml transfer pipettes, and 40 μM cell straine....

Discussion

There are various approaches toward culture fresh tissue and producing human cell lines. Historically, fresh tissue has been harvested and cultured immediately to generate various cell types in the central nervous system. This approach however is clearly limited by the number of samples that can be obtained- which in the case for human samples, is usually quite small. Given the minimal degree of manipulation, freshly cultured neural cells provide the most reliable experimental system by limiting potential artifacts from .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
KNOCKOUT DMEM/F12	Invitrogen	12660-012	Dissociation medium
Stem Pro NSC SFM	Invitrogen	A10509-01	Culture medium
Fetal Bovine Serum	Invitrogen	10091-148	Frozen medium
Hanks solution (-Ca2+, -Mg2+)	Invitrogen	14175-095	Dissociation medium
DMSO	Sigma-Aldrich	D2650	Frozen medium
EDTA	Sigma-Aldrich	431788	Dissociation medium
Paraformaldehyde	Sigma-Aldrich	158127	Fixation solution
bFGF	R&D Systems	234-FSE	Differentiation medium
SHH	R&D Systems	1845-SH	Differentiation medium
PDGF-AA	R&D Systems	221-AA	Differentiation medium
B27	Invitrogen	17504-044	Differentiation medium
Mouse Anti-MAP2	Sigma-Aldrich	M2320	1:200
Rabbit Anti-DCX	Cell Signaling Technology	4604s	1:200
Rabbit Anti-GFAP	Dako	Z0334	1:200
Rabbit Anti-S100B	Dako	Z0311	1:200
Rabbit Anti-O1	gifts of Professor Timothy Vartanian*		1:50
Rabbit Anti-O4	Gifts of Professor Timothy Vartanian*		1:50
40μm cell strainer	BD Biosciences	352340
* Timothy Vartanian, MD, PhD, Department of Neurology and Neuroscience, Weill Cornell Medical College, New York, USA