Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Summary

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

Abstract

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.

This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

Protocol

Part 1: Basic set up before proceeding to dissection:

1. Appropriate volume of complete NSC medium is prepared by mixing NeuroCult NSC Basal Medium and NeuroCult NSC Proliferation Supplements at a 9:1 ratio, respectively. NSC medium can also be made in laboratory based on NSC growth medium composition published in the literature¹. If your lab does not have much experience with making medium, we strongly recommend to use commercially available medium that has been quality controled for NSCs growth prior to being sold (which is the case for NeuroCult, Stem Cell Technolgies).
2. The medium is warmed up in a 37°C water bath.
3. Cold H....

Discussion

The neurosphere assay ² has gained broad attention in the research community not only for the isolation and study of NSCs from CNS ^4-5 but also for the isolation of other types of putative stem cells from numerous tissues, such as breast ⁶ and heart ⁷ and for the identification of brain, breast and colon tumor stem cells ^8-9 suggesting that this culture system can be applied to a number of somatic and tumor precursor cell populations throughout the body. Some of the .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
NeuroCult NSC Basal Medium	Medium	Stem Cell Technologies	05700
NeuroCult NSC Proliferation Supplements	Medium supplement	Stem Cell Technologies	05701
%0.05 trypsin-EDTA	Reagent	GIBCO, by Life Technologies	25300-062
Soybean trypsin inhibitor	Reagent	Sigma-Aldrich	T6522
Cell strainer	Sieve	BD Biosciences	352340
T25 flask	Culture ware	Nalge Nunc international	136196
T80 flask	Culture ware	Nalge Nunc international	178905
EGF	Growth factor	R&D Systems	2028-EG
Pen/Strep	Reagent	GIBCO, by Life Technologies	15140-122
*MEM	Reagent	GIBCO, by Life Technologies	41500-018	HEM component
*HEPES	Reagent	Sigma-Aldrich	H4034	HEM component
*Distilled water	Reagent	GIBCO, by Life Technologies	15230-147
15 ml tubes	Culture ware	BD Biosciences	352096
50 ml tubes	Culture ware	BD Biosciences	352070
Fine curved forceps	Surgical tools	Fine Science Tools	11251‐35
Small fine forceps	Surgical tools	Fine Science Tools	11272‐30
Small forceps	Surgical tools	Fine Science Tools	11050‐10
b-FGF	Growth factor	R&D Systems	3139-FB
Heparin	Growth factor	Sigma-Aldrich	H4784	Reconstituted in PBS
*To make HEM, mix 1×10L packet of MEM and160ml of 1M HEPES and bring the volume to 8.75 L using distilled water. Set the final PH to 7.4 and store it at 4°C.