Isolation and Culture of Embryonic Mouse Neural Stem Cells

Summary

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E¹³ mouse embryo ganglionic eminence.

Abstract

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E¹³ mouse embryo ganglionic eminence. Surgical procedures include harvesting E¹³ mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Introduction

Neural stem cells (NSCs) reside in different regions of the adult and embryo brain and they have a tendency to generate different types of neurons and glial cell¹. The subventricular zones in the adult mammalian brain² and ganglionic eminence in the embryo brain are NSC rich regions³. In the developing brain, the ganglionic eminence delivers most of the cortical interneurons and particularly GABAergic interneurons³. There are also less invasive methods for neural stem cell generation from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) that reduce the ....

Protocol

All the surgeries and procedures on animal were approved by the animal ethics committee of the Royan Institute, Tehran, Iran.

1. Preparation of Surgical Instruments, Washing Buffer (HEPES-MEM), Cell Culture Media and Cell Culture Plates

1. Sterilize the surgical tools (scissors, scalpel blade handle and forceps) by autoclaving them based on the routine sterilization guidelines.
2. Prepare an adequate volume of HEPES-MEM buffer (around 100 mL is enough for each pregnant mice). Add high concentrations of antibiotics (10% penicillin/streptomycin) to the HEPES-MEM buffer. This concentration of antibiotics is necessary to reduce ....

Results

Micro-Dissection, Cell Isolation and Neurosphere Culture. Here, we presented a rapid and efficient method for mouse E¹³ brain microsurgery and isolation of neural stem cells. This article shows that it is possible to remove the whole brain from an E¹³ mouse embryo skull through a fine rupture in frontal fontanelle. With this method, the brain endures less damage and it is possible to isolate cells from different parts of the brain. The harvested cell.......

Discussion

Using a suitable source of neural stem cells is very important for neuroscientists. Neural stem cells could be harvested from different areas of the embryo brain and they can generate specific types of neurons and glial cells. There are several methods for induction of differentiation of neural stem cells to induce them to generate mature neurons and glial cells. There are numerous reports indicating that under specific culture conditions and trophic factor regiments, they could generate neurons¹⁴.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
15 mL tubes	Falcon	352097
50 mL tubes	Falcon	352235
Adson Forceps, 12 cm, Straight	WPI	14226
B27	Gibco	17504-44
bFGF	Sigma	F0291
bovine serum albumin (BSA)	Sigma	A2153
dish 10cm	Falcon	353003
Dressing Forceps, 12.5 cm, Straight	WPI	15908
Dumont Tweezers, 11 cm, Straight	WPI	500342
EGF	Sigma	E9644
Ethanol	Merck	100983
Glutamate	Sigma	G3291
Glutamax	Gibco	35050061
goat anti rabbit FITC conjugated secondary antibody	Sigma	AP307F
goat serum	Sigma	G9023
Heparin	Sigma	h3149
HEPES	Sigma	83264
HEPES	Sigma	90909C
insulinsyringe with 25-27 gauge Needle	SUPA medical	A1SNL127
laminin	sigma	L2020
MEM	Sigma	M2279
N2 supplement	Gibco	17502048
NB medium	Gibco	21103-31
Non-essential amino acid (NEAA)	Gibco	11140050
PBS without Ca and Mg	Gibco	20012050
Penicilin/ Streptomycin	Gibco	5140122
Poly-L-ornithine	Sigma	P4957
rabbit anti mouse beta tubulin-III antibody	Sigma	T2200
rabbit anti mouse GFAP antibody	Sigma	G4546
rabbit anti mouse Nestin antibody	Sigma	N5413
Scalpel Handle #3	WPI	500236
Scissors curve	WPI	14396
Scissors sharp straight	WPI	14192
Soybean trypsin inhibitor	Roche	10109886001
Tissue culture flasks, T25	BD	353014
Tissue culture flasks, T75	BD	353024
Tween 20	Sigma	P1379
Vannas Scissors,  8 cm, Straight	WPI	14003
β-mercaptoethanol	Sigma	M6250