Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

Summary

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

Abstract

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

Introduction

Embryonic stem cells are pluripotent cells derived from the early embryo with the capacity to proliferate indefinitely in vitro while retaining the ability to differentiate into all adult cell types following reintroduction into an appropriate stage embryo (by forming a chimaera), injection into syngeneic or immunocompromised hosts (by forming a teratoma) or in vitro subject to appropriate cues¹. The in vitro differentiation of mouse embryonic stem cells into neural lineages was first described in 1995 and involved the formation of multicellular suspension aggregates (embryoid bodies, EBs) in serum-containing media supplemented wi....

Protocol

1. Media Preparation

NOTE: The protocol relies on the use of a mix of two separate media: DMEM/F12 supplemented with modified N2 supplement and Neurobasal supplemented with B27 supplement, typically in a 1:1 ratio.

1. Prepare the modified N2 supplement by mixing the components in a 15 ml tube. Do not vortex or filter this; mix by inverting the tube until the solution is clear.
   1. Start by pipetting 7.2 ml of DMEM/F12, then add 1 ml of 25 mg/ml insulin (made up in 0.01 M HCl) and mix well by inverting the tube. It takes a couple of minutes until the solution is clear.
   2. Add 1 ml of 100 mg/ml apo-transferrin (made u....

Results

In this experiment, we used the 46C cell line¹⁴, mouse embryonic stem cells with an endogenous Sox1-GFP reporter, to track neural differentiation. By using this cell line, expression of Sox1, a marker for neural progenitor, can be detected by green fluorescence. Plating density is a critical factor to achieve neuronal differentiation. Mouse embryonic stem cells were plated in 6-well plate at different densities varying from 10,500 to 88,500 cells/cm². Figure 1A shows differentiation.......

Discussion

The monolayer neural differentiation protocol has been in use for over a decade⁶. The protocol is highly efficient, composed of defined medium, and done in a monolayer system which makes the system more applicable for preclinical (e.g., drug screening) uses. However, there are some critical factors that determine differentiation efficiency. This article points out those factors and the solution for each obstacle.

Density of the cells after plating in the differentiation con.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fetal bovine serum	Life Technologies	10270-106
DMEM/F12	Life Technologies	11320-074
Neurobasal medium	Life Technologies	21103-049
StemPro Accutase Cell Dissociation Reagent	Life Technologies	A11105-01
B-27 Supplement, serum free	Life Technologies	17504-044
Insulin	Sigma	I6634	Reconstitute with sterile 0.01 M HCl
Apo-transferrin	Sigma	T1147	Reconstitute with sterile water
Progesterone	Sigma	P8783	Reconstitute with ethanol
Putrescine	Sigma	P5780	Reconstitute with sterile water
Sodium selenite	Sigma	S5261	Reconstitute with sterile water
Bovine albumin fraction V	Life Technologies	15260-037
L-Glutamine	Life Technologies	25030-081	Make sure it is completely dissolved before use as glutamine is usually sedimented
Gelatine	Sigma	G1890
6-well tissue culture dish	Thermo Scientific	140675
24-well tissue culture dish	Thermo Scientific	142475
96-well tissue culture dish	Thermo Scientific	167008
GMEM	Life Technologies	11710-035
MEM Non-essential amino acids solution	Life Technologies	11140-050
Sodium pyruvate	Life Technologies	11360-070
2-mercaptoethanol	Sigma	M7522
Leukaemia inhibitory factor	prepared in-house as in Smith 1991 Journal of Tissue Culture Methods
Tween-20	Sigma	P1379
4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI)	Sigma	D9542	Protect from light
Mouse anti βIII tubulin IgG antibody	Covance	MMS-435P
Fluorescence-labelled anti-mouse IgG antibody	Life Technologies	A31571	Protect from light
25 cm2 tissue culture flask	Thermo Scientific	156367