Generating Cortical Pyramidal Neurons from Human Neural Stem Cells

Abstract

Source: Gouder, L., et al,. Three-dimensional quantification of dendritic spines from pyramidal neurons derived from human induced pluripotent stem cells. J. Vis. Exp. (2015)

This video describes a method for preparing glass coverslips coated with polyornithine and laminin to enhance the adhesion and growth of neural stem cells. The technique involves sequential incubations and washes, facilitating the formation of a supportive matrix that promotes neuronal cell growth and differentiation, mimicking structures found in the neurons.

Protocol

1. Neuronal Culture
   Note: Fibroblast reprogramming in pluripotent stem cells, commitment to the dorsal telencephalon lineage, derivation, amplification, and banking of late cortical progenitors (LCP) were described in Boissart et al. Neuronal differentiation of LCP-like cells was also performed according to Boissart et al with slight modifications. Other procedures have been developed for direct reprogramming of fibroblasts into induced pluripotent stem cells, followed by their differentiation into neurons. This protocol was retained since it allows the selective production of pyramidal glutamatergic neurons.
   1. Treat 6-well culture plates with glass coverslips with poly-ornithine (diluted to 1/6 in DPBS (Dulbecco s Phosphate Buffered Saline), stock concentration 0.01%) O/N, followed by three washes in DPBS. Then add laminin (stock concentration 1 mg/ml, diluted 500 times in DPBS) for at least 10 hr under the flow hood.
   2. Plate and dispatch NSC at low density (50,000 cells/cm²) in 6-well culture plates with glass coverslips in 3 ml of culture medium consisting of DMEM (Dulbecco′s Modified Eagle′s Medium)/F12 (500 ml), 2 vials (5 ml each) of N2 supplement, 2 vials (10 ml each) of B27 supplement, 10 ml of Pen-Streptomycin (Penicillin = 10,000 units/ml and Streptomycin = 10,000 units/ml), 1 ml of 2-mercaptoethanol (Stock solution: 50 mM) and laminin (1/500), without growth factors. CRITICAL STEP: Carry out this step carefully by adding cells with slow rotating movements to reduce cell clustering.
   3. Remove the culture medium. Add fresh N2B27 medium containing 2 µg/ml of fresh laminin solution to keep the neuron attached to the glass coverslips and avoid clumping. Change the medium every 3 days. Keep some of the remaining medium (200 µl) before adding fresh medium to prevent the cell from drying. Alternatively, proceed rapidly and change the total volume (3 ml).

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PD-PBS (1X), sans Calcium, Magnesium et Phenol Red	Gibco/ Life Technologies	14190169
Poly-L-Ornithine Solution Bioreagent	Sigma Aldrich	P4957
Mouse laminin	Dutscher Dominique	354232
N2 Supplement	Gibco/ Life Technologies	17502048
B-27 Supplement w/o vit A (50X)	Gibco/ Life Technologies	12587010
DMEM/NUT.MIX F-12 W/GLUT-I	Gibco/ Life Technologies	31331028
Neurobasal Med SFM	Gibco/ Life Technologies	21103049
2-mercaptoethanol	Gibco/ Life Technologies	31350-010
Pen-Steptomycin	Gibco/ Life Technologies	15140-122
Coverglass 13 mm	VWR	631-0150
Prolong Gold Antifade Reagent avec DAPI	Gibco/ Life Technologies	P36931
Tween(R) 20 Bioextra, Viscous Liquid	Sigma Aldrich Chimie	P7949
Human Fibroblasts	Coriell Cell Line Biorepository	GM 4603 and GM 1869	Coriell Institute for Medical Research, Camden, NJ, USA
CO2 incubator
6 well culture plates
Piptette