Generating Cortical Pyramidal Neurons from Human Neural Stem Cells
Published: August 30, 2024
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
- 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.- 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.
- 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.
- 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).
開示
The authors have nothing to disclose.
Materials
| 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 |
