Differentiation of Neural Progenitor Cells into Neurons
Published: August 30, 2024
Abstract
Source: Mao, X., et al. Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro. J. Vis. Exp. (2020).
The video showcased a cell culture method for differentiating neural progenitor cells into neurons. By incubating cells in a nutrient-rich medium supplemented with growth factors, the progenitor cells matured into neurons with developed axons and dendrites. A stabilizing agent ensured cell viability and protected against oxidative damage throughout the differentiation process.
Protocol
1. Differentiation from NPCs to neurons (Phase II)
- Seed about 5 x 105 mouse embryonic stem cells or mESC derivatives within 2 mL of basal differentiation medium I per well onto the 0.1% gelatin-coated 6-well plates. Randomly divide the mESC derivatives into 3 groups, as Phase II protocol 1, protocol 2, and protocol 3, respectively.
- Place the plate into the 5% CO2 incubator at 37 °C to allow for attachment for 6 h. Wash them twice with 2 mL of PBS.
- Add 2 mL of basal differentiation medium I, N2B27 medium I and N2B27 medium II (Table 1), respectively, to each well of the above groups.
- Place the plates into the incubator and allow them to differentiate for another 10 days. Change the corresponding medium every 2 days.
- Check the differentiation status and record the morphological changes as mentioned in step 3.
- On Day 18, evaluate the generation of neurons (β-Tubulin III positive) and determine the differentiation efficiency of the 3 protocols used in step 4.
Representative Results
Table 1: Details of the 3 protocols used in phase II differentiation.
| Differentiation Phase II (10d) | ||
| Protocols | Media | |
| Protocol 1 | Differentiation naturally: With basal differentiation medium I only | Basal differentiation medium I: DMEM/F12 +15%FBS +1%NEAA +0.1mM 2ME+ 1%P/S |
| Protocol 2 | Differentiation with N2B27 medium I | N2B27 medium I: DMEM/F12 + 1%N2 + 2%B27 + 1%GlutaMAX +0.1mM 2ME |
| Protocol 3 | Differentiation with N2B27 medium II | N2B27 medium II: 49% DMEM/F12+ 1% N2 + 48% Neurobasal medium + 2% B27 +1%GlutaMAX+ 0.1mM 2ME |
Disclosures
The authors have nothing to disclose.
Materials
| Anti-β-Tubulin III antibody produced in rabbit | Sigma Aldrich | T2200 | stored at -80 °C, avoid repeated freezing and thawing |
| B-27 Supplement (50X), serum free | Gibco | 17504044 | stored at -20 °C, and protect from light |
| DME/F-12 1:1 (1x) | HyClone | SH30023.01B | stored at 4 °C |
| Fetal bovine serum | HyClone | SH30084.03 | stored at -20 °C, avoid repeated freezing and thawing |
| Fluorescence microscopy | Olympus | CKX53 | |
| Gelatin | Gibco | CM0635B | stored at room temperature |
| GlutaMAX Supplement | Gibco | 35050061 | stored at 4 °C |
| Immunol Staining Primary Antibody dilution Buffer | Beyotime | P0103 | stored at 4 °C |
| KnockOut DMEM/F-12 | Gibco | 12660012 | stored at 4 °C |
| MEM Non-essential amino acids solution | Gibco | 11140076 | stored at 4 °C |
| N-2 Supplement (100X) | Gibco | 17502048 | stored at -20 °C and protect from light |
| Normal goat serum | Jackson | 005-000-121 | stored at -20 °C |
| Neurobasal Medium | Gibco | 21103049 | stored at 4 °C |
| Nonadhesive bacterial dish | Corning | 3262 | |
| Phosphate Buffered Saline (1X) | HyClone | SH30256.01B | stored at 4 °C |
| Penicillin/ Streptomycin Solution | HyClone | SV30010 | stored at 4 °C |
| PD0325901(Mirdametinib) | Selleck | S1036 | stored at -20 °C |
| Retinoic acid | Sigma | R2625 | stored at -80 °C and protect from light |
| Strain 129 Mouse Embryonic Stem Cells | Cyagen | MUAES-01001 | Maintained in feeder-free culture system |
| 2-Mercaptoethanol | Gibco | 21985023 | stored at 4 °C and protect from light |
| 4% paraformaldehyde | Beyotime | P0098 | stored at -20 °C |
| 6 – well plate | Corning | 3516 | |
| 60 mm cell culture dish | Corning | 430166 | |
| 15 ml centrifuge tube | NUNC | 339650 |
Cite This Article
Differentiation of Neural Progenitor Cells into Neurons. J. Vis. Exp. (Pending Publication), e22410, doi: (2024).