Differentiating Neural Stem Cells into Neurons
Title |
Differentiating Neural Stem Cells into Neurons |
Date Submitted |
May 5, 2012 |
Submitted by - |
Efthymiou, Anastasia - anastasia.efthymiou@nih.gov |
Adapted from - |
Gibco Protocol |
Contributors - |
Efthymiou, Anastasia |
Affiliation(s) - |
NIH CRM - NIAMS – Laboratory of Stem Cell Biology |
Introduction:
Human iPSC-derived neuronal culture: Neurons/Tuj1 (Green); Glia/GFAP (Red) Nuclei/DAPI (Blue) - Rakesh Karmacharya, MD, PhD (Broad/McLean) from Harvard Stem Cell Institute
Protocol:
Neural stem cells (NSCs) will proliferate as progenitors a few times even after the complete growth medium is replaced with the appropriate differentiation medium. If the cells reach 90% confluency, it might be necessary to split the cells at a 1:2 ratio. However, do not split the cells once they reach day 9-10 of differentiation when they can get damaged during the passaging process.
- Plate neural stem cells on a polyornithine and laminin - coated culture dish in complete StemPro NSC SFM at 2.5 × 10^4 or 5 × 10^4 cells/cm2.
- After 2 days, change the medium to neural differentiation medium. Change the spent medium every 3 to 4 days.
- If expedited differentiation is desired, add 0.5 mM of dibutyryl cAMP (Sigma, Cat. no. D0627) to the differentiation medium daily starting at day 7 of differentiation for 3 days.
IMPORTANT! Do not expose cells to air at any time after they have differentiated into neurons.
Materials:
polyornithine and laminin-coated culture dish |
||
complete StemPro NSC SFM medium |
||
| neural differentiation medium | ||
dibutyryl cAMP |
||
StemPro NSC SFM Complete Media |
||
Component |
Final concentration |
Amount |
KnockOutTM D-MEM/F-12 |
1X |
97 mL |
GlutaMAXTM-I Supplement |
2 mM |
1 mL |
bFGF (prep as 100 µg/mL stock) |
20 ng/mL |
20 μL |
EGF (prep as 100 µg/mL stock) |
20 ng/mL |
20 μL |
StemPro® Neural Supplement |
2% |
2 mL |
| Neural Differentiation Medium | ||
Component |
Final concentration |
Amount |
| Neurobasal® Medium | 1X | 97 mL |
| B-27® Serum-Free Supplement | 2% | 12 mL |
| GlutaMAXTM-I Supplement | 2 mM | 1 mL |
