Feb 16, 2025
IPSC Neuron Development
- 1Caltech
Protocol Citation: Chelsie Steele, Yujie Fan 2025. IPSC Neuron Development . protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4wrzovo5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 14, 2025
Last Modified: February 16, 2025
Protocol Integer ID: 118317
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-020495
Abstract
IPSC Neuron Development
Materials
E8 medium (Thermo Fisher, A1517001)
Vitronectin (Thermo Fisher, A14700)
Matrigel (Corning, 354262)
Accutase (Fisher Scientific, A1110501)
Y-27632 - (Bio-Techne, 1254/10)
SB (R&D #1614)
LDN (Stemgent, 04-0074)
XAV (R&D , 3748)
N2 supplement (Stem Cell Technologies, 07156)
B27 (Fisher Scientific,17-504-044)
NAEE (Fisher Scientific,11-140-050)
Glutamax (ThermoFisher, 35050061)
PenStrep (ThermoFisher, 15140122)
Poly-L-ornithine (Sigma Aldrich, P4957-50ML)
GDNF (Recombinant Human Glial Derived Neurotrophic Factor) (Peptrotech, 450-10)
BDNF (Recombinant Human/Mouse BDNF) (R&D, 248-BDB)
AA (Ascorbic Acid) (Sigma, 4034-100g)
dbCAMP(Cyclic Monophosphate Sodium Salt)(Sigma,D0627)
before day -1
before day -1
hESCs were cultured on 6-well coated with Vitronectin (1:100 diluted in DPBS and coated in cold room overnight) and maintained in E8 medium (Thermo Fisher, A1517001).
The E8 medium was changed daily, and the cells were split every 3-5 days at 70-85% confluence. then seeded into the matrigel plates for day -1
Day-1 (Cortical differentiation (H9 /E8-E6))
Day-1 (Cortical differentiation (H9 /E8-E6))
1d
1d
Thaw a 10mL Matrigel container overnight on ice, then aliquot & freeze.
12h
Dilute Matrigel 1:100 in cold DMEM/F12 and coat a 24 well with 1/2ml per well overnight at 4°C
12h
Aspirate E8 medium from hESCs and wash 1X with PBS. Add sufficient Accutase to cover the surface of the cells. In a 6-well plate, this is about 1mL of Accutase.
Incubate the plate at 37°C for 10 minutes. Then, triturate the cells in Accutase 10X using a P1000 to break them into single cells. Collect the cells into a 15mL tube, leaving one well for maintaining.
Spin down the 15mL canonical tube at 300g for 5 minutes. Aspirate the supernatant, and re-suspend the pellet in 1mL of E6 medium.
Count the cells by taking out 10uL of the re-suspended cells and mixing it thoroughly with 10uL of trypan blue in a separate 1.5mL Eppendorf tube. Load 10uL of this mixture onto a counting plate to count the cells. When you count, it will give you a concentration. Based on this concentration, plate approximately 100,000 cells/cm^2. For instance, in a 6-well plate, this is approximately 1,000,000 cells per well, and so you would need N = 1,000,000 cells/well x 6 wells = 6 million cells.
Using V = N/C, calculate the volume, V, needed from the 15mL tube of re-suspended cells for seeding. In a 50mL falcon tube, E6 media+ Y-27632 (10 μM, 1:1000) + the appropriate volume (V) from the 15mL tube.
Add 2mL of the mixture from the 50mL plate onto the geltrex or matrigel new well plates.
Day 0 to day 3
Day 0 to day 3
Check the cell confluence. The cells need to be 100% confluent before initiating differentiation.
Feed cells daily with E6 media supplemented as follows with: SB (10μM - 1:1000 of the stock, stock at 10mM); LDN (100nM - 1:5000 of the stock, stock at 500 μM); XAV ( 500nM 1:2000 of the stock, stock at 1mM)
Day 4 to day 10
Day 4 to day 10
Feed cells daily with E6 media supplemented with: SB (10μM - 1:1000 of the stock, stock at 10mM);
LDN (100nM - 1:5000 of the stock, stock at 500 μM)
Day 10 to day 20
Day 10 to day 20
Feed cells daily with Neurobasal media supplemented with the following: N2(1:100) + B27(1:50)+NAEE+GluMax (1:1000)+P/S (1:1000)
D18 to D19 Prepare PO/LM/FN plates
D18 to D19 Prepare PO/LM/FN plates
1d 16h
1d 16h
Prep the plates with a poly-L-ornithine solution diluted in DPBS for 24 hours.
1d
Aspirate the poly-L-ornithine solution, and wash each well twice with 0.5 mL DPBS.
Add 12 μL Laminin (LM, 1 μg/mL) and 12 μL Fibronectin (FN, 1 μg /mL) to 12 mL DPBS. Mix and add 0.5 mL of the mixture to each well. Incubate at 37°C for ≥ 16 h.
16h
Day 20 - Passage Cells
Day 20 - Passage Cells
20m
20m
Wash each well with DPBS then add Accutase for 20 min / 37° C in the incubator. Follow the steps from 6-9 to re-plate the cells. However, plate the cells at a density of 150, 000 cells/cm2 in the pre-prepared PO/LM/FN plates, and use the following media mixture to feed the cells instead of E6: Neurobasal media supplemented with N2(1:100) + B27(1:50)+NAEE+GluMax (1:1000)+P/S (1:1000)+ ROCK inhibitor (1:1000, Y-27632).
20m
Day 21
Day 21
Feed the cells on with Neurobasal basic media supplemented with: N2(1:100) + B27(1:50)+NAEE+GluMax (1:1000)+P/S (1:1000)+ ROCK inhibitor (1:1000, Y-27632) + GDNF (10ng/ml, 1X1000 dilution from 10ug/ml stock)+BDNF (10ng/ml, 1X1000 dilution from 10ug/ml stock) + AA (100uM, 1X1000 dilution from 100mM stock) + dbCAMP (100uM, 1x1000 dilution from 100mM stock).
Day 30 - end point
Day 30 - end point
Maintain in the same media mentioned in day 21 changing ½ of the media every 5 days approx.