1.2. Differentiation of iPSCs into neural progenitor cells (NPCs)

Differentiation of iPSCs into neural progenitor cells (NPCs)

iPSCs maintained in mTeSR Plus media with routine removal of differentiated cells

Day -1: Passage iPSCs using ReLeSR following vendor’s protocol;

Day 1: Refresh media using PSC Neural Induction Medium;

Day 2: Refresh media using PSC Neural Induction Medium;

Day 3: Refresh media using PSC Neural Induction Medium;

Day 4: Refresh media using PSC Neural Induction Medium. Check cells and remove non-neuronal cells;

Day 5: Refresh media using PSC Neural Induction Medium. Check cells and remove non-neuronal cells;

Day 6: Refresh media using PSC Neural Induction Medium. Check cells and remove non-neuronal cells;

Day 7: Refresh media using PSC Neural Induction Medium. Check cells and remove non-neuronal cells;

Day 8: Refresh media using PSC Neural Induction Medium. Check cells and remove non-neuronal cells;

Day 9: Refresh media using PSC Neural Induction Medium. Check cells and remove non-neuronal cells;

Day 10: Passage P0 NPCs using Rosette Selection Reagents following vendor’s protocol. NPCs will detach after treatment of Rossette Selection Reagents. Collect and replate NPCs in Neural Expansion Medium with 5 uM ROCK inhibitor in new Matrigel-coated dishes;

Day 11: Withdraw ROCK inhibitor and refresh media using Neural Expansion Medium;

Day 12-: NPCs should be maintained in Neural Expansion Medium with media refreshed every other day. Passage NPCs at 1:4 – 1:6 when reach 100% confluence.

5 uM ROCK inhibitor should be supplemented for passaging and withdrawn 24 hr post passaging.

From P0-P3, NPCs should be passaged using Rosette Selection Reagents to remove non-neuronal cells; from P4, NPCs can be passaged using Accutase.

NPCs can be maintained for no more than ten passages.

Refer to vendor’s manual (https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2FMAN0008031.pdf&title=UHJvdG9jb2w6IEluZHVjdGlvbiBvZiBOU0NzIFVzaW5nIEdpYmNvIE5ldXJhbCBJbmR1Y3Rpb24gTWVkaXVt ) for non-neuronal impurity cells morphology.

iPSCs and NPCs are maintained in Matrigel coated tissue culture dishes.

Reagent list:

mTeSR Plus (StemCell# 100-0276)

ReLeSR (StemCell# 05872)

PSC Neural Induction Medium (ThermoFisher# A1647801): 490 ml Basal medium + 10 ml 50x supplement

Neural Expansion Medium: 245 ml Basal medium (from PSC Neural Induction Medium kit) + 245 ml advanced DMEM/F12 (ThermoFisher# 12634010) + 10 ml 50x supplement (from PSC Neural Induction Medium kit)

Rosette Selection Reagent (StemCell# 05832)

ROCK inhibitor (R&D Systems# 1254/1)

Accutase (StemCell# 07920)

Matrigel (Corning# 354277)