All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.
1. Isolation of Putative Induced Neural Progenitor Cell (iNPC) Colonies
- One day before picking iNPC colonies, coat one 48 well-plate with laminin: dilute laminin to 1 µg/ml in Dulbecco's Phosphate-Buffered Saline (DPBS), add 300 µl of the solution onto plates, and keep at 4 °C for 24 hr. One day later, aspirate laminin from plates and add 200 µl of neuroinduction media (1:1 DMEM/F-12:Neurobasal, 1x N2, 1x B27, 1% GlutaMAX, 10 ng/ml human leukemia inhibitory factor, 3 μM CHIR99021, 2 μM SB431542) to the wells.
- Wash the 6 well-plates containing the colonies to be picked once with DPBS and add 2 ml neuroinduction media per well of 6 well-plate. Pick the colonies mechanically: scrape around colonies using a thin needle to get rid of surrounding cells; set the 200 µl pipetman on 50 µl and pick the colonies using the respective pipette tips.
- Transfer one colony each into one well of a laminin-coated 48 well-plate and mechanically generate a single cell suspension by pipetting up and down 10 times. Add ROCK inhibitor Y27632 in a final concentration of 10 µM to the cells.
- Grow the cells on the 48 well-plate at 37 °C, 5% CO2 for 2 days. Change the media every second day until cells reach 80 - 90% confluency (approx. 3 - 4 days).
