Isolating Induced Neural Progenitor Cell Colonies Derived From Adult Human Fibroblasts

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

1. 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.
2. 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.
3. 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.
4. 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).