A Co-culture Technique for Differentiating Human Neural Progenitor Cells into Neurons

1. Generation of Induced Pluripotent Stem Cells

1. Culture human fibroblasts in DMEM supplemented with 10% fetal bovine serum (FBS) in 6-well plates.
2. Prior to commencing reprogramming of fibroblasts, coat 6-wells with Matrigel and incubate for at least an hour at room temperature. At 80% confluence, remove media from fibroblast cultures and wash once with 1x phosphate-buffered saline (PBS).
3. Add sufficient 0.25% trypsin-EDTA to cover entire plate and incubate at 37 °C for 10 min.
4. Once cells have dislodged from wells, add twice the amount of DMEM with 10% FBS as that of trypsin to quench trypsin digestion. Gently pipette cell suspension up and down to break up cells. Count the number of single cells with a hemocytometer.
5. Transfer 7 x 10⁵ cells into a 15 ml centrifuge tube and centrifuge the cell suspension at 200 x g for 5 min.
6. Remove supernatant and resuspend cell pellet in electroporation solution. Electroporate the fibroblasts according to manufacturer's instructions. Resuspend cells in 100 µl of electroporation buffer and add 1 µg of each episomal vector. Electroporate cells with settings of 1,650 V, 10 ms and 3 time pulses.
7. Transfer cell suspension into Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS and plate 5 x 10⁴ cells per well of a 6-well plate. Incubate plates at 37 °C and 5% CO₂.
8. After 48 hr, remove media from transfected fibroblast cultures and replace with mTeSR medium. Replace culture media daily until induced pluripotent stem cell (iPSC) colonies are ready to be isolated. iPSC colonies can be observed after 28-35 days.
9. When ready for isolation, mechanically cut an iPSC colony and reseed in mTeSR medium with 10 µM Rho-associated protein kinase (ROCK) inhibitor (Y-27632) onto a Matrigel coated 6-well plate. Replace culture medium daily.

2. Neural Induction and Maintenance of Human NPCs

1. When iPSC cultures are around 20% confluence, remove mTeSR medium and replace with neural induction medium. Replenish medium every second day.
2. After 7 days, remove neural induction medium and replace with human NPC maintenance medium. Replenish medium every second day up to 7 days before passaging cultures.
3. Prior to passaging cultures, coat plates/flasks with Matrigel at room temperature for at least an hr.
4. Remove media from confluent human NPC cultures and wash once with 1x PBS then aspirate PBS off.
5. Add enough cell detachment solution to cover all wells then incubate for 5 min at 37 °C.
6. Ensure that all cells have detached. Add twice the amount of DMEM/F-12 (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12) as that of cell detachment solution to the wells/flasks.
7. Transfer DMEM/F-12 containing cells into a 15 ml centrifuge tube and centrifuge at 200 x g for 5 min.
8. Remove supernatant and resuspend cells in 5 ml human NPC maintenance medium. Gently pipette cell suspension up and down to break up cells.
9. Plate a third of total cells into Matrigel coated culture wells/flasks and top up sufficient medium supplemented with 10 µM Y-27632. Incubate culture vessels at 37 °C and 5% CO₂.
   1. Split cultures 1:3 every 3 to 4 days and replenish medium every other day. Maintain human NPC cultures at high density to prevent differentiation.

3. Differentiation of Human NPCs into Neurons in Co-cultures

1. Add the lentiviral vector Synapsin1-tdTomato to human NPC culture before initiating differentiation into neurons. Prepare astrocyte/cortical neuron co-cultures a day in advance before differentiating human NPCs.
2. Wash human NPC cultures once with 1x PBS and add enough cell detachment solution to cover all wells/flasks then incubate for 5 min at 37 °C.
3. Ensure that all cells have detached. Add twice the amount of DMEM/F-12 as that of cell detachment solution to the wells/flasks. Transfer into a 15 ml centrifuge tube. Centrifuge at 200 x g for 5 min.
4. Remove supernatant and resuspend cells in 5 ml neuronal differentiation medium. Gently pipette cell suspension up and down to break up cell pellet into single cells. Count the number of single cells with a hemocytometer.
5. Carefully aspirate medium from astrocyte/cortical neuron cultures. Plate human NPCs at 5 x 10³ cells/cm² in 500 µl of neuronal differentiation medium supplemented with 10 µM Y-27632 for each 24-well. Gently add medium containing human NPCs into each well to avoid disturbing the astrocytes and cortical neurons.
6. Incubate plates at 37 °C and 5% CO₂. Replace half of the medium with fresh medium every two to three days for at least 28 days.