Differentiation of Induced Pluripotent Stem Cells into Neural Progenitor Cells

1. Generation of Neural Progenitor Cells

1. Maintain hiPSCs derived from fibroblasts and peripheral blood mononuclear cells (PBMCs) in 6-well plates on a γ-irradiated mouse embryonic feeder (MEF) cell layer in human iPSC medium supplemented with small-molecules.
   NOTE: Daily maintenance is a modified version of previously reported procedures.
   1. Plate 6 x 10⁵ MEFs onto each well of a 6-well tissue culture grade plate in 300 μL/well recommended medium (following manufacturer's protocol).
   2. After 48 h, replace MEFs medium with 300 μL/well hiPSC medium for 1 h before adding hiPSCs.
   3. Quickly thaw hiPSCs in a 37 °C water bath and slowly add 1 mL hiPSC medium dropwise. Transfer the hiPSCs and medium to a 15 mL conical tube. Add medium to bring the total volume to 5 mL. Centrifuge for 4 min at 129 x g, aspirate the supernatant, gently resuspend the pellet in 1 mL hiPSC medium with Rho-associated protein kinase (ROCK) inhibitor at 1:1,000 concentration.
   4. Plate the cell suspension (typically seeded at 300,000 cells/well) onto the MEF cell layer and incubate at 37 °C, 5% CO₂, 95% humidity.
   5. Monitor iPSC culture closely using a light microscope. After 48 h, remove cultures that have uneven edges, have grown to become cystic-like or appear yellowish-brown under the light microscope to minimize spontaneous differentiation.
      NOTE: After 7 – 10 days, hiPSC colonies should form. Colonies should be round, with clearly defined borders and uniform cell densities, and devoid of loosely packed non-uniform cells.
   6. Manually select round hiPSC colonies to clear the culture of spontaneously differentiated cells. Expand the colonies by placing them on fresh MEF layers. Expand 1:3 every 7 days when cultures near confluency and freeze.
2. After expansion and freezing cells (for backup), grow hiPSC colonies on plates until they reach 50 – 75% confluence.
3. Seven-days post plating, use mild enzymatic treatment (5-10 min with 300 μL of enzyme solution) and gentle trituration (2-4 times with a 1,000 µL pipet) to harvest and prepare hiPSCs for neural precursor cell differentiation.
4. Centrifuge the cell suspension at 129 x g for 4 min, aspirate the supernatant, and resuspend the pellet in 5 mL human iPSC medium. Transfer the cell suspension to a 0.1% gelatin coated 5 cm cell culture dish for 1 h at 37 °C to eliminate MEFs and to maximize the hiPSC yield. Transfer the medium (with non-adherent cells) to another 5 cm gelatin coated dish.
5. Transfer the non-adherent cells to a 15 mL centrifuge tube using a transfer pipet. Gently rinse the plates with 3 mL medium and add it to the 15 mL tube.
6. Centrifuge the cell suspension at 129 x g for 4 min, aspirate the supernatant, and gently resuspend the pellet in medium. Determine the cell count using an automated cell counter. Plate 9,000 cells/well in a 96-well low-adhesion V-bottom plate.
7. Centrifuge the plate at 163 x g for 3 min. Incubate the plate at 37 °C, 95% humidity and 5% CO₂. Using a pipette, change 50% medium every other day by removing half of the medium and replacing it with fresh chemically-defined differentiating medium 1 (DM1; Figure 1; Table of Materials) for 14 days.