This article outlines a protocol for differentiating human induced pluripotent stem cells (hiPSCs) into neural progenitor cells using mouse embryonic fibroblasts (MEFs) as a feeder layer. The process involves several steps including cell culture, detachment, and incubation to promote cell aggregation and differentiation.
Take a microplate containing an adherent culture of mouse embryonic fibroblasts, or MEFs, in a medium.
The adhered MEFs function as a feeder layer, providing a supportive cell culture microenvironment.
Remove the medium and introduce human induced pluripotent stem cells, or hiPSCs, in an hiPSC medium supplemented with small molecules that enhance cell survival.
Incubate to allow hiPSCs to adhere to the feeder layer, which secretes growth factors that facilitate hiPSC proliferation.
Remove the medium and add an enzyme solution to detach the cells. Transfer the cells and centrifuge them, then remove the supernatant and resuspend them in the hiPSC medium.
Transfer the cells to a biopolymer-coated plate. Incubate to allow the MEFs to adhere.
Transfer the suspended hiPSCs to a V-bottom microplate. Centrifuge the cells and incubate them, inducing cell aggregate formation.
Replenish with a differentiation medium. The medium's nutrients and cell-cell interactions within the aggregate facilitate hiPSC differentiation into neural progenitor cells.
For this protocol, begin with establishing feeder cells. To establish this culture, plate 600,000 feeder cells into each well of a six-well plate, with 300 milliliters of medium per well. After culturing the feeder cells for 48 hours, replace the medium, and incubate the plates for an hour, while preparing the stem cells.
Thaw the stem cells in a 37 degrees Celsius water bath. Once thawed, transfer the cells to a 15-milliliter conical tube and fill the tube to 5 milliliters with culture medium, added dropwise. Next, gently centrifuge the cells for four minutes. Aspirate the supernatant, and gently resuspend the pellet in 1 milliliter of stem cell medium with ROCK inhibitor.
After quantifying the cell density, plate 300,000 stem cells onto the feeder cells. Then, grow and expand the cultures, periodically selecting for healthy cells. After expansion and freezing cells for backup, grow the stem cells' colonies on standard plates until they reach 50% to 70% confluence.
Then, use a mild enzymatic treatment and gentle titration with a 1-milliliter pipette tip to harvest the HIPSCs for neural precursor cell differentiation. Gently centrifuge the suspension, aspirate the supernatant, and resuspend the pellet in 5 milliliters human iPSC medium. Then, transfer the cell suspension to a 0.1% gelatin-coated 5-centimeter cell culture dish, and incubate the plate for one hour.
After an hour, transfer the non-adherent cells to a 15-milliliter centrifuge tube. Then, gently rinse the plate with 3 milliliters of medium, and transfer it to the same tube. Next, repeat the resuspension step with gentle centrifugation.
Then, determine the cell density and plate the cells into a 96-well, low-adhesion V-bottom plate at 9,000 cells per well. Now, spin the plate for three minutes and start culturing the cells. Over the next 14 days, every other day, replace half the medium with fresh differentiating medium one.
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