Generating Schwann Cell-Like Cells from Bone Marrow Stromal Cells

For hypoxic preconditioning, first, release the ring clamp and clean the exposed parts with 70% ethanol. Further, disassemble the hypoxia chamber into components and place them into a laminar flow hood for sterilization under UV light for 15 minutes.

Next, wash an 80% to 90% confluent MSC culture with 10 milliliters of PBS as just demonstrated, and treat the cultures with fresh MSC growth medium supplemented with 25 millimolar HEPES. Place the treated culture into the reassembled hypoxia chamber and tighten the ring clamp. Seal the connecting ends of the chamber to ensure that there is no gas leakage and flush a gas mixture of 99% nitrogen and 1% oxygen into the chamber at a flow rate of 10 liters per minute.

After five minutes, place the chamber into the cell culture incubator for 16 hours. The next morning, detach the cells as just demonstrated, and collect them by centrifugation. Resuspend the pellet in neural progenitor medium and seed 6 x 10³ cells per square centimeter onto low-attachment six-well plates for their culture at 37 degrees Celsius and 5% CO₂ for 12 days.

Sizable non-adherent spheres of cells should be observed by day 6 to 7 with more neurospheres apparent in hypoxia-conditioned cultures than those under normoxia by days 10 to 12. On day 12, use a 10-milliliter pipette to transfer the neurospheres into a 15-milliliter conical tube and collect the neurospheres by centrifugation.

Resuspend the spheres in DMEM/F12 and plate 5 to 10 neurospheres per square centimeter onto Poly-D-Lysine laminin-coated coated six-well plates in 1.5 milliliters of glial induction medium per well. Maintain the sphere cell culture for at least seven days, refreshing the medium every three days.