This study investigates the differentiation of bone marrow stromal cells into neuronal progenitors and Schwann cell-like cells under hypoxic conditions. The methodology includes hypoxia-preconditioning followed by culture in specific media to promote cell growth and differentiation.
Begin with an adherent culture of bone marrow stromal cells.
Subject the cells to hypoxia, a condition characterized by reduced oxygen levels, for a required duration.
Isolate the hypoxia-preconditioned cells, then centrifuge and remove the supernatant. Resuspend the cells in neuronal progenitor media containing neuronal growth factors.
Transfer the cells to a low-attachment culture plate and incubate.
Hypoxia-preconditioning and neuronal growth factors induce stromal cell differentiation into neuronal progenitors.
The low-attachment plate surface maintains the progenitors in suspension, facilitating their proliferation and aggregation into three-dimensional neurospheres.
Harvest the neurospheres, centrifuge, and remove the supernatant. Resuspend the cells in glial induction media containing glia-specific growth factors.
Transfer the neurospheres to a culture plate coated with laminin, an extracellular matrix component, and poly-D-lysine, a positively-charged synthetic polymer.
The coated surface promotes cell adhesion, facilitating neurosphere attachment.
The glia-specific growth factors stimulate neuronal progenitor differentiation into Schwann cell-like cells that exhibit the characteristic tapered morphology of Schwann 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.
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