Generating Schwann Cell-Like Cells from Bone Marrow Stromal Cells

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.