Dorsal root ganglia of embryonic day 15 rats were dissociated with TrypLE Express and collected by centrifugation (250× g, 5 min, 4 °C) [16 (link),17 (link),18 (link),19 (link),20 ,21 (link)]. Cells were resuspended in neuron maintenance medium comprising Neurobasal medium (Thermo Fisher Scientific) supplemented with 2% B27 and NGF (20 ng/mL, Millipore, Burlington, MA, USA) and then seeded at 100,000 cells/cm2 onto poly-d -lysine/laminin-coated plates. Endogenous Schwann cells and fibroblasts were eliminated following brief treatment with fluorodeoxyuridine and uridine (10 μM each, Sigma-Aldrich), whereas dorsal root ganglion neurons and neurite network remained adherent on the coated plates.
aMSC-derived OPs were seeded at 10,000 cells/cm2 onto the axonal network of dorsal root ganglion neurons and the co-culture was maintained in myelination medium comprising Neurobasal medium supplemented with B27, NGF neutralizing antibody (5 μg/mL, Abcam, Cambridge, UK), thyroid hormone (10 ng/mL, Sigma-Aldrich), and DAPT (10 ng/mL, Sigma) for 14 days. Myelin-forming OLs and axons in co-cultures were immunostained for myelin basic protein (MBP) and neurofilament 200 (NF200) respectively. Co-cultures were also prepared for examination of compact myelin under transmission electron microscopy. aMSCs in co-culture with dorsal root ganglion neurons were prepared in parallel as controls.
aMSC-derived OPs were seeded at 10,000 cells/cm2 onto the axonal network of dorsal root ganglion neurons and the co-culture was maintained in myelination medium comprising Neurobasal medium supplemented with B27, NGF neutralizing antibody (5 μg/mL, Abcam, Cambridge, UK), thyroid hormone (10 ng/mL, Sigma-Aldrich), and DAPT (10 ng/mL, Sigma) for 14 days. Myelin-forming OLs and axons in co-cultures were immunostained for myelin basic protein (MBP) and neurofilament 200 (NF200) respectively. Co-cultures were also prepared for examination of compact myelin under transmission electron microscopy. aMSCs in co-culture with dorsal root ganglion neurons were prepared in parallel as controls.
Free full text: Click here
