MPCs were seeded at 1 × 103 cells/cm2 on tissue culture plastic or NFs and differentiated neurotrophically using a modified 10-day protocol [27 (link), 29 (link), 42 (link)]. Twenty-four hours after seeding, MPCs were incubated with GM supplemented with 10 mM β-mercaptoethanol (Sigma) for 24 h, followed by 48 h in GM supplemented with 10 mM β-mercaptoethanol + 35 ng/ml retinoic acid (Sigma). For the 6 following days, MPCs were incubated with neurotrophic medium (DMEM/Ham’s F12 + PSF (Invitrogen) supplemented with 2% FBS, 2% B-27 (Invitrogen), 6 mg/ml retinoic acid, 1 ng/ml FGF-2 (Sigma), 10 ng/ml platelet-derived growth factor (PDGF; Sigma), 150 ng/ml heregulin (an isoform of neuregulin-1; Sigma), and 10 μM forskolin (Sigma)).
Following neurotrophic differentiation, MPCs (designated nMPCs) were washed with PBS and either lysed with TRiZol, or incubated with basal medium (see above) for 48 h to produce conditioned medium (nMPC-CM). For DRG coculture experiments, tissue culture plastic-cultured nMPCs were trypsinized and transferred to DRG-containing fibers at a concentration of 1 × 103 cells/cm2.
Following neurotrophic differentiation, MPCs (designated nMPCs) were washed with PBS and either lysed with TRiZol, or incubated with basal medium (see above) for 48 h to produce conditioned medium (nMPC-CM). For DRG coculture experiments, tissue culture plastic-cultured nMPCs were trypsinized and transferred to DRG-containing fibers at a concentration of 1 × 103 cells/cm2.
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