While Plp1 and Sox10 are generic markers for cochlear glial cells, Sox2 is only expressed in a subset of glial cells. Degeneration of SGNs is usually a major cause of sensorineural hearing loss and severely affects the effectiveness of cochlear implant therapy. Cochlear MZP-54 glial cells are able to form MZP-54 spheres and differentiate into neurons and for 5C10 min. The pellet was resuspended in culture media and filtered through a 70 m cell strainer. Cells were counted, plated and grown to full confluency (5C7 days). Media was then removed and replaced with SCM media for suspension culture. Neuronal Differentiation To induce neuronal differentiation, cochlear glial spheres were plated on 96-well plate (Thermo 310109008) or MZP-54 glass slides (Thermo Fisher 12-545-80) coated with Poly-L-ornithine (Sigma P4957) and 10 ng/ml Laminin (Corning 354232) in SCM for 12C24 h, and then replaced with SCDM made up of DMEM/F12 (HyClone 36254) supplemented with B27 (Thermo 17504044) and N2 (Thermo 17502048) supplement, 50 ng/ml BDNF (Stemcell 78005), 50 ng/ml NT3 (Stemcell 78074). Half of the medium was replaced every 2C3 days. Differentiated cells were analyzed after 9 days or more for immunocytochemistry and qPCR. Additional control SCDM/FGF referred to SCDM supplemented with bFGF (100 ng/ml). The induction media (IM) for small molecule reprogramming contains Neurobasal Medium (Thermo 21103049), supplemented with B27 and N2, GlutaMax (Thermo 35050061), penicillin-streptomycin and bFGF MZP-54 (100 ng/ml), with or without small molecules Forskolin (20 M), ISX9 (20 M), I-BET Rabbit polyclonal to SLC7A5 (1C2 M), Chir99021 (10 M) (all from Selleck), and LIF (1000 U/ml) (Novus Biologicals). Real-Time Quantitative PCR RNA was isolated using Trizol (Takara 9108) and reverse transcription of total RNA was performed with the Primescript RT reagent kit (Takara RR047A) according to the manufacturers protocol. The Quantitative PCR reactions were performed with the Hieff UNICON? qPCR SYBR Green Grasp Mix (YEASEN 11198ES03) on LightCycler 96 (Roche LightCycler? 96 Instrument). Details of the primers were in Table 1. Data are normalized to GAPDH, and fold changes are calculated by using 2CCT method. TABLE 1 Primers used for real-time qPCR. and were able to proliferate for more than five generations (Figures 1A,B). We then optimized the growth media by evaluating the sphere numbers with diameters larger than 50 m. The result showed that bFGF is the primary factor for spheres growth, consistent with the previous report (Diensthuber et al., 2014a). Heparan sulfate has been reported to promote the binding and activation of FGF (Loo and Salmivirta, 2002). Therefore, we cultured the spheres with serum-free media made up of IGF, EGF, FGF, and heparan sulfate (Physique 1C). Real-time quantitative PCR (RT-qPCR) results showed increased expression of neuronal stem cell markers such as and in spheres compared to cochlear modiolus (Physique 1D). These results indicated that postnatal cochlear spheres were able to proliferate and preserve the stemness following passages (div). (B) Diameters of G1CG4 spheres during propagation. (C) Quantification of sphere diameters at 5 div with supplementations of E, I, F, H. E, EGF; I, IGF; F, FGF, and H, heparan sulfate. = 21C48. and in cochlear spheres compared to controls (dissociate modiolus cells). = 3, error bars represent mean SD. (E) Representative TUJ1-positive cells after differentiation at 18 div with BDNF and NT3 (B-NT3). (F) The ratio of TUJ1 positive cells. = 5 and 4, error bars represent mean SD. (G) Axon lengths of the induced neuron-like cells. = 35 and 220, error bars represent mean SD. (H) mRNA expression of of either spheres or differentiated neuron-like cells (Diff). = 3 or 4 4, error bars represent mean SD. * 0.05, ** 0.01, *** 0.001 by one-way ANOVA (C) or unpaired students (Wise et al., 2011; Li et al., 2016; Suzuki et al., 2016; Akil et al., 2019). We found that BDNF and NT3 treatment not only increased the differentiation efficiency (Figures 1E,F) but also promoted neurite extension (Figures 1E,G) during induced differentiation of the spheres. Therefore, BDNF and NT3 were added in the subsequent differentiation experiments. RT-qPCR results also showed that neuronal markers expression were increased, while neuronal stemness markers and were decreased after induced neuronal differentiation (Physique 1H). These results indicate that postnatal cochlear spheres are able to differentiate into neuron-like cells. Sox2 Expression Identifies a Subpopulation.