EFFECTS OF ROCK INHIBITOR Y27632 AND EGFR INHIBITOR PD168393 ON HUMAN NEURAL PRECURSORS CO-CULTURED WITH RAT AUDITORY BRAINSTEM EXPLANT E. NOVOZHILOVA, a * U. ENGLUND-JOHANSSON, b A. KALE, a Y. JIAO a,c AND P. OLIVIUS a,d * a Center for Hearing and Communication Research and the Department of Clinical Sciences, Intervention and Technology (CLINTEC), Karolinska Institute, Karolinska University Hospital, Stockholm 17176, Sweden b Department of Ophthalmology, Institution of Clinical Sciences in Lund, Lund University, 221 84 Lund, Sweden c Department of Otolaryngology, Head and Neck Surgery, Beijing Tongren Hospital, Capital Medical University, 100730 Beijing, China d Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linko ¨ping University, SE-58183, Sweden Abstract—Hearing function lost by degeneration of inner ear spiral ganglion neurons (SGNs) in the auditory nervous sys- tem could potentially be compensated by cellular replace- ment using suitable donor cells. Donor cell-derived neuronal development with functional synaptic formation with auditory neurons of the cochlear nucleus (CN) in the brainstem is a prerequisite for a successful transplantation. Here a rat auditory brainstem explant culture system was used as a screening platform for donor cells. The explants were co-cultured with human neural precursor cells (HNPCs) to determine HNPCs developmental potential in the presence of environmental cues characteristic for the auditory brainstem region in vitro. We explored effects of pharmacological inhibition of GTPase Rho with its effector Rho-associated kinase (ROCK) and epidermal growth factor receptor (EGFR) signaling on the co-cultures. Pharmacolog- ical agents ROCK inhibitor Y27632 and EGFR blocker PD168393 were tested. Effect of the treatment on explant penetration by green fluorescent protein (GFP)-labeled HNPCs was evaluated based on the following criteria: num- ber of GFP-HNPCs located within the explant; distance migrated by the GFP-HNPCs deep into the explant; length of the GFP+/neuronal class III b-tubulin (TUJ1)+ processes developed and phenotypes displayed. In a short 2-week co- culture both inhibitors had growth-promoting effects on HNPCs, prominent in neurite extension elongation. Signifi- cant enhancement of migration and in-growth of HNPCs into the brain slice tissue was only observed in Y27632-treated co-cultures. Difference between Y27632- and PD168393- treated HNPCs acquiring neuronal fate was significant, though not different from the fates acquired in control co-culture. Our data suggest the presence of inhibitory mechanisms in the graft–host environment of the auditory brainstem slice co-culture system with neurite growth arresting properties which can be modulated by administra- tion of signaling pathways antagonists. Therefore the co-culture system can be utilized for screens of donor cells and compounds regulating neuronal fate determination. Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: hearing, organotypic culture, neuronal restora- tion, Y27632, PD168393. INTRODUCTION Therapeutic strategies to protect hearing Mechanisms contributing to sensorineural hearing impairment resulting in a slow but progressive loss of spiral ganglion neurons (SGNs) are still under debate (Van de Heyning and Kleine Punte, 2010). SGNs degen- eration arises from the damage to either their peripheral (dendrites) or central (axons forming the auditory nerve, AN) processes (Spoendlin, 1987). Recovering damaged neuronal circuitry is therefore crucial in developing thera- peutics for people with sensorineural hearing loss. Attempts to preserve SGNs from further deterioration have been made (Shibata et al., 2011). Mostly they are confined to the introduction of neurotrophic factors. The factors can be overexpressed (Husseman and Raphael, 2009) using a viral vector to transduce cells (e.g. brain- derived neurotrophic factor (Rejali et al., 2007; Shibata et al., 2010); glial cell-derived neurotrophic factor (Kanzaki et al., 2002) or introduced via a passive release matrix (e.g. neurotrophin-3 (Richardson et al., 2007). Stimulating residual SGNs electrically directly (Buchman http://dx.doi.org/10.1016/j.neuroscience.2014.12.009 0306-4522/Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved. * Corresponding authors. Address: Center for Hearing and Commu- nication Research, Karolinska Universitetssjukhuset, Solna, Byggnad M1, 17176 Stockholm, Sweden. Tel: +46-8-51-773833; fax: +46-8- 348546. E-mail addresses: 1035bms@gmail.com (E. Novozhilova), petri. olivius@ki.se (P. Olivius). Abbreviations: AN, auditory nerve; ANOVA, analysis of variance; BSA, bovine serum albumin; CI, cochlear implant; CM, conditioned medium; CN, cochlear nucleus; CSPGs, chondroitin sulfate proteoglycans; DAPI, 4 0 ,6-diamidino-2-phenylindole; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulfoxide; DRG, dorsal root ganglion; ECM, extracellular matrix; EGFR, epidermal growth factor receptor; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; HBSS, Hank’s balanced salt solution; Hepes, 4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid; hESC, human embryonic stem cells; HNPCs, human neural precursor cells; MEF, mouse embryonic fibroblast; MOI, multiplicity of infection; NC, neural- crest; PBS, phosphate-buffered saline; PDL, poly-D-lysine; PFA, paraformaldehyde; RGC, retinal ganglion cell; ROCK, Rho- associated kinase; RT, room temperature; SGNs, spiral ganglion neurons; TUJ1, neuronal class III b-tubulin. Neuroscience 287 (2015) 43–54 43