title: Effects of ROCK inhibitor Y27632 and EGFR inhibitor PD168393 on human neural precursors co-cultured with rat auditory brainstem explant.
abstract: Hearing function lost by degeneration of inner ear spiral ganglion neurons (SGNs) in the auditory nervous system could potentially be compensated by cellular replacement 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. Pharmacological 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: number of GFP-HNPCs located within the explant; distance migrated by the GFP-HNPCs deep into the explant; length of the GFP+/neuronal class III β-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. Significant 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 administration of signaling pathways antagonists. Therefore the co-culture system can be utilized for screens of donor cells and compounds regulating neuronal fate determination.
authors.affiliation: Center for Hearing and Communication Research and the Department of Clinical Sciences, Intervention and Technology (CLINTEC), Karolinska Institute, Karolinska University Hospital, Stockholm 17176, Sweden. Electronic address: 1035bms@gmail.com.
authors.forename: E
authors.initials: E
authors.lastname: Novozhilova
authors.affiliation: Department of Ophthalmology, Institution of Clinical Sciences in Lund, Lund University, 221 84 Lund, Sweden.
authors.forename: U
authors.initials: U
authors.lastname: Englund-Johansson
authors.affiliation: Center for Hearing and Communication Research and the Department of Clinical Sciences, Intervention and Technology (CLINTEC), Karolinska Institute, Karolinska University Hospital, Stockholm 17176, Sweden.
authors.forename: A
authors.initials: A
authors.lastname: Kale
authors.affiliation: Center for Hearing and Communication Research and the Department of Clinical Sciences, Intervention and Technology (CLINTEC), Karolinska Institute, Karolinska University Hospital, Stockholm 17176, Sweden; Department of Otolaryngology, Head and Neck Surgery, Beijing Tongren Hospital, Capital Medical University, 100730 Beijing, China.
authors.forename: Y
authors.initials: Y
authors.lastname: Jiao
authors.affiliation: Center for Hearing and Communication Research and the Department of Clinical Sciences, Intervention and Technology (CLINTEC), Karolinska Institute, Karolinska University Hospital, Stockholm 17176, Sweden; Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, SE-58183, Sweden. Electronic address: petri.olivius@ki.se.
authors.forename: P
authors.initials: P
authors.lastname: Olivius
mesh_terms: D000577:Amides / Q000494:pharmacology; D000818:Animals; D001933:Brain Stem / Q000166:cytology / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D017626:Cochlear Nucleus / Q000166:cytology* / Q000378:metabolism*; D018920:Coculture Techniques; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D058953:Neural Stem Cells / Q000166:cytology* / Q000378:metabolism*; D009457:Neuroglia / Q000166:cytology / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D011799:Quinazolines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D046509:Tissue Culture Techniques; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D000577:Amides; D004791:Enzyme Inhibitors; C509204:PD168393; D011725:Pyridines; D011799:Quinazolines; C108830:Y 27632; D066246:ErbB Receptors; D054460:rho-Associated Kinases
keywords: PD168393; Y27632; hearing; neuronal restoration; organotypic culture