title: A comparison of differentiation protocols for RGC-5 cells.
abstract: PURPOSE. Although the RGC-5 cell line is widely used in retinal ganglion cell (RGC) research, recent data have raised questions about the nature of these cells. The authors performed a systematic analysis of RGC-5 cells to determine which RGC or neuronal markers are expressed after treatment with known differentiating agents, thus providing further insight into the nature of these cells and assisting in defining their future use. METHODS. RGC-5 cells were treated for 5 days with staurosporine (STSN; 316 nM), trichostatin A (TSA; 500 nM), or succinyl-concanavalin A (sConA; 50 microg/mL), after which they were assayed for specific marker antigen/mRNA expression. Treated cells were also assayed for excitotoxic responsiveness. RESULTS. Neither treated nor untreated RGC-5 cells expressed any specific RGC marker mRNAs or proteins (Brn-3, neurofilaments, Thy-1) or calbindin, calretinin, synaptophysin, PKCalpha, or glial fibrillary acidic protein. However, control RGC-5 cells did express the neuronal markers tau, betaIII-tubulin, microtubule-associated protein (MAP)-1b, MAP2, and PGP9.5. Although treatment with sConA had no effect on the expression of these markers, STSN and (dose dependently) TSA increased their expression and induced excitotoxic responsiveness. All cells, treated or not, expressed high levels of nestin but no other progenitor cell markers. All cells also expressed cone-specific, but not rod-specific, opsin indicative of cone photoreceptor lineage. CONCLUSIONS. RGC-5 cells expressed neuronal, but not RGC-specific, markers that were dose dependently upregulated by TSA. Hence, TSA provided the best tested means to terminally differentiate the cells to a neuronal phenotype from a precursor-like lineage.
authors.affiliation: Ophthalmic Research Laboratories, South Australian Institute of Ophthalmology, Hanson Institute Centre for Neurological Diseases, Adelaide, Australia. john.wood@imvs.sa.gov.au
authors.forename: John P M
authors.initials: JP
authors.lastname: Wood
authors.forename: Glyn
authors.initials: G
authors.lastname: Chidlow
authors.forename: Tuyet
authors.initials: T
authors.lastname: Tran
authors.forename: Jonathan G
authors.initials: JG
authors.lastname: Crowston
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Casson
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D019070:Cell Lineage; D003208:Concanavalin A / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006877:Hydroxamic Acids / Q000494:pharmacology*; D007381:Intermediate Filament Proteins / Q000378:metabolism; D051379:Mice; D009419:Nerve Tissue Proteins / Q000378:metabolism; D064231:Nestin; D055355:Opsins / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012165:Retinal Ganglion Cells / Q000166:cytology* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019311:Staurosporine / Q000494:pharmacology*
chemical_list: D015415:Biomarkers; D006877:Hydroxamic Acids; D007381:Intermediate Filament Proteins; D009419:Nerve Tissue Proteins; C577485:Nes protein, mouse; D064231:Nestin; D055355:Opsins; D012333:RNA, Messenger; D003208:Concanavalin A; C012589:trichostatin A; C019558:succinylconcanavalin A; D019311:Staurosporine