Photoreceptor loss causes irreversible blindness in many retinal diseases. Repair of such damage by cell transplantation is one of the most feasible types of central nervous system treatment. Retinal stem cells (RSC) are a substrate for cell-replacement therapy, and previous studies have shown that RSCs from different developmental stages have distinct properties in proliferative capacity and differentiation potential. The tailbud stage is of special interest in retinogenesis, because RSCs commence differentiation after this period. However, no information about the characteristics of RSCs from the tailbud stage is available. In this study, the characteristics of cell cultures from the rat optic cup (referred to as optic-cup-derived RSCs; OC-RSCs) at embryonic day 12.5 (tailbud stage) were analyzed. OC-RSCs grew either as monolayers or as neurospheres in the presence of basic fibroblast growth factor and could be dissociated into a single cell suspension. Using the MTT assay, immunochemistry, cytogenetic analysis, and flow cytometry, we found that OC-RSCs were easily enriched to 92% by three passages, had a normal diploid karyotype, and exhibited no obvious differences in proliferative rate during eight passages (doubling time: 36 h). OC-RSCs produced retinal specific cells after the addition of serum to the medium, but the differentiation potential was affected by serum concentration. Preliminary results showed that transplanted OC-RSCs were incorporated into the degenerated retina of RCS rats and differentiated into rhodopsin-positive cells. Thus, OC-RSCs, after suitable enrichment, provide a population of stem cells with distinct growth and differentiation properties that make them suitable for research into RSC differentiation and transplantation.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
Ader M, Meng J, Schachner M, Bartsch U (2000) Formation of myelin after transplantation of neural precursor cells into the retina of young postnatal mice. Glia 30:301–310
Adler R, Hatlee M (1989) Plasticity and differentiation of embryonic retinal cells after terminal mitosis. Science 243:391–393
Ahmad I, Das AV, James J, Bhattacharya S, Zhao X (2004) Neural stem cells in the mammalian eye: types and regulation. Semin Cell Dev Biol 15:53–62
Bhattacharya S, Jackson JD, Das AV, Thoreson WB, Kuszynski C, James J, Joshi S, Ahmad I (2003) Direct identification and enrichment of retinal stem cells/progenitors by Hoechst dye efflux assay. Invest Ophthalmol Vis Sci 44:2764–2773
Bochkov NP, Voronina ES, Kosyakova NV, Liehr T, Rzhaninova AA, Katosova LD, Platonova VI, Gol’dshtein DV (2007) Chromosome variability of human multipotent mesenchymal stromal cells. Bull Exp Biol Med 143:122–126
Canola K, Angenieux B, Tekaya M, Quiambao A, Naash MI, Munier FL, Schorderet DF, Arsenijevic Y (2007) Retinal stem cells transplanted into models of late stages of retinitis pigmentosa preferentially adopt a glial or a retinal ganglion cell fate. Invest Ophthalmol Vis Sci 48:446–454
Cepko CL, Austin CP, Yang X, Alexiades M, Ezzeddine D (1996) Cell fate determination in the vertebrate retina. Proc Natl Acad Sci USA 93:589–595
Chacko DM, Rogers JA, Turner JE, Ahmad I (2000) Survival and differentiation of cultured retinal progenitors transplanted in the subretinal space of the rat. Biochem Biophys Res Commun 268:842–846
Coles BL, Angenieux B, Inoue T, Del Rio Tsonis K, Spence JR, McInnes RR, Arsenijevic Y, van der Kooy D (2004) Facile isolation and the characterization of human retinal stem cells. Proc Natl Acad Sci USA 101:15772–15777
Cui YY, Xie H, Qi KB, He YM, Wang JF (2004) Effects of Pinus massoniana bark extract on cell proliferation and apoptosis of human hepatoma BEL-7402 cells. World J Gastroenterol 11:5277–5282
Das AV, James J, Rahnenfuhrer J, Thoreson WB, Bhattacharya S, Zhao X, Ahmad I (2005a) Retinal properties and potential of the adult mammalian ciliary epithelium stem cells. Vision Res 45:1653–1666
Das AV, Edakkot S, Thoreson WB, James J, Bhattacharya S, Ahmad I (2005b) Membrane properties of retinal stem cells/progenitors. Prog Retin Eye Res 24:663–681
Djojosubroto MW, Arsenijevic Y (2008) Retinal stem cells: promising candidates for retina transplantation. Cell Tissue Res 331:347–357
Engelhardt M, Bogdahn U, Aigner L (2005) Adult retinal pigment epithelium cells express neural progenitor properties and the neuronal precursor protein doublecortin. Brain Res 1040:98–111
Hegde GV, James J, Das AV, Zhao X, Bhattacharya S, Ahmad I (2007) Characterization of early retinal progenitor microenviroment: presence of activities selective for the differentiation of retinal ganglion cells and maintenance of progenitors. Exp Eye Res 84:577–590
Hojo M, Abe T, Sugano E, Yoshioka Y, Saigo Y, Tomita H, Wakusawa R, Tamai M (2004) Photoreceptor protection by iris pigment epithelial transplantation transduced with AAV-mediated brain-derived neurotrophic factor gene. Invest Ophthalmol Vis Sci 45:3721–3726
James J, Das AV, Bhattacharya S, Chacko DM, Zhao X, Ahmad I (2003) In vitro generation of early-born neurons from late retinal progenitors. J Neurosci 23:8193–8203
James J, Das AV, Rahnenfuhrer J, Ahmad I (2004) Cellular and molecular characterization of early and late retinal stem cells/progenitors: differential regulation of proliferation and context dependent role of Notch signaling. J Neurobiol 61:359–376
Klassen H, Ziaeian B, Kirov II, Young MJ, Schwartz PH (2004a) Isolation of retinal progenitor cells from post-mortem human tissue and comparison with autologous brain progenitors. J Neurosci Res 77:334–343
Klassen H, Sakaguchi DS, Young MJ (2004b) Stem cells and retinal repair. Prog Retin Eye Res 23:149–181
Kobayashi N, Rivas-Carrillo JD, Soto-Gutierrez A, Fukazawa T, Chen Y, Navarro-Alvarez N, Tanaka N (2005) Gene delivery to embryonic stem cells. Birth Defects Res C Embryo Today 75:10–18
Lakshmipathy U, Pelacho B, Sudo K, Linehan JL, Coucouvanis E, Kaufman DS, Verfaillie CM (2004) Efficient transfection of embryonic and adult stem cells. Stem Cells 22:531–543
Layer PG, Robitzki A, Rothermel A, Willbold E (2002) Of layers and spheres: the reaggregate approach in tissue engineering. Trends Neurosci 25:131–134
Levine EM, Green ES (2004) Cell-intrinsic regulators of proliferation in vertebrate retinal progenitors. Semin Cell Dev Biol 15:63–74
Lillien L (1995) Changes in retinal cell fate induced by overexpression of EGF receptor. Nature 377:158–162
Livesey FJ, Young TL, Cepko CL (2004) An analysis of the gene expression program of mammalian neural progenitor cells. Proc Natl Acad Sci USA 101:1374–1379
MacLaren RE, Pearson RA, MacNeil A, Douglas RH, Salt TE, Akimoto M, Swaroop A, Sowden JC, Ali RR (2006) Retinal repair by transplantation of photoreceptor precursors. Nature 444:203–207
Mizumoto H, Mizumoto K, Shatos MA, Klassen H, Young MJ (2003) Retinal transplantation of neural progenitor cells derived from the brain of GFP transgenic mice. Vision Res 43:1699–1708
Qiu G, Seiler MJ, Mui C, Arai S, Aramant RB, de Juan E Jr, Sadda S (2005) Photoreceptor differentiation and integration of retinal progenitor cells transplanted into transgenic rats. Exp Eye Res 80:515–525
Qiu G, Seiler MJ, Thomas BB, Wu K, Radosevich M, Sadda S (2007) Revisiting nestin expression in retinal progenitor cells in vitro and after transplantation in vivo. Exp Eye Res 84:1047–1059
Reh TA, Fischer AJ (2001) Stem cells in the vertebrate retina. Brain Behav Evol 58:296–305
Reh TA, Kljavin IJ (1989) Age of differentiation determines rat retinal germinal cell phenotype: induction of differentiation by dissociation. J Neurosci 9:4179–4189
Salasznyka RM, Kleesa RF, Williamsa WA, Boskeyb A, Plopper GE (2007) Focal adhesion kinase signaling pathways regulate the osteogenic differentiation of human mesenchymal stem cells. Exp Cell Res 313:22–37
Schwartz PH, Bryant PJ, Fuja TJ, Su H, O’Dowd DK, Klassen H (2003) Isolation and characterization of neural progenitor cells from post-mortem human cortex. J Neurosci Res 74:838–851
Schwartz PH, Nethercott H, Kirov II, Ziaeian B, Young MJ, Klassen H (2005) Expression of neurodevelopmental markers by cultured porcine neural precursor cells. Stem Cells 23:1286–1294
Tropepe V, Coles BL, Chiasson BJ, Horsford DJ, Elia AJ, McInnes RR, van der Kooy D (2000) Retinal stem cells in the adult mammalian eye. Science 287:2032–2036
Wojciechowski AB, Englund U, Lundberg C, Wictorin K, Warfvinge K (2002) Subretinal transplantation of brain-derived precursor cells to young RCS rats promotes photoreceptor cell survival. Exp Eye Res 75:23–37
Yanagi Y, Inoue Y, Kawase Y, Uchida S, Tamaki Y, Araie M, Okochi H (2006) Properties of growth and molecular profiles of rat progenitor cells from ciliary epithelium. Exp Eye Res 82:471–478
Yang P, Seiler MJ, Aramant RB, Whittemore S (2002) Differential lineage restriction of rat retinal progenitor cells in vitro and in vivo. J Neurosci Res 69:466–476
Yu DY, Cringle SJ, Su EN, Yu PK (2000) Intraretinal oxygen levels before and after photoreceptor loss in the RCS rat. Invest Ophthalmol Vis Sci 41:3999–4006
Zahir T, Klassen H, Young MJ (2005) Effects of ciliary neurotrophic factor on differentiation of late retinal progenitor cells. Stem Cells 23:424–432
Zahir T, Klassen H, Tomita M, Young MJ (2006) Sorbitol causes preferential selection of Muller glial precursors from late retinal progenitor cells in vitro. Mol Vis 12:1606–1614
Zhao S, Thornquist SC, Barnstable CJ (1995) In vitro transdifferentiation of embryonic rat retinal pigment epithelium to neural retina. Brain Res 677:300–310
We thank Professor Wenqin Cai and Huaizhen Ruan for useful comments on this research. We are also grateful to Professor Sheila G. Crewther and Dr. Thomas FitzGibbon for comments and discussion regarding earlier versions of the manuscript and to Mr. Shijun Wang and Ms. Yuxiao Zeng for their technical assistance.
This work was supported by grants from the National Basic Research Program of China (no. 2007CB512203) and the Nature Science Foundation of Chongqing, China (no. 2006BA5002).
Xiao Yong Huang and Zheng Qin Yin contributed equally to the work.
Commercial disclosure: none.
About this article
Cite this article
Huang, X.Y., Yin, Z.Q. & Tan, X.L. Characteristics of retinal stem cells from rat optic cup at embryonic day 12.5 (tailbud stage). Cell Tissue Res 333, 381–393 (2008). https://doi.org/10.1007/s00441-008-0653-1
- Retinal cultures
- Rat (RCS; Long Evans)