Differentiation of retinal mice neurospheres by trophic and contact factors: Functional identification using single cell calcium imaging

Abstract

Event Abstract Back to Event Differentiation of retinal mice neurospheres by trophic and contact factors: Functional identification using single cell calcium imaging De Melo-Reis1* 1 UFRJ, Instituto de Biofisica CCF, Brazil The retina is a complex tissue composed of six types of neurons and one type of glial cell (Müller glia). Several retinal diseases affect millions of people worldwide and understanding the mechanisms related to the differentiation of photoreceptors or retinal ganglion cells (RGCs) from progenitors might allow a cell-based therapy to particular disorders. Mitogenic factors and extrinsic cues direct the choice of cell fate in the retina, controlling the production of proper ratios of cells and their differentiation and synaptic connection. We used progenitor cells isolated from postnatal mice and expanded them into neurospheres in the presence of 20ng/ml EGF (epidermal growth factor) in DMEM medium for 5-7days, when cultures contained cells clonal aggregated into tightly packed colonies. Differentiation of neurospheres for 3 days in control condition (DMEM + B27 in 2.5μg/ml laminin + poly-D-lysine) revealed the presence of stem cells (immunolabeled for sox-2 and cd133), progenitors (nestin), neuronal (β3-tubulin, doublecortin, PSA-NCAM, GluR1, MAP-2), and glial (GFAP and glutamine synthase) cells. Single Cell Ca2+ Imaging (SCCI) analysis showed that approximately 50% of the differentiated cells responded to 10μM AMPA or 40mM K+ (mature neurons), 15 % responded to 100μM muscimol (immature neurons) and < 15% responded to 1mM ATP (glia). Incubation with 50ng/ml CNTF (ciliary neurotrophic factor) or BDNF (brain-derived neurotrophic factor) increased the number of cells responding to K+ or AMPA, while the cells responding to muscimol dropped to less than 5%; however, CNTF, increased the number of cells responding to ATP. Whereas purified retinal neuronal cultures were essentially responsive to K+ and AMPA, but not muscimol or ATP, purified Muller cultures exposed to 1mM ATP or 100μM BzATP (benzoylbenzoylATP) released [14C]glutamate and [3H]dopamine in a BBG (brilliant blue G) dependent manner, indicating the possible involvement of P2X7 receptor activation. Interestingly, although laminin is an essential factor to differentiation, we found that undifferentiated neurospheres expressed GluR1 subunit of AMPA receptors and their activation induced the release of [3H]GABA and [14C]glutamate, but not [3H]dopamine, in a process inhibited by CNQX.Our data show that postnatal retinal mice neurospheres differentiate selectively into neurons and glia cells depending on the extracellular cues. At the long term we envision the use of this knowledge in the treatment of neurodegenerative diseases such as glaucoma, diabetes and retinitis pigmentosa.Financial Support CAPES-Grices Project, CNPq and FAPERJ. PTDC/SAU-NEU/68465/2006 Conference: 11th Meeting of the Portuguese Society for Neuroscience, Braga, Portugal, 4 Jun - 6 Jun, 2009. Presentation Type: Poster Presentation Topic: Abstracts Citation: Melo-Reis D (2009). Differentiation of retinal mice neurospheres by trophic and contact factors: Functional identification using single cell calcium imaging. Front. Neurosci. Conference Abstract: 11th Meeting of the Portuguese Society for Neuroscience. doi: 10.3389/conf.neuro.01.2009.11.046 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 06 Aug 2009; Published Online: 06 Aug 2009. * Correspondence: De Melo-Reis, UFRJ, Instituto de Biofisica CCF, Janeiro, Brazil, ramreis@biof.ufrj.br Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers De Melo-Reis Google De Melo-Reis Google Scholar De Melo-Reis PubMed De Melo-Reis Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.