title: Down regulation of pRb in cultures of avian neuroretina cells promotes proliferation of reactive Müller-like cells and emergence of retinal stem/progenitors.
abstract: The aim of this work was to define the role of pRb depletion in the proliferation and differentiation of avian retinoblasts in vitro. For this purpose vectors expressing pRb short hairpin RNA were used to deplete pRb in cultures of avian neuroretinal cells. Down regulation of pRb was observed by Western blot and quantification of nuclear pRb. Cell proliferation and differentiation were studied following BrdU labeling and immunostaining. Transfection significantly down-regulated pRb in neuroretinal cells. Long-term effect of pRb depletion mainly induced proliferation of epithelial-like cells that expressed markers of reactive Müller glial cells. A minority of these cells that survived passaging could be maintained as neurosphere-like aggregates with low pRb, not observed in control cultures. BrdU labeling followed by a two week chase showed the presence of cells still remained labelled, indicating low cell cycling. Under appropriate conditions, these aggregates differentiate in precursors of amacrine interneurons shown by the expression of AP2, in absence of the photoreceptors marker visinin and the late neuronal marker MAP2. Taken together these data show that decrease pRb level in cultures of avian neuroretinal cells promotes the emergence and proliferation of stem cell/progenitors from reactive-like Muller cells.
authors.affiliation: Institut Curie, Centre National de Recherche Scientifique (CNRS) UMR3347, Institut National de la Santé et de Recherche Médicale (INSERM) U1021, Université Paris-Sud 11, Centre Universitaire Orsay, Orsay, France.
authors.forename: Maria
authors.initials: M
authors.lastname: Marx
authors.forename: Céline
authors.initials: C
authors.lastname: Lebuhotel
authors.forename: Danielle
authors.initials: D
authors.lastname: Laugier
authors.forename: Audrey
authors.initials: A
authors.lastname: Chapelle
authors.forename: Georges
authors.initials: G
authors.lastname: Calothy
authors.forename: Simon
authors.initials: S
authors.lastname: Saule
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000502:physiology; D049109:Cell Proliferation; D002642:Chick Embryo; D015536:Down-Regulation / Q000502:physiology*; D019084:Fluorescent Antibody Technique, Indirect; D020868:Gene Silencing; D005822:Genetic Vectors; D017403:In Situ Hybridization; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009457:Neuroglia / Q000166:cytology* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D055351:Retinal Neurons / Q000166:cytology* / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D014162:Transfection; D014746:Vimentin / Q000378:metabolism
chemical_list: D015415:Biomarkers; D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016160:Retinoblastoma Protein; D014746:Vimentin; C045140:visinin