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The purpose of the present work was to generate, from retinal stem cells (RSCs), a large number of cells committed toward the photoreceptor fate in order to provide an unlimited cell source for neurogenesis and transplantation studies. We expanded RSCs (at least 34 passages) sharing characteristics of radial glial cells and primed the cells in vitro with fibroblast growth factor (FGF)-2 for 5 days, after which cells were treated with the B27 supplement to induce cell differentiation and maturation. Upon differentiation, cells expressed cell type-specific markers corresponding to neurons and glia. We show by immunocytochemistry analysis that a subpopulation of differentiated cells was committed to the photoreceptor lineage given that these cells expressed the photoreceptor proteins recoverin, peripherin, and rhodopsin in a same ratio. Furthermore, cells infected during the differentiation procedure with a lentiviral vector expressing green fluorescent protein (GFP) under the control of either the rhodopsin promoter or the interphotoreceptor retinoidbinding protein (IRBP) promoter, expressed GFP. FGF-2 priming increased neuronal differentiation while decreasing glia generation. Reverse transcription-polymerase chain reaction analyses revealed that the differentiated cells expressed photoreceptor-specific genes such as Crx, rhodopsin, peripherin, IRBP, and phosphodiesterase-α. Quantification of the differentiated cells showed a robust differentiation into the photoreceptor lineage: Approximately 25%–35% of the total cells harbored photoreceptor markers. The generation of a significant number of nondifferentiated RSCs as well as differentiated photoreceptors will enable researchers to determine via transplantation studies which cells are the most adequate to integrate a degenerating retina. [ABSTRACT FROM AUTHOR]

The polycomb transcriptional repressor Bmi1 promotes cell cycle progression, controls cell senescence, and is implicated in brain development. Loss of Bmi1 leads to a decreased brain size and causes progressive ataxia and epilepsy. Recently, Bmi1 was shown to control neural stem cell (NSC) renewal. However, the effect of Bmi1 loss on neural cell fate in vivo and the question whether the action of Bmi1 was intrinsic to the NSCs remained to be investigated. Here, we show that Bmi1 is expressed in the germinal zone in vivo and in NSCs as well as in progenitors proliferating in vitro, but not in differentiated cells. Loss of Bmi1 led to a decrease in proliferation in zones known to contain progenitors: the newborn cortex and the newborn and adult subventricular zone. This decrease was accentuated in vitro, where we observed a drastic reduction in NSC proliferation and renewal because of NSC-intrinsic effects of Bmi1 as shown by the means of RNA interference. Bmi1-/- mice also presented more astrocytes at birth, and a generalized gliosis at postnatal day 30. At both stages, colocalization of bromodeoxyuridine and GFAP demonstrated that Bmi1 loss did not prevent astrocyte precursor proliferation. Supporting these observations, Bmi1-/- neurospheres generate preferentially astrocytes probably attributable to a different responsiveness to environmental factors. Bmi1 is therefore necessary for NSC renewal in a cell-intrinsic mode, whereas the altered cell pattern of the Bmi1-/- brain shows that in vivo astrocyte precursors can proliferate in the absence of Bmi1. [ABSTRACT FROM AUTHOR]

Gene transfer is a promising approach for single gene defects occurring in several retinal degenerations, such as Leber congenital amaurosis (LCA), a group of severe retinal dystrophies affecting children. About 10% of LCA cases are due to a mutation in RPE65, a key enzyme for the metabolism of Vitamin A during the visual cycle. The consequence of RPE65 deficiency is a lack of the 11-cis- retinal chromophore at the photoreceptor level and an associated retinal degeneration. Gene therapy in dogs lacking RPE65 protein restores visual function on the long term (Acland et al., Nat Genet 28: 92; Narfstrom et al., Invest Ophthalmol Vis Sci 44: 1663). However no confirmed indications on cone rescue and cone function restoration, necessary for day vision and high visual acuity, have been reported in the different animal models used in gene therapy studies. To evaluate the efficiency of Rpe65 replacement by gene transfer in rodent models of LCA, we have constructed a lentiviral vector expressing the Rpe65 mouse cDNA under the control of a promoter fragment from the human RPE65 gene. We studied the therapeutic efficiency of this vector in the Rpe65 knockout mouse as well as in the Rpe65/Gnat1a double knockout mouse, to reveal the effect of Rpe65 gene transfer on the cone photoreceptors. Gnat1a-/- mice do not have any rod function. Thus, any photoreceptor activity occurring in the Rpe65/Gnat1a double knockout background after Rpe65 gene replacement should arise from cone photoreceptors.The therapeutic lentiviral vector (LV-RPE65) as well as a control vector encoding GFP (LV-GFP) were delivered by subretinal injection. We evaluated the retinal function by electroretinogram recordings at different times after vector injection, and following the last ERG, the animals were sacrificed to study transgene expression and photoreceptor survival at the histological level. Treatment of Rpe65-/- mice at postnatal day 5 revealed that lentiviral-mediated transfer of Rpe65 cDNA restores the ERG response. Furthermore, after treatment at the same age, Rpe65/Gnat1a double knockout mice displayed a clear ERG response, thus demonstrating that LV-RPE65 treatment allows to rescue the cone function. At the histological level, analysis of Rpe65-/- treated mice revealed a robust cone rescue exceeding the transduced area of the retina, and lasting until at least four months of age, the latest time-point examined. This study demonstrates for the first time that Rpe65 gene transfer in rodent models of LCA improves the function and the survival of cone photoreceptors. Furthermore, this provides evidence that HIV-1-derived vectors are suitable for such gene replacement strategy.Molecular Therapy (2006) 13, S161–S161; doi: 10.1016/j.ymthe.2006.08.483 [ABSTRACT FROM AUTHOR]

An abstract of the article "Lentiviral Gene Transfer of RPE65 cDNA in Knock-Out Mouse Models of Leber Congenital Amaurosis," by Corinne Kostic and colleagues is presented.