Retinal stem cells transplanted into models of late stages of retinitis pigmentosa preferentially adopt a glial or a retinal ganglion cell fate.

PURPOSE: To characterize the potential of newborn retinal stem cells (RSCs) isolated from the radial glia population to integrate the retina, this study was conducted to investigate the fate of in vitro expanded RSCs transplanted into retinas devoid of photoreceptors (adult rd1 and old VPP mice and rhodopsin-mutated transgenic mice) or partially degenerated retina (adult VPP mice) retinas. METHODS: Populations of RSCs and progenitor cells were isolated either from DBA2J newborn mice and labeled with the red lipophilic fluorescent dye (PKH26) or from GFP (green fluorescent protein) transgenic mice. After expansion in EGF+FGF2 (epidermal growth factor+fibroblast growth factor), cells were transplanted intravitreally or subretinally into the eyes of adult wild-type, transgenic mice undergoing slow (VPP strain) or rapid (rd1 strain) retinal degeneration. RESULTS: Only limited migration and differentiation of the cells were observed in normal mice injected subretinally or in VPP and rd1 mice injected intravitreally. After subretinal injection in old VPP mice, transplanted cells massively migrated into the ganglion cell layer and, at 1 and 4 weeks after injection, harbored neuronal and glial markers expressed locally, such as beta-tubulin-III, NeuN, Brn3b, or glial fibrillary acidic protein (GFAP), with a marked preference for the glial phenotype. In adult VPP retinas, the grafted cells behaved similarly. Few grafted cells stayed in the degenerating outer nuclear layer (ONL). These cells were, in rare cases, positive for rhodopsin or recoverin, markers specific for photoreceptors and some bipolar cells. CONCLUSIONS: These results show that the grafted cells preferentially integrate into the GCL and IPL and express ganglion cell or glial markers, thus exhibiting migratory and differentiation preferences when injected subretinally. It also appears that the retina, whether partially degenerated or already degenerated, does not provide signals to induce massive differentiation of RSCs into photoreceptors. This observation suggests that a predifferentiation of RSCs into photoreceptors before transplantation may be necessary to obtain graft integration in the ONL.

Generation of cells committed towards the photoreceptor fate for retinal transplantation.

Cell transplantation is an active field of research to replace lost cells in retinal dystrophies to potentially restore visual function. We hypothesized that in-vitro differentiated retinal stem cells would integrate the appropriate retinal layer and differentiate into photoreceptors when transplanted during development. Here we show that retinal stem cells driven to the photoreceptor fate start to incorporate the retina and express photoreceptor markers but do not survive. Nevertheless surviving grafted cells express the glial marker glial fibrillary acidic protein and incorporate the ganglion cell layer as well as the inner plexiform layer. These results suggest that the maturation state of the photoreceptors is primordial to obtain robust incorporation and that a fine tuning of retinal stem cells differentiation should provide adequate cells for transplantation.

The control of UCP1 is dissociated from that of PGC-1alpha or of mitochondriogenesis as revealed by a study using beta-less mouse brown adipocytes in culture.

In rodent brown adipose tissue, the beta-adrenergic signaling is believed, by an action on PGC-1alpha, to control UCP1 expression and mitochondriogenesis. We addressed this hypothesis using beta(1)/beta(2)/beta(3)-adrenoceptor knockout (beta-less) brown adipocytes in primary culture. In these cells: (a) proliferation and differentiation into multilocular cells were normal; (b) UCP1 mRNA expression was dramatically decreased (by 93%), whereas PGC-1alpha and mtTFA mRNA expressions were not; (c) UCP1, PGC-1alpha and COX IV protein expressions were decreased by 97%, 62% and 22%, respectively. Altogether the data show a dissociation between the control of UCP1, which is mostly beta-adrenoceptor-dependent and that of PGC-1alpha and of mitochondriogenesis which are not.

Beta(1)/beta(2)/beta(3)-adrenoceptor knockout mice are obese and cold-sensitive but have normal lipolytic responses to fasting.

Catecholamines are viewed as major stimulants of diet- and cold-induced thermogenesis and of fasting-induced lipolysis, through the beta-adrenoceptors (beta(1)/beta(2)/beta(3)). To test this hypothesis, we generated beta(1)/beta(2)/beta(3)-adrenoceptor triple knockout (TKO) mice and compared them to wild type animals. TKO mice exhibited normophagic obesity and cold-intolerance. Their brown fat had impaired morphology and lacked responses to cold of uncoupling protein-1 expression. In contrast, TKO mice had higher circulating levels of free fatty acids and glycerol at basal and fasted states, suggesting enhanced lipolysis. Hence, beta-adrenergic signalling is essential for the resistance to obesity and cold, but not for the lipolytic response to fasting.

Remaining rod activity mediates visual behavior in adult Rpe65-/- mice.

PURPOSE: C57/Bl6, Cpfl1(-/-) (cone photoreceptors function loss 1; pure rod function), Gnat1a(-/-) (rod α-transducin; pure cone function), and Rpe65(-/-);Rho(-/-) double-knockout mice were studied to distinguish the respective contributions of the different photoreceptor (PR) systems that enable light perception and mediate a visual reflex in adult Rpe65(-/-) mice, with a simple behavioral procedure. METHODS: Visual function was estimated using a rotating automated optomotor drum covered with vertical black-and-white stripes at spatial frequencies of 0.025 to 0.5 cycles per degree (cyc/deg) in both photopic and scotopic conditions. Mouse strains with different luminances were tested to evaluate the contribution and the light-intensity threshold of each PR system. RESULTS: Stripe rotation elicited head movements in the wild-type (WT) animals in photopic and scotopic conditions, depending on the spatial frequency, whereas the Cpfl1(-/-) mice show a reduced activity in the photopic condition and the Gnat1a(-/-) mice an almost absent response in the scotopic condition. A robust visual response was obtained with Rpe65(-/-) knockout mice at 0.075 and 0.1 cyc/deg in the photopic condition. The residual rod function in the Rpe65(-/-) animals was demonstrated by testing Rpe65(-/-);Rho(-/-) mice that present no response in photopic conditions. CONCLUSIONS: The optomotor test is a simple method of estimating the visual function and evaluating the respective contributions of rod and cone systems. This test was used to demonstrate that in Rpe65(-/-) mice, devoid of functional cones and of detectable 11-cis-retinal protein, the rods mimic cone function in part, by mediating vision in photopic conditions.

High yield of cells committed to the photoreceptor fate from expanded mouse retinal stem cells.

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 retinoid-binding 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-alpha. 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.

D-TAT transporter as an ocular peptide delivery system.

BACKGROUND: Future treatment for genetic diseases may involve the replacement of malfunctioning genes through virus-mediated gene therapy. However, this approach is plagued with many problems, both ethical and scientific. Therefore, alternative treatments based on new molecules may represent a safer option. Molecular treatment of many eye diseases will need to bring active molecules into the photoreceptors. Recently, the trans-activator protein (TAT) human immunodeficiency virus type 1 (HIV-1) transcriptional factor has proven to be effective in transporting molecules across cellular membranes. The half-life of these molecules does not exceed 48 hours. The potential use of the retro-inverso form of the TAT (D-TAT) peptide, the protein transducing domain of the HIV-1 transcriptional factor, as a molecular transporter was investigated. METHODS: FITC-labelled D-TAT (D-TAT FITC) was applied to the 661W murine photoreceptor cell line in culture. The labelled peptide was also injected into the vitreous body or the subretinal space of adult mice. Cells and cryosections of eyes were analysed under fluorescence microscopy at various time points after peptide treatment. Coimmunostaining with various antibodies was performed in order to characterize the transduces cells. RESULTS: D-TAT was effective in transducing photoreceptor cells in culture. Transduction of D-TAT FITC was also effective when injected into the vitreous or subretinal space and was observed for a longer period of time than L-TAT FITC. CONCLUSIONS: The retro-inverso form of the TAT sequence is effective in transducing cells from various compartments of the eye. After 14 days, the D-TAT FITC was clearly visible in the retina whereas L-TAT FITC had almost disappeared. The D-TAT peptide represents an interesting molecular transporter that, when coupled to a specific effector, may have potential therapeutic future, especially when a long-lasting action is needed.