RESUMO
The retina is subject to degenerative conditions leading to blindness. Although retinal regeneration is possible in lower vertebrates, it does not occur in the adult mammalian retina. Retinal stem cell (RSC) research offers unique opportunities for developing clinical application for therapy. The ciliary body of adult mammals represents a source of quiescent RSC. These neural progenitors have a limited self-renewal potential in vitro but this can be improved by mitogens. Pigment Epithelium Derived Factor (PEDF), a member of the serpin gene family, is synthesized and secreted by retinal pigment epithelium (RPE) cells. We tested combinations of PEDF with fibroblast growth factor (FGF) during RSC growth to evaluate self-renewal and subsequent differentiation into retinal-like neuronal cell types. Medium supplemented with FGF + PEDF enhanced the RSC yield and more interestingly allowed expansion of the culture by increasing secondary retinal neurospheres after the 1st passage. This effect was accompanied by cell proliferation as revealed by BrdU incorporation. PEDF usage did not affect rod-like differentiation potential. This was demonstrated by immunofluorescence analysis of Rhodopsin and Pde6b that were found similarly expressed in cells derived from FGF or FGF + PEDF cultured RSC. Our studies suggest a possible application of PEDF in Retinal Stem Cell culture and transplantation.
Assuntos
Proteínas do Olho/farmacologia , Fatores de Crescimento Neural/farmacologia , Neurônios/citologia , Retina/citologia , Serpinas/farmacologia , Esferoides Celulares/citologia , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Retina/efeitos dos fármacos , Esferoides Celulares/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacosRESUMO
PURPOSE: The developmental processes that mediate differentiation from retinal stem cells (RSC) to different retinal neuronal types remain unclear. During retinal development, progenitor cells modify expression of growth factor (GF) receptors and their differentiation potentials. Similarly, RSC in culture may exhibit alternative molecular characteristics in response to different GF stimuli. METHODS: RSC were purified from the adult ciliary margin and exposed to fibroblast growth factor (FGF), epidermal growth factor (EGF), or FGF+EGF. Proliferation was analyzed by bromodeoxyuridine (BrdU) labeling. Differentiation was evaluated by immunofluorescence with antibodies recognizing specific markers of different retinal cell types. RESULTS: In the absence of GF stimuli, RSC in culture expressed FGFR1, similar to early progenitors in vivo. Treatment with GFs up-regulated the expression of both fibroblast growth factor receptor 1 (FGFR1) and epidermal growth factor receptor (EGFR). Exposure to either FGF, EGF, or FGF+EGF strongly affected retinal stem cell-renewal and differentiation. Specifically, expression of progenitor/stem cell markers and stem cell-renewal was higher in the presence of FGF than in that of EGF. FGF favored differentiation of RSC into photoreceptor-like cells. Finally, we showed that the treatment of the primary culture with FGF+EGF imprinted the cells and limited plasticity in subsequent differentiation. CONCLUSIONS: We provide evidence that conditions of the primary culture have a strong influence on cell-renewal and differentiation potentials of RSC.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Fatores de Crescimento de Fibroblastos/farmacologia , Retina/citologia , Células-Tronco/citologia , Animais , Biomarcadores/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Sinergismo Farmacológico , Receptores ErbB/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células Fotorreceptoras de Vertebrados/citologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Retina/metabolismo , Células-Tronco/metabolismo , Regulação para CimaRESUMO
Multiple sclerosis (MS) is a progressive, inflammatory, demyelinating disease. An altered cytokine network has been reported to occur during the disease, and its pathogenetic role has been hypothesized. To date, interferon-beta (IFN-beta) is the most effective and reliable therapy in the majority of MS patients, although the mechanisms underlying its therapeutic effects are not fully understood. Breakdown of the blood-brain barrier (BBB) with consequent extravasation of the T cells and their invasion of the brain parenchyma seems to be one of the most important steps in the pathogenesis of the disease. Matrix metalloproteinease-2 (MMP-2) and MMP-9 are enzymes with proteolytic activities toward extracellular matrix ECM components. They are physiologically balanced by the MMP tissue inhibitors TIMP-2 and TIMP-1, so that proteolysis occurs as the result of increased MMP or decreased TIMP levels. In 38 patients with MS, MMP-2 and TIMP-1 levels were similar before and after 9 months of IFN-beta therapy, whereas MMP-9 levels significantly decreased and TIMP-2 levels significantly increased in comparison to values obtained before treatment. These results suggest that IFN-beta modulates T cell activities, including MMP and TIMP production, thus contributing either to maintaining the integrity of the BBB or to slowing the progression of the disease.
Assuntos
Interferon beta/uso terapêutico , Esclerose Múltipla Recidivante-Remitente/enzimologia , Esclerose Múltipla Recidivante-Remitente/terapia , Adolescente , Feminino , Humanos , Injeções Intramusculares , Interferon beta/administração & dosagem , Masculino , Metaloproteinase 2 da Matriz/sangue , Metaloproteinase 9 da Matriz/sangue , Pessoa de Meia-Idade , Esclerose Múltipla Recidivante-Remitente/genética , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/uso terapêutico , Inibidor Tecidual de Metaloproteinase-1/sangue , Inibidor Tecidual de Metaloproteinase-2/biossíntese , Inibidor Tecidual de Metaloproteinase-2/sangueRESUMO
In vitro generation of photoreceptors from stem cells is of great interest for the development of regenerative medicine approaches for patients affected by retinal degeneration and for high throughput drug screens for these diseases. In this study, we show unprecedented high percentages of rod-fated cells from retinal stem cells of the adult ciliary epithelium. Molecular characterization of rod-like cells demonstrates that they lose ciliary epithelial characteristics but acquire photoreceptor features. Rod maturation was evaluated at two levels: gene expression and electrophysiological functionality. Here we present a strong correlation between phototransduction protein expression and functionality of the cells in vitro. We demonstrate that in vitro generated rod-like cells express cGMP-gated channels that are gated by endogenous cGMP. We also identified voltage-gated channels necessary for rod maturation and viability. This level of analysis for the first time provides evidence that adult retinal stem cells can generate highly homogeneous rod-fated cells.
Assuntos
Células-Tronco Adultas/citologia , Cílios/metabolismo , Células Epiteliais/citologia , Retina/citologia , Células Fotorreceptoras Retinianas Bastonetes/citologia , 1-Metil-3-Isobutilxantina/farmacologia , Células-Tronco Adultas/efeitos dos fármacos , Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/efeitos da radiação , Animais , Canais de Cloro CLC-2 , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Diferenciação Celular/efeitos da radiação , Canais de Cloreto/metabolismo , GMP Cíclico/metabolismo , Dependovirus/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/efeitos da radiação , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Proteínas de Fluorescência Verde/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/efeitos da radiação , Luz , Transdução de Sinal Luminoso/efeitos dos fármacos , Transdução de Sinal Luminoso/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Células Fotorreceptoras Retinianas Bastonetes/efeitos dos fármacos , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/efeitos da radiação , Rodopsina/metabolismo , Esferoides Celulares/citologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/efeitos da radiaçãoRESUMO
One of the limitations in molecular and functional studies of the retinal pigment epithelium (RPE) has been the lack of an in vitro system retaining all the features of in vivo RPE cells. Retinal pigment epithelium cell lines do not show characteristics typical of a functional RPE, such as pigmentation and expression of specific markers. The present study was aimed at the development of culture conditions to differentiate, in vitro, retinal stem cells (RSC), derived from the adult ciliary body, into a functional RPE. Retinal stem cells were purified from murine eyes, grown as pigmented neurospheres and induced to differentiate into RPE on an extracellular matrix substrate using specific culture conditions. After 7-15 days of culture, pigmented cells with an epithelial morphology showed a polarized organization and a capacity for phagocytosis. We detected different stages of melanogenesis in cells at 7 days of differentiation, whereas RPE at 15 days contained only mature melanosomes. These data suggest that our protocol to differentiate RPE in vitro can provide a useful model for molecular and functional studies.