RESUMEN

The retina is a central nervous tissue essential to visual perception and highly susceptible to environmental damage. Lower vertebrate retinas activate intrinsic regeneration mechanisms in response to retinal injury regulated by a specialized population of progenitor cells. The mammalian retina does not have populations of progenitor/stem cells available to activate regeneration, but contains a subpopulation of differentiated cells that can be reprogrammed into retinal stem cells, the ciliary epithelium (CE) cells. Despite the regenerative potential, stem cells derived from CE exhibit limited reprogramming capacity probably associated with the expression of intrinsic regulatory mechanisms. Platelet-activating factor (PAF) is a lipid mediator widely expressed in many cells and plays an important role in stem cell proliferation and differentiation. During mammalian development, PAF receptor signaling showed important effects on retinal progenitors' cell cycle regulation and neuronal differentiation that need to be further investigated. In this study, our findings suggested a dynamic role for PAF receptor signaling in CE cells, impacting stem cell characteristics and neurosphere formation. We showed that PAF receptors and PAF-related enzymes are downregulated in retinal progenitor/stem cells derived from PE cells. Blocking PAFR activity using antagonists increased the expression of specific progenitor markers, revealing potential implications for retinal tissue development and maintenance.

Asunto(s)

Glicoproteínas de Membrana Plaquetaria , Receptores Acoplados a Proteínas G , Retina , Células Madre , Animales , Proliferación Celular , Células Madre/metabolismo , Epitelio , Mamíferos

RESUMEN

A growing wealth of data suggest that reactive oxygen species (ROS) signalling might be crucial in conferring embryonic or adult stem cells their specific properties. However, how stem cells control ROS production and scavenging, and how ROS in turn contribute to stemness, remain poorly understood. Using the Xenopus retina as a model system, we first investigated the redox status of retinal stem cells (RSCs). We discovered that they exhibit higher ROS levels compared with progenitors and retinal neurons, and express a set of specific redox genes. We next addressed the question of ROS functional involvement in these cells. Using pharmacological or genetic tools, we demonstrate that inhibition of NADPH oxidase-dependent ROS production increases the proportion of quiescent RSCs. Surprisingly, this is accompanied by an apparent acceleration of the mean division speed within the remaining proliferating pool. Our data further unveil that such impact on RSC cell cycling is achieved by modulation of the Wnt/Hedgehog signalling balance. Altogether, we highlight that RSCs exhibit distinctive redox characteristics and exploit NADPH oxidase signalling to limit quiescence and fine-tune their proliferation rate.

Asunto(s)

Células Madre Adultas , Células-Madre Neurales , Animales , Xenopus laevis/metabolismo , Especies Reactivas de Oxígeno , Proliferación Celular , Proteínas Hedgehog , Retina/metabolismo , Células Madre Adultas/metabolismo , NADPH Oxidasas/genética , Vía de Señalización Wnt

RESUMEN

The discovery and study of adult stem cells have revolutionized regenerative medicine by offering new opportunities for treating various medical conditions. Anamniote stem cells, which retain their full proliferative capacity and full differentiation range throughout their lifetime, harbour a greater potential compared to mammalian adult stem cells, which only exhibit limited stem cell potential. Therefore, understanding the mechanisms underlying these differences is of significant interest. In this review, we examine the similarities and differences of adult retinal stem cells in anamniotes and mammals, from their embryonic stages in the optic vesicle to their residence in the postembryonic retinal stem cell niche, the ciliary marginal zone located in the retinal periphery. In anamniotes, developing precursors of retinal stem cells are exposed to various environmental cues during their migration in the complex morphogenetic remodelling of the optic vesicle to the optic cup. In contrast, their mammalian counterparts in the retinal periphery are primarily instructed by neighbouring tissues once they are in place. We explore the distinct modes of optic cup morphogenesis in mammals and teleost fish and highlight molecular mechanisms governing morphogenesis and stem cells instruction. The review concludes with the molecular mechanisms of ciliary marginal zone formation and offers a perspective on the impact of comparative single cell transcriptomic studies to reveal the evolutionary similarities and differences.

Asunto(s)

Células Madre Adultas , Disco Óptico , Animales , Retina , Peces , Desarrollo Embrionario , Mamíferos

RESUMEN

Zika virus (ZIKV) causes microcephaly and congenital eye disease. The cellular and molecular basis of congenital ZIKV infection are not well understood. Here, we utilized a biologically relevant cell-based system of human fetal retinal pigment epithelial cells (FRPEs), hiPSC-derived retinal stem cells (iRSCs), and retinal organoids to investigate ZIKV-mediated ocular cell injury processes. Our data show that FRPEs were highly susceptible to ZIKV infection exhibiting increased apoptosis, whereas iRSCs showed reduced susceptibility. Detailed transcriptomics and proteomics analyses of infected FRPEs were performed. Nucleoside analogue drug treatment inhibited ZIKV replication. Retinal organoids were susceptible to ZIKV infection. The Asian genotype ZIKV exhibited higher infectivity, induced profound inflammatory response, and dysregulated transcription factors involved in retinal organoid differentiation. Collectively, our study shows that ZIKV affects ocular cells at different developmental stages resulting in cellular injury and death, further providing molecular insight into the pathogenesis of congenital eye disease.

Asunto(s)

Oftalmopatías , Células Madre Pluripotentes Inducidas , Infección por el Virus Zika , Virus Zika , Humanos , Virus Zika/fisiología , Retina/patología , Replicación Viral , Organoides , Células Epiteliales/patología , Pigmentos Retinianos/metabolismo

RESUMEN

Objective:Glaucoma is a leading cause of irreversible blindness,and effective therapies to reverse the visual system damage caused by glaucoma are still lacking.Recently,the stem cell therapy enable the repair and regeneration of the damaged retinal neurons,but challenges regarding the source of stem cells remain.This study aims to investigate a protocol that allows the dedifferentiation of Müller cells into retinal stem cells,following by directed differentiation into retinal ganglion cells with high efficiency,and to provide a new method of cellular acquisition for retinal stem cells. Methods:Epidermal cell growth factor and fibroblast growth factor 2 were used to induce the dedifferentiation of rat retinal Müller cells into retinal neural stem cells.Retinal stem cells derived from Müller cells were infected with a Trim9 overexpression lentiviral vector(PGC-FU-Trim9-GFP),and the efficiency of viral infection was assessed by fluorescence microscopy and flow cytometry.Retinoic acid and brain-derived neurotrophic factor treatments were used to induce the differentiation of the retinal stem cells into neurons and glial cells with or without the overexpression of Trim9.The expressions of each cellular marker(GLAST,GS,rhodopsin,PKC,HPC-1,Calbindin,Thy1.1,Brn-3b,Nestin,Pax6)were detected by immunofluorescence,PCR/real-time RT-PCR or Western blotting. Results:Rat retinal Müller cells expressed neural stem cells markers(Nestin and Pax6)with the treatment of epidermal cell growth factor and fibroblast growth factor 2.The Thy1.1 positive cell rate of retinal stem cells overexpressing Trim9 was significantly increased,indicating their directional differentiation into retinal ganglion cells after treatment with retinoic acid and brain-derived neurotrophic factor. Conclusion:In this study,rat retinal Müller cells are dedifferentiated into retinal stem cells successfully,and Trim9 promotes the directional differentiation from retinal stem cells to retinal ganglion cells effectively.

RESUMEN

OBJECTIVES: Glaucoma is a leading cause of irreversible blindness, and effective therapies to reverse the visual system damage caused by glaucoma are still lacking. Recently, the stem cell therapy enable the repair and regeneration of the damaged retinal neurons, but challenges regarding the source of stem cells remain. This study aims to investigate a protocol that allows the dedifferentiation of Müller cells into retinal stem cells, following by directed differentiation into retinal ganglion cells with high efficiency, and to provide a new method of cellular acquisition for retinal stem cells. METHODS: Epidermal cell growth factor and fibroblast growth factor 2 were used to induce the dedifferentiation of rat retinal Müller cells into retinal neural stem cells. Retinal stem cells derived from Müller cells were infected with a Trim9 overexpression lentiviral vector (PGC-FU-Trim9-GFP), and the efficiency of viral infection was assessed by fluorescence microscopy and flow cytometry. Retinoic acid and brain-derived neurotrophic factor treatments were used to induce the differentiation of the retinal stem cells into neurons and glial cells with or without the overexpression of Trim9. The expressions of each cellular marker (GLAST, GS, rhodopsin, PKC, HPC-1, Calbindin, Thy1.1, Brn-3b, Nestin, Pax6) were detected by immunofluorescence, PCR/real-time RT-PCR or Western blotting. RESULTS: Rat retinal Müller cells expressed neural stem cells markers (Nestin and Pax6) with the treatment of epidermal cell growth factor and fibroblast growth factor 2. The Thy1.1 positive cell rate of retinal stem cells overexpressing Trim9 was significantly increased, indicating their directional differentiation into retinal ganglion cells after treatment with retinoic acid and brain-derived neurotrophic factor. CONCLUSIONS: In this study, rat retinal Müller cells are dedifferentiated into retinal stem cells successfully, and Trim9 promotes the directional differentiation from retinal stem cells to retinal ganglion cells effectively.

Asunto(s)

Glaucoma , Células Ganglionares de la Retina , Animales , Ratas , Factor Neurotrófico Derivado del Encéfalo , Células Ependimogliales , Factor 2 de Crecimiento de Fibroblastos , Nestina , Tretinoina

RESUMEN

The present manuscript stems from evidence, which indicates that specific wavelength produce an activation of the autophagy pathway in the retina. These effects were recently reported to synergize with the autophagy-inducing properties of specific phytochemicals. The combined administration of photo-modulation and phytochemicals was recently shown to have a strong potential in eliciting the recovery in the course of retinal degeneration and it was suggested as a non-invasive approach named "Lugano protocol" to treat age-related macular degeneration (AMD). Recent translational findings indicate that the protective role of autophagy may extend also to acute neuronal injuries including traumatic neuronal damage. At the same time, very recent investigations indicate that autophagy activation and retinal anatomical recovery may benefit from sound exposure. Therefore, in the present study, the anatomical rescue of a traumatic neuronal loss at macular level was investigated in a patient with idiopathic macular hole by using a combined approach of physical and chemical non-invasive treatments. In detail, light exposure was administered in combination with sound pulses to the affected retina. This treatment was supplemented by phytochemicals known to act as autophagy inducers, which were administered orally for 6 months. This combined administration of light and sound with nutraceuticals reported here as Advanced Lugano's Protocol (ALP) produced a remarkable effect in the anatomical architecture of the retina affected by the macular hole. The anatomical recovery was almost complete at roughly one year after diagnosis and beginning of treatment. The structural healing of the macular hole was concomitant with a strong improvement of visual acuity and the disappearance of metamorphopsia. The present findings are discussed in the light of a synergism shown at neuronal level between light and sound in the presence of phytochemicals to stimulate autophagy and promote proliferation and neuronal differentiation of retinal stem cells.

Asunto(s)

Perforaciones de la Retina , Suplementos Dietéticos , Humanos , Retina , Perforaciones de la Retina/cirugía , Agudeza Visual , Vitrectomía/métodos

RESUMEN

In multicellular eukaryotic organisms, the initiation of DNA replication occurs asynchronously throughout S-phase according to a regulated replication timing program. Here, using Xenopus egg extracts, we showed that Yap (Yes-associated protein 1), a downstream effector of the Hippo signalling pathway, is required for the control of DNA replication dynamics. We found that Yap is recruited to chromatin at the start of DNA replication and identified Rif1, a major regulator of the DNA replication timing program, as a novel Yap binding protein. Furthermore, we show that either Yap or Rif1 depletion accelerates DNA replication dynamics by increasing the number of activated replication origins. In Xenopus embryos, using a Trim-Away approach during cleavage stages devoid of transcription, we found that either Yap or Rif1 depletion triggers an acceleration of cell divisions, suggesting a shorter S-phase by alterations of the replication program. Finally, our data show that Rif1 knockdown leads to defects in the partitioning of early versus late replication foci in retinal stem cells, as we previously showed for Yap. Altogether, our findings unveil a non-transcriptional role for Yap in regulating replication dynamics. We propose that Yap and Rif1 function as brakes to control the DNA replication program in early embryos and post-embryonic stem cells.

Asunto(s)

Origen de Réplica , Proteínas de Unión a Telómeros , Animales , Replicación del ADN , Momento de Replicación del ADN , Fase S/genética , Proteínas de Unión a Telómeros/genética , Xenopus laevis/genética , Xenopus laevis/metabolismo

RESUMEN

BACKGROUND: The adult mammalian retina does not have the capacity to regenerate cells lost due to damage or disease. Therefore, retinal injuries and blinding diseases result in irreversible vision loss. However, retinal stem cells (RSCs), which participate in retinogenesis during development, persist in a quiescent state in the ciliary epithelium (CE) of the adult mammalian eye. Moreover, RSCs retain the ability to generate all retinal cell types when cultured in vitro, including photoreceptors. Therefore, it may be possible to activate endogenous RSCs to induce retinal neurogenesis in vivo and restore vision in the adult mammalian eye. METHODS: To investigate if endogenous RSCs can be activated, we performed combinatorial intravitreal injections of antagonists to BMP and sFRP2 proteins (two proposed mediators of RSC quiescence in vivo), with or without growth factors FGF and Insulin. We also investigated the effects of chemically-induced N-methyl-N-Nitrosourea (MNU) retinal degeneration on RSC activation, both alone and in combination withthe injected factors. Further, we employed inducible Msx1-CreERT2 genetic lineage labeling of the CE followed by stimulation paradigms to determine if activated endogenous RSCs could migrate into the retina and differentiate into retinal neurons. RESULTS: We found that in vivo antagonism of BMP and sFRP2 proteins induced CE cells in the RSC niche to proliferate and expanded the RSC population. BMP and sFRP2 antagonism also enhanced CE cell proliferation in response to exogenous growth factor stimulation and MNU-induced retinal degeneration. Furthermore, Msx1-CreERT2 genetic lineage tracing revealed that CE cells migrated into the retina following stimulation and/or injury, where they expressed markers of mature photoreceptors and retinal ganglion cells. CONCLUSIONS: Together, these results indicate that endogenous adult mammalian RSCs may have latent regenerative potential that can be activated by modulating the RSC niche and hold promise as a means for endogenous retinal cell therapy to repair the retina and improve vision.

Asunto(s)

Retina , Células Madre , Animales , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Mamíferos , Retina/metabolismo , Células Madre/metabolismo

RESUMEN

Implementing different tools and injury mechanisms in multiple animal models of retina regeneration, researchers have discovered the existence of retinal stem/progenitor cells. Although they appear to be distributed uniformly across the vertebrate lineage, the reparative potential of the retina is mainly restricted to lower vertebrates. Regenerative repair post-injury requires the creation of a proliferative niche, vital for proper stem cell activation, propagation, and lineage differentiation. This seems to be lacking in mammals. Hence, in this review, we first discuss the many forms of retinal injuries that have been generated using animal models. Next, we discuss how they are utilized to stimulate regeneration and mimic eye disease pathologies. The key to driving stem cell activation in mammals relies on the information we can gather from these models. Lastly, we present a brief update about the genes, growth factors, and signaling pathways that have been brought to light using these models.

Asunto(s)

Retina , Enfermedades de la Retina , Animales , Proliferación Celular , Enfermedades de la Retina/genética , Células Madre , Vertebrados

RESUMEN

The zebrafish retina grows for a lifetime. Whether embryonic and postembryonic retinogenesis conform to the same developmental program is an outstanding question that remains under debate. Using single-cell RNA sequencing of ∼20,000 cells of the developing zebrafish retina at four different stages, we identified seven distinct developmental states. Each state explicitly expresses a gene set. Disruption of individual state-specific marker genes results in various defects ranging from small eyes to the loss of distinct retinal cell types. Using a similar approach, we further characterized the developmental states of postembryonic retinal stem cells (RSCs) and their progeny in the ciliary marginal zone. Expression pattern analysis of state-specific marker genes showed that the developmental states of postembryonic RSCs largely recapitulated those of their embryonic counterparts, except for some differences in rod photoreceptor genesis. Thus, our findings reveal the unifying developmental program used by the embryonic and postembryonic retinogenesis in zebrafish.

Asunto(s)

Embrión no Mamífero/metabolismo , Neurogénesis/genética , Retina/metabolismo , Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente/crecimiento & desarrollo , Animales Modificados Genéticamente/metabolismo , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Desarrollo Embrionario , Proteínas de Unión a Ácidos Grasos/deficiencia , Proteínas de Unión a Ácidos Grasos/genética , Proteínas de Unión a Ácidos Grasos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Retina/citología , Retina/crecimiento & desarrollo , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Células Madre/citología , Células Madre/metabolismo , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Pez Cebra/crecimiento & desarrollo , Proteínas de Pez Cebra/deficiencia , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo

RESUMEN

PURPOSE: Retinal degenerative diseases lead to the death of retinal neurons causing visual impairment and blindness. In lower order vertebrates, the retina and its surrounding tissue contain stem cell niches capable of regenerating damaged tissue. Here we examine these niches and review their capacity to be used as retinal stem/progenitor cells (RSC/RPCs) for retinal repair. RECENT FINDINGS: Exogenous factors can control the in vitro activation of RSCs/PCs found in several niches within the adult eye including cells in the ciliary margin, the retinal pigment epithelium, iris pigment epithelium as well as the inducement of Müller and amacrine cells within the neural retina itself. Recently, factors have been identified for the activation of adult mammalian Müller cells to a RPC state in vivo. SUMMARY: Whereas cell transplantation still holds potential for retinal repair, activation of the dormant native regeneration process may lead to a more successful process including greater integration efficiency and proper synaptic targeting.

RESUMEN

Purpose: The sub-retinal injections are not very commonly performed procedures in vitreoretina, but form a crucial step in any cell replacement therapy for retinal diseases. The purpose of this study is to describe the learning curve of a trained vitreo-retinal surgeon in sub-retinal injections in a rat model and its implications in future clinical trials. Methods: This is an in-vivo retrospective animal study using Wistar rats. All ARVO guidelines regarding animal handling were followed. After anesthetization, aspectic preparation and dilating the pupils with 1% tropicamide eye drops, subretinal injection of 10 µl saline was done via a limbal entry. Data recorded included time taken for the procedure, success of injection, associated complications, post-operative infections and complications. The rats were followed up for 1 month post procedure. A trend analysis was done for the above factors to look for improvement in ease of procedure, reduction in procedure time and reduction in complications for the clinician using a novel objective scale. Results: About 20 eyes were studied. Mean weight of the rats was 188 ± 12.82 gram. Mean time taken for the procedure was 14.1 ± 5.07 minutes. There was a significant inverse co-relation between the serial number of the eye and time taken for the procedure (r = -0.89, P < 0.0001). Comparative complications noted between the first ten and the last ten eyes were: conjunctival tear 30% versus 10% (P = 0.27), lens touch 50% versus 10% (P = 0.05), subretinal hemorrhage 40% versus 0% (P = 0.13), vitreous loss 30% versus 0% (P = 0.06). The successful subretinal injection without intraocular complications was achieved in 40% versus 90% (P = 0.02). There was a significant co-relation between the serial number of the eye and ease of the procedure (r = 0.87, P < 0.0001). Post operatively none of the eyes had any infection. Six eyes (12%) developed cataract and 3 eyes (6%) had non-resolving retinal detachment at the last examination visit. Conclusion: Subretinal injections in rats have a definite learning curve even for a trained vitreoretinal surgeon. This should be accounted for and resources allocated accordingly to achieve good technical comfort and negate confounding by the surgeon factor in the results of future clinical trials.

Asunto(s)

Educación de Postgrado en Medicina/métodos , Curva de Aprendizaje , Oftalmología/educación , Hemorragia Retiniana/cirugía , Cirujanos/educación , Grabación en Video , Animales , Modelos Animales de Enfermedad , Humanos , Inyecciones , Ratas , Ratas Wistar , Hemorragia Retiniana/tratamiento farmacológico , Estudios Retrospectivos

RESUMEN

Retinopathy has become one of the major factors that lead to blindness worldwide. Although many clinical therapies are concerned about such disease, most of them focus on symptoms alleviation. In this study, we aim to investigate whether coculture retinal stem cells (RSCs) with bone marrow mesenchymal stem cells transfected with angiogenin-1 (Ang-1-BMSCs) affects the damaged retinal tissue of oxygen-induced retinopathy of prematurity (OIR-ROP) mice. After OIR-ROP mouse model establishment, Ang-1-BMSCs, RSCs, and OIR-ROP retinal tissues were cocultured in a a transwell chamber. RSCs proliferation and the expression of Ang-1, insulin-like growth factor-1 (IGF-1) in the supernatant of RSCs, as well as ß-tubulin and protein kinase C (PKC) expression were evaluated. Finally, the repair of OIR-ROP mice retinal tissues was observed by injecting Ang-1-BMSCs + RSCs. In the OIR-ROP mouse model, RSCs cocultured with OIR-ROP retinal tissues could be induced to differentiate into cells expressing ß-tubulin and PKC and promote the expression of Ang-1 and IGF-1. coculture of Ang-1-BMSCs further enhanced the proliferation and differentiation of RSCs by promoting the expression of Ang-1 and IGF-1. Coculture of RSCs + Ang-1-BMSCs induced differentiation of Ang-1-BMSCs through interaction among intercellular factors and restored the damaged retinal tissue of OIR-ROP mice. Collectively, our study provided evidence that coculture of Ang-1-BMSCs and RSCs could promote the proliferation and differentiation of RSCs and improve the treatment for the damaged retina tissue of OIR-ROP mice.

Asunto(s)

Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/metabolismo , Células-Madre Neurales/metabolismo , Retinopatía de la Prematuridad , Ribonucleasa Pancreática/metabolismo , Animales , Células de la Médula Ósea/metabolismo , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Técnicas de Cocultivo , Ratones , Células-Madre Neurales/citología , Retina/citología , Retina/metabolismo , Transfección

RESUMEN

Retinal diseases are characterized by the degeneration of retinal neural cells, and are the main cause of blindness. Although the development of stem cell including retinal stem cell therapies raises hope for retinal neuron replacement, currently, there is still no efficient method to regenerate retinal neurons. To realize the potential roles of the production of retinal neurons, neurotrophic factor direct the differentiation of retinal stem cells should be extensively identified. In this article, we characterized growth/differentiation 5 (GDF5), which caused the activation of Smad signaling, can induce neurogenesis and neurite outgrowth in retinal stem cell differentiation. Moreover, a bHLH transcription factor, Atoh8 modulates the effects stimulated by GDF5. These data suggested that GDF5 regulates neuron differentiation through mediating Atoh8 and help us to understand the pathophysiological function of GDF5 in retinal regeneration.

Asunto(s)

Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular/fisiología , Factor 5 de Diferenciación de Crecimiento/metabolismo , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , Retina/metabolismo , Animales , Ratones , Células-Madre Neurales/citología , Neurogénesis/fisiología , Neuronas/citología , Retina/citología

RESUMEN

Retinal stem cells (RSCs) are promising candidates for patient-derived cell therapy to repair damage to the eye; however, RSCs are rare in retinal samples and lack validated markers, making cell sorting a significant challenge. Here we report a high-resolution deterministic lateral displacement microfluidic device that profiles RSCs in distinct size populations. Only by developing a chip that promotes cell tumbling do we limit cell deformation through apertured channels and thereby increase the size-sorting resolution of the device. We systematically explore a spectrum of microstructures, including optimized notched pillars, to study and then rationally promote cell tumbling. We find that RSCs exhibit larger diameters than most ciliary epithelial cells, an insight into RSC morphology that allows enrichment from biological samples.

Asunto(s)

Diferenciación Celular , Proliferación Celular , Células Epiteliales/metabolismo , Dispositivos Laboratorio en un Chip , Retina/metabolismo , Células Madre/metabolismo , Animales , Células Cultivadas , Células Epiteliales/citología , Humanos , Ratones , Retina/citología , Células Madre/citología

RESUMEN

BACKGROUNDS: The plasticity of retinal stem cells (RSCs), a type of cells that can differentiate into neuron cells and photoreceptor cells, endows them with potential therapeutic properties that can be applied to regenerative medicine. Gene modification of these stem cells before trans-differentiation and transplantation enhances their survival and increases their therapeutic function. The different ways to effectively deliver gene into RSCs are still discussed. This study aimed to use the acoustic waves to improve the efficacy of gene delivery for RSCs. METHODS: RSCs were obtained from non-fetal human ocular pigmented ciliary margin tissues. The enhanced green fluorescent protein-encoded murine stem cell retroviruses (MSCV) were prepared and used to infect RSCs. Glass chambers containing RSCs, retroviruses, and various concentrations of polybrene (0, 0.8, 2, 4 and 8 µg/mL) were exposed under 20 or 25 Vp-p ultrasonic standing wave fields (USWF) for 5 min. The percentage of green fluorescent protein positive cells in each sample was calculated and compared to test the efficacy of gene transduction. RESULTS: Our results showed that the efficiency of gene transduction by MSCV infection was enhanced following the concentration of polybrene and the energy of USWF. The percentage of green fluorescent protein positive cells was significantly higher in chambers that contained 8 µg/mL of polybrene and was exposed to 20Vp-p of USWF for 5 min. In addition, the percentage increased in chambers contained 2, 4 and 8 µg/mL of polybrene when they were exposed to 25Vp-p of USWF. Comparing to those did not treated with ultrasound, the efficiency of retroviral transduction to RSCs increased 4-fold after exposed to USWF for 5 min. CONCLUSION: We demonstrated the ability of ultrasound standing waves to improve retroviral transduction into RSCs. We believe that this may be applied to the experimental designs of future studies and may have possible therapeutic uses.

Asunto(s)

Retroviridae/genética , Sonido , Células Madre/metabolismo , Transducción Genética/métodos , Adulto , Anciano , Agregación Celular , Separación Celular , Células Cultivadas , Bromuro de Hexadimetrina/farmacología , Humanos , Lactante , Retina

RESUMEN

Objective To investigate the effects of retinal ganglion cell-conditioned medium on the differentiation of retinal stem cells.Methods Rettial stem cells and retinal ganglion cells were isolated from rats,and immunofluorescence staining was applied to identify rat retinal stem cells and retinal ganglion cells with Nestin and Thy-1 antibody,respectively.Retinal stem cells were cultured in presence or absence of retinal ganglion cell-conditioned medium for 72 h,followed by detection of Nestin,PAX6,Thy-1 and Bin-3 gene expression in retinal stem cells by qPCR.Results isolated retinal stem cells were Nestin positive,and retinal ganglion cells were Thy-1 positive,indicating the success of isolation.Compared to retinal stem cells cultured without ganglion cellconditioned medium,ones cultured with ganglion-conditioned medium had significantly downregulated expression of Nestin and PAX6 (both P ＜ 0.000 1),and markedly upregulated expression of Thy-1 and Brn-3 (both P ＜ 0.05).Conclusion Retinal ganglion cell-conditioned medium can induce the differentiation of retinal progenitor cells into retinal ganglion-like cells.

RESUMEN

PURPOSE OF REVIEW: Retinal degenerative diseases have immense socio-economic impact. Studying animal models that recapitulate human eye pathologies aids in understanding the pathogenesis of diseases and allows for the discovery of novel therapeutic strategies. Some non-mammalian species are known to have remarkable regenerative abilities and may provide the basis to develop strategies to stimulate self-repair in patients suffering from these retinal diseases. RECENT FINDINGS: Non-mammalian organisms, such as zebrafish and Xenopus, have become attractive model systems to study retinal diseases. Additionally, many fish and amphibian models of retinal cell ablation and cell lineage analysis have been developed to study regeneration. These investigations highlighted several cellular sources for retinal repair in different fish and amphibian species. Moreover, major differences in repair mechanisms have been reported in these animal models. SUMMARY: This review aims to emphasize first on the importance of zebrafish and Xenopus models in studying the pathogenesis of retinal diseases and, second, on the different modes of regeneration processes in these model organisms.

RESUMEN

AIM:To investigate the combination therapeutic effect of Ginkgobalide B (GKB) and retinal stem cells (RSCs)transplantation on glaucoma in rats.METHODS:Rats were divided randomly into five groups:control group,glaucoma group,RSCs group,GKB group and RSCs combination therapy group.A chronic glaucoma model was established in rats,accordingly.The morphological changes in ocular tissues were analyzed by HE staining.Retinal ganglion cells apoptosis were analyzed by TUNEL staining.The protein expressions of Bcl-2,Bax,Cleaved caspase-3 and Cleaved caspase-9 were determined by Western blot.The mRNA levels of Bcl-2 and Bax were determined by qPCR.RESULTS:HE staining revealed that RSCs transplantation or GKB treatment decreased fiber interstitial edema and vacuole,as compared to glaucoma group.Furthermore,this improvement was more pronounced in combination therapy group than in single treatment alone.Combination therapy significantly inhibited retinal ganglion cells apoptosis,increased Bcl-2 mRNA and protein expression,but decreased Bax mRNA and protein expression.Moreover,the protein expression of Cleaved caspase-3 and Cleaved caspase-9 expression were decreased after combination therapy.CONCLUSION:Our data demonstrate that combination of Ginkgobalide B and retinal stem cells transplantation can inhibit retinal ganglion cells apoptosis and protect against glaucoma.These effects may be associated with the regulation of Bcl-2,Bax,Cleaved caspase-3 and Cleaved caspase-9 expression.