RESUMO
Cone-rod degeneration (CRD) belongs to the disease spectrum of retinal degenerations, a group of hereditary disorders characterized by an extreme clinical and genetic heterogeneity. It mainly differentiates from other retinal dystrophies, and in particular from the more frequent disease retinitis pigmentosa, because cone photoreceptors degenerate at a higher rate than rod photoreceptors, causing severe deficiency of central vision. After exome analysis of a cohort of individuals with CRD, we identified biallelic mutations in the orphan gene CEP78 in three subjects from two families: one from Greece and another from Sweden. The Greek subject, from the island of Crete, was homozygous for the c.499+1G>T (IVS3+1G>T) mutation in intron 3. The Swedish subjects, two siblings, were compound heterozygotes for the nearby mutation c.499+5G>A (IVS3+5G>A) and for the frameshift-causing variant c.633delC (p.Trp212Glyfs(∗)18). In addition to CRD, these three individuals had hearing loss or hearing deficit. Immunostaining highlighted the presence of CEP78 in the inner segments of retinal photoreceptors, predominantly of cones, and at the base of the primary cilium of fibroblasts. Interaction studies also showed that CEP78 binds to FAM161A, another ciliary protein associated with retinal degeneration. Finally, analysis of skin fibroblasts derived from affected individuals revealed abnormal ciliary morphology, as compared to that of control cells. Altogether, our data strongly suggest that mutations in CEP78 cause a previously undescribed clinical entity of a ciliary nature characterized by blindness and deafness but clearly distinct from Usher syndrome, a condition for which visual impairment is due to retinitis pigmentosa.
Assuntos
Proteínas de Ciclo Celular/genética , Cílios/patologia , Distrofias de Cones e Bastonetes/complicações , Distrofias de Cones e Bastonetes/genética , Perda Auditiva Neurossensorial/genética , Perda Auditiva Neurossensorial/patologia , Mutação/genética , Idoso , Alelos , Animais , Cadáver , Proteínas de Ciclo Celular/metabolismo , Estudos de Coortes , Distrofias de Cones e Bastonetes/patologia , Distrofias de Cones e Bastonetes/fisiopatologia , Exoma/genética , Olho/embriologia , Olho/metabolismo , Proteínas do Olho/metabolismo , Feminino , Fibroblastos/patologia , Grécia , Perda Auditiva Neurossensorial/complicações , Perda Auditiva Neurossensorial/fisiopatologia , Heterozigoto , Homozigoto , Humanos , Íntrons/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Linhagem , Ligação Proteica , RNA Mensageiro/análise , Suécia , Transcriptoma , Síndromes de Usher/patologiaRESUMO
The cone function is essential to mediate high visual acuity, color vision, and daylight vision. Inherited cone dystrophies and age-related macular degeneration affect a substantial percentage of the world population. To identify and isolate the most competent cells for transplantation and integration into the retina, cone tracing during development would be an important added value. To that aim, the Chrnb4-EGFP mouse line was characterized throughout retinogenesis. It revealed a sub-population of early retinal progenitors expressing the reporter gene that is progressively restricted to mature cones during retina development. The presence of the native CHRNB4 protein was confirmed in EGFP-positive cells, and it presents a similar pattern in the human retina. Sub-retinal transplantations of distinct subpopulations of Chrnb4-EGFP-expressing cells revealed the embryonic day 15.5 high-EGFP population the most efficient cells to interact with host retinas to provoke the appearance of EGFP-positive cones in the photoreceptor layer. Importantly, transplantations into the DsRed retinas revealed material exchanges between donor and host retinas, as >80% of transplanted EGFP-positive cones also were DsRed positive. Whether this cell material fusion is of significant therapeutic advantage requires further thorough investigations. The Chrnb4-EGFP mouse line definitely opens new research perspectives in cone genesis and retina repair.
Assuntos
Rastreamento de Células/métodos , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas do Tecido Nervoso/genética , Receptores Nicotínicos/genética , Proteínas Recombinantes de Fusão/genética , Células Fotorreceptoras Retinianas Cones/metabolismo , Animais , Humanos , Degeneração Macular , Camundongos , Fatores de Transcrição Otx/genética , Fatores de Transcrição Otx/metabolismo , Retina/embriologia , Retina/metabolismo , Receptor X Retinoide gama/genética , Receptor X Retinoide gama/metabolismo , Transplante de Células-Tronco , Células-Tronco/citologia , Células-Tronco/metabolismoAssuntos
Câmara Anterior/efeitos dos fármacos , Câmara Anterior/patologia , Antineoplásicos Alquilantes/uso terapêutico , Melfalan/uso terapêutico , Neoplasias da Retina/tratamento farmacológico , Retinoblastoma/tratamento farmacológico , Adolescente , Câmara Anterior/diagnóstico por imagem , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Microscopia Acústica , Invasividade Neoplásica , Neoplasias da Retina/patologia , Retinoblastoma/patologia , Estudos Retrospectivos , Terapia de SalvaçãoRESUMO
The authors determined the effect of the GLP-1 receptor agonist liraglutide on endothelial surface expression of vascular cell adhesion molecule (VCAM)-1 in murine apolipoprotein E knockout atherosclerosis. Contrast-enhanced ultrasound molecular imaging using microbubbles targeted to VCAM-1 and control microbubbles showed a 3-fold increase in endothelial surface VCAM-1 signal in vehicle-treated animals, whereas in the liraglutide-treated animals the signal ratio remained around 1 throughout the study. Liraglutide had no influence on low-density lipoprotein cholesterol or glycated hemoglobin, but reduced TNF-α, IL-1ß, MCP-1, and OPN. Aortic plaque lesion area and luminal VCAM-1 expression on immunohistology were reduced under liraglutide treatment.
RESUMO
Cell identity is acquired in different brain structures according to a stereotyped timing schedule, by accommodating the proliferation of multipotent progenitor cells and the generation of distinct types of mature nerve cells at precise times. However, the molecular mechanisms coupling the identity of a specific neuron and its birth date are poorly understood. In the neural retina, only late progenitor cells that divide slowly can become bipolar neurons, by the activation of otx2 and vsx1 genes. In Xenopus, we found that Xotx2 and Xvsx1 translation is inhibited in early progenitor cells that divide rapidly by a set of cell cycle-related microRNAs (miRNAs). Through expression and functional screenings, we selected 4 miRNAs--mir-129, mir-155, mir-214, and mir-222--that are highly expressed at early developmental stages in the embryonic retina and bind to the 3' UTR of Xotx2 and Xvsx1 mRNAs inhibiting their translation. The functional inactivation of these miRNAs in vivo releases the inhibition, supporting the generation of additional bipolar cells. We propose a model in which the proliferation rate and the age of a retinal progenitor are linked to each other and determine the progenitor fate through the activity of a set of miRNAs.
Assuntos
Linhagem da Célula , MicroRNAs/fisiologia , Retina/crescimento & desenvolvimento , Animais , Proliferação de Células , Proteínas do Olho/antagonistas & inibidores , Proteínas do Olho/genética , MicroRNAs/metabolismo , Neurônios/citologia , Fatores de Transcrição Otx/antagonistas & inibidores , Fatores de Transcrição Otx/genética , RNA Mensageiro/metabolismo , Retina/embriologia , Células Bipolares da Retina/citologia , Células-Tronco/citologia , Xenopus , Proteínas de Xenopus/antagonistas & inibidores , Proteínas de Xenopus/genéticaRESUMO
Initiation of PINK1- and PRKN-dependent mitophagy is a highly regulated process involving the activity of the AAA-ATPase VCP/p97, a cofactor-guided multifunctional protein central to handling ubiquitinated client proteins. Removal of ubiquitinated substrates such as the mitofusin MFN2 from the outer mitochondrial membrane by VCP is critical for PRKN accumulation on mitochondria, which drives mitophagy. Here we characterize the role of the UBA and UBX-domain containing VCP cofactor UBXN1/SAKS1 during mitophagy. Following mitochondrial depolarization and depending on PRKN, UBXN1 translocated alongside VCP to mitochondria. Prior to mitophagy, loss of UBXN1 led to mitochondrial fragmentation, diminished ATP production, and impaired ER-mitochondrial apposition. When mitophagy was induced in cells lacking UBXN1, mitochondrial translocation of VCP and PRKN was impaired, diminishing mitophagic flux. In addition, UBXN1 physically interacted with PRKN in a UBX-domain depending manner. Interestingly, ectopic expression of the pro-mitophagic VCP cofactor UBXN6/UBXD1 fully reversed impaired PRKN recruitment in UBXN1-/- cells. Mechanistically, UBXN1 acted downstream of PINK1 by facilitating MFN2 removal from mitochondria. In UBXN1-/- cells exposed to mitochondrial stress, MFN2 formed para-mitochondrial blobs likely representing blocked intermediates of the MFN2 removal process partly reversible by expression of UBXN6. Presence of these MFN2 blobs strongly correlated with impaired PRKN translocation to depolarized mitochondria. Our observations connect the VCP cofactor UBXN1 to the initiation and maintenance phase of PRKN-dependent mitophagy, and indicate that, upon mitochondrial stress induction, MFN2 removal from mitochondria occurs through a specialized process.
Assuntos
Mitofagia , Ubiquitina-Proteína Ligases , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagia , GTP Fosfo-Hidrolases/metabolismo , Humanos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteína com Valosina/metabolismoRESUMO
The ciliary margin in lower vertebrates is a site of continual retinal neurogenesis and a stem cell niche. By contrast, the human eye ceases retinal neuron production before birth and loss of photoreceptors during life is permanent and a major cause of blindness. The discovery of a proliferative cell population in the ciliary epithelium (CE) of the adult mammalian eye, designated retinal stem cells, raised the possibility that these cells could help to restore sight by replacing lost photoreceptors. We previously demonstrated the feasibility of photoreceptor transplantation using cells from the developing retina. CE cells could provide a renewable source of photoreceptors for transplantation. Several laboratories reported that these cells generate new photoreceptors, whereas a recent report questioned the existence of retinal stem cells. We used Nrl.gfp transgenic mice that express green fluorescent protein in rod photoreceptors to assess definitively the ability of CE cells to generate new photoreceptors. We report that CE cells expanded in monolayer cultures, lose pigmentation, and express a subset of eye field and retinal progenitor cell markers. Simultaneously, they continue to express some markers characteristic of differentiated CE and typically lack a neuronal morphology. Previously reported photoreceptor differentiation conditions used for CE cells, as well as conditions used to differentiate embryonic retinal progenitor cells (RPCs) and embryonic stem cell-derived RPCs, do not effectively activate the Nrl-regulated photoreceptor differentiation program. Therefore, we conclude that CE cells lack potential for photoreceptor differentiation and would require reprogramming to be useful as a source of new photoreceptors.
Assuntos
Células-Tronco Adultas/citologia , Retina/citologia , Células Fotorreceptoras Retinianas Bastonetes/citologia , Células-Tronco Adultas/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Retina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismoRESUMO
Dominant optic atrophy (DOA) is an inherited mitochondrial disease leading to specific degeneration of retinal ganglion cells (RGCs), thus compromising transmission of visual information from the retina to the brain. Usually, DOA starts during childhood and evolves to poor vision or legal blindness, affecting the central vision, whilst sparing the peripheral visual field. In 20% of cases, DOA presents as syndromic disorder, with secondary symptoms affecting neuronal and muscular functions. Twenty years ago, we demonstrated that heterozygous mutations in OPA1 are the most frequent molecular cause of DOA. Since then, variants in additional genes, whose functions in many instances converge with those of OPA1, have been identified by next generation sequencing. OPA1 encodes a dynamin-related GTPase imported into mitochondria and located to the inner membrane and intermembrane space. The many OPA1 isoforms, resulting from alternative splicing of three exons, form complex homopolymers that structure mitochondrial cristae, and contribute to fusion of the outer membrane, thus shaping the whole mitochondrial network. Moreover, OPA1 is required for oxidative phosphorylation, maintenance of mitochondrial genome, calcium homeostasis and regulation of apoptosis, thus making OPA1 the Swiss army-knife of mitochondria. Understanding DOA pathophysiology requires the understanding of RGC peculiarities with respect to OPA1 functions. Besides the tremendous energy requirements of RGCs to relay visual information from the eye to the brain, these neurons present unique features related to their differential environments in the retina, and to the anatomical transition occurring at the lamina cribrosa, which parallel major adaptations of mitochondrial physiology and shape, in the pre- and post-laminar segments of the optic nerve. Three DOA mouse models, with different Opa1 mutations, have been generated to study intrinsic mechanisms responsible for RGC degeneration, and these have further revealed secondary symptoms related to mitochondrial dysfunctions, mirroring the more severe syndromic phenotypes seen in a subgroup of patients. Metabolomics analyses of cells, mouse organs and patient plasma mutated for OPA1 revealed new unexpected pathophysiological mechanisms related to mitochondrial dysfunction, and biomarkers correlated quantitatively to the severity of the disease. Here, we review and synthesize these data, and propose different approaches for embracing possible therapies to fulfil the unmet clinical needs of this disease, and provide hope to affected DOA patients.
Assuntos
Atrofia Óptica Autossômica Dominante , Animais , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Humanos , Camundongos , Mitocôndrias , Atrofia Óptica Autossômica Dominante/genética , Atrofia Óptica Autossômica Dominante/metabolismo , Nervo Óptico/metabolismo , Células Ganglionares da Retina/metabolismoRESUMO
The retinal pigment epithelium (RPE) is a monolayer of cobblestone-like epithelial cells that accomplishes critical functions for the retina. Several protocols have been published to differentiate pluripotent stem cells into RPE cells suitable for disease modelling and therapy development. In our study, the RPE identity of human induced pluripotent stem cell (hiPSC)-derived RPE (iRPE) was extensively characterized, and then used to test a lentiviral-mediated RPE65 gene augmentation therapy. A dose study of the lentiviral vector revealed a dose-dependent effect of the vector on RPE65 mRNA levels. A marked increase of the RPE65 mRNA was also observed in the iRPE (100-fold) as well as in an experimental set with RPE derived from another hiPSC source and from foetal human RPE. Although iRPE displayed features close to bona fide RPE, no or a modest increase of the RPE65 protein level was observed depending on the protein detection method. Similar results were observed with the two other cell lines. The mechanism of RPE65 protein regulation remains to be elucidated, but the current work suggests that high vector expression will not produce an excess of the normal RPE65 protein level.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Epitélio Pigmentado da Retina/citologia , cis-trans-Isomerases/genética , cis-trans-Isomerases/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular , Células Cultivadas , Técnicas de Transferência de Genes , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Lentivirus/genética , RNA Mensageiro/metabolismo , Epitélio Pigmentado da Retina/embriologia , Epitélio Pigmentado da Retina/metabolismo , Regulação para CimaRESUMO
The reason why different types of vertebrate nerve cells are generated in a particular sequence is still poorly understood. In the vertebrate retina, homeobox genes play a crucial role in establishing different cell identities. Here we provide evidence of a cellular clock that sequentially activates distinct homeobox genes in embryonic retinal cells, linking the identity of a retinal cell to its time of generation. By in situ expression analysis, we found that the three Xenopus homeobox genes Xotx5b, Xvsx1, and Xotx2 are initially transcribed but not translated in early retinal progenitors. Their translation requires cell cycle progression and is sequentially activated in photoreceptors (Xotx5b) and bipolar cells (Xvsx1 and Xotx2). Furthermore, by in vivo lipofection of "sensors" in which green fluorescent protein translation is under control of the 3' untranslated region (UTR), we found that the 3' UTRs of Xotx5b, Xvsx1, and Xotx2 are sufficient to drive a spatiotemporal pattern of translation matching that of the corresponding proteins and consistent with the time of generation of photoreceptors (Xotx5b) and bipolar cells (Xvsx1 and Xotx2). The block of cell cycle progression of single early retinal progenitors impairs their differentiation as photoreceptors and bipolar cells, but is rescued by the lipofection of Xotx5b and Xvsx1 coding sequences, respectively. This is the first evidence to our knowledge that vertebrate homeobox proteins can work as effectors of a cellular clock to establish distinct cell identities.
Assuntos
Diferenciação Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Homeodomínio/fisiologia , Retina/citologia , Animais , Relógios Biológicos/genética , Ciclo Celular/fisiologia , Diferenciação Celular/genética , Linhagem da Célula , Células Cultivadas , Fatores de Transcrição E2F/metabolismo , Fatores de Transcrição E2F/fisiologia , Proteínas do Olho/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Dados de Sequência Molecular , Fatores de Transcrição Otx/metabolismo , Biossíntese de Proteínas , Células Bipolares da Retina/metabolismo , Transfecção , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Xenopus laevis/fisiologia , Proteínas GADD45RESUMO
Maturation of miRNAs by dicer is required in vertebrates for normal neural development. Here we report that dicer inactivation in Xenopus affects cell cycle progression, survival and timing of the generation of retinal cells, resulting in small retinas with lamination defects. In particular, dicer inactivation delays the exit from the cell cycle and the translation of key genes of late neurogenesis, highlighting a crucial role of miRNAs in retinal development.
Assuntos
Retina/crescimento & desenvolvimento , Ribonuclease III/antagonistas & inibidores , Xenopus laevis/crescimento & desenvolvimento , Xenopus laevis/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , Ciclo Celular , Sobrevivência Celular , Inativação Gênica , MicroRNAs/genética , MicroRNAs/metabolismo , Oligodesoxirribonucleotídeos Antissenso/genética , Retina/anormalidades , Retina/citologia , Retina/metabolismo , Ribonuclease III/genética , Células-Tronco/citologia , Células-Tronco/metabolismo , Xenopus laevis/metabolismoRESUMO
BACKGROUND: The anterior chamber has been shown by pharmacokinetic studies to represent a sanctuary never achieving a tumoricidal dose with the present administration routes, such as systemic, intra-arterial, or intravitreal injections. METHOD: A novel intracameral chemotherapy technique is described to control aqueous seeding in a pilot unilateral group E retinoblastoma case with primary aqueous seeding. Anterior segment toxicity was carefully monitored. RESULTS: Control of the retinal tumor and vitreous seeding was achieved by intra-arterial and intravitreal chemotherapies. Sterilization of the aqueous was achieved after a first cycle of 7 melphalan injections in the anterior chamber, but relapse was noted 3.5 months later. This relapse was finally controlled with a second cycle of 6 intracameral injections targeting the posterior chamber. Corneal endothelial cell density remained stable over the injection period. Heterochromia and a progressive cataract developed, which required cataract surgery. At 5 years' follow-up, the patient is tumor free with normal vision (20/20 in both eyes), full binocularity, and no metastasis. CONCLUSIONS: The present bicameral injection technique appears to be safe and effective with limited toxicity. Melphalan-induced side effects were noted on the iris and lens but with no impact on the final visual function.
RESUMO
The loss of cone photoreceptors that mediate daylight vision represents a leading cause of blindness, for which cell replacement by transplantation offers a promising treatment strategy. Here, we characterize cone differentiation in retinas derived from mouse embryonic stem cells (mESCs). Similar to in vivo development, a temporal pattern of progenitor marker expression is followed by the differentiation of early thyroid hormone receptor ß2-positive precursors and, subsequently, photoreceptors exhibiting cone-specific phototransduction-related proteins. We establish that stage-specific inhibition of the Notch pathway increases cone cell differentiation, while retinoic acid signaling regulates cone maturation, comparable with their actions in vivo. MESC-derived cones can be isolated in large numbers and transplanted into adult mouse eyes, showing capacity to survive and mature in the subretinal space of Aipl1-/- mice, a model of end-stage retinal degeneration. Together, this work identifies a robust, renewable cell source for cone replacement by purified cell suspension transplantation.
Assuntos
Células-Tronco Embrionárias Murinas/transplante , Células Fotorreceptoras Retinianas Cones/citologia , Degeneração Retiniana/terapia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Fatores de Transcrição de Zíper de Leucina Básica/antagonistas & inibidores , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Diferenciação Celular/efeitos dos fármacos , Modelos Animais de Doenças , Proteínas do Olho/antagonistas & inibidores , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Fator 6 Nuclear de Hepatócito/metabolismo , Fator Inibidor de Leucemia/farmacologia , Camundongos , Camundongos Knockout , Células-Tronco Embrionárias Murinas/citologia , Fator de Transcrição 2 de Oligodendrócitos/metabolismo , Opsinas/metabolismo , Receptores Nucleares Órfãos/antagonistas & inibidores , Receptores Nucleares Órfãos/genética , Receptores Nucleares Órfãos/metabolismo , Fatores de Transcrição Otx/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores Notch/antagonistas & inibidores , Receptores Notch/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Degeneração Retiniana/patologia , Transdução de Sinais , Tretinoína/metabolismo , Tretinoína/farmacologiaRESUMO
Retinal degenerative diseases resulting in the loss of photoreceptors are one of the major causes of blindness. Photoreceptor replacement therapy is a promising treatment because the transplantation of retina-derived photoreceptors can be applied now to different murine retinopathies to restore visual function. To have an unlimited source of photoreceptors, we derived a transgenic embryonic stem cell (ESC) line in which the Crx-GFP transgene is expressed in photoreceptors and assessed the capacity of a 3D culture protocol to produce integration-competent photoreceptors. This culture system allows the production of a large number of photoreceptors recapitulating the in vivo development. After transplantation, integrated cells showed the typical morphology of mature rods bearing external segments and ribbon synapses. We conclude that a 3D protocol coupled with ESCs provides a safe and renewable source of photoreceptors displaying a development and transplantation competence comparable to photoreceptors from age-matched retinas.
Assuntos
Células-Tronco Embrionárias/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Animais , Células Cultivadas , Feminino , Citometria de Fluxo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos SCID , Células Fotorreceptoras , Transativadores/genética , Transativadores/metabolismo , Transgenes/genéticaRESUMO
The protocols described in this unit provide detailed information on how to isolate and expand, in culture, ciliary epithelial cells (CECs), previously identified as retinal stem cells, from the adult mouse eye, and embryonic retinal progenitor cells (RPCs) from the developing retina. CECs are initially cultured in floating conditions as neurospheres and then expanded in monolayer cultures. RPCs are cultured in floating conditions. Detailed protocols for retinal differentiation, as well as exogenous gene expression using lentivirus are also described.
Assuntos
Envelhecimento/fisiologia , Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Corpo Ciliar/citologia , Retina/citologia , Células-Tronco/citologia , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Embrião de Mamíferos/citologia , Células Epiteliais/citologia , Lentivirus/genética , Camundongos , Esferoides Celulares , Transgenes/genéticaRESUMO
Photoreceptors have recently been generated from mouse and human embryonic stem cells (ESCs), although ethics concerns impede their utilization for cell replacement therapy for retinal disease. Extra-embryonic tissues have received attention as alternative therapeutic sources of stem cells. Human and mouse amniotic fluid-derived stem cells (AFCs) have been reported to be multipotent and express embryonic and adult stem cell markers. Here, in vitro conditions that generate retinal cells from ESCs were used to analyze and compare the retinal potential of murine AFCs and ESCs. We show that AFCs express pluripotency markers (Nanog, Sox2, and Oct3/4) as well as retinal transcription factor genes (Et, Lhx2, Tll1, Six6, Otx2, Pax6, and Fgf15). AFCs from amniotic fluid of Fgf15.gfp, Nrl.gfp, and Crx.gfp embryos cultured in retinal proliferation and differentiation conditions failed to switch on these retinal transgenes. AFCs cultured in retinal-promoting conditions, effective on ESCs, showed reduced expression of retinal markers. Retinal co-cultures activated retinal genes in ESCs but not in AFCs, and migration assays in retinal explants showed limited migration of AFCs compared with ESCs. Unlike ESCs, AFCs do not express the early embryonic ectodermal gene Utf1 and Western analysis of AFCs identified only the B isoform of Oct3/4, rather than the isoform A present in ESCs. We conclude that AFCs have restricted potential and differ considerably from ESCs and retinal progenitor cells. Reprogramming to induce pluripotency or new differentiation protocols will be required to confer retinal potential to AFCs as expression of a subset of pluripotency and retinal markers is not sufficient.
Assuntos
Líquido Amniótico/citologia , Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes/citologia , Retina/citologia , Transplante de Células-Tronco/métodos , Engenharia Tecidual/métodos , Líquido Amniótico/metabolismo , Animais , Biomarcadores/análise , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular , Movimento Celular , Proteínas Cromossômicas não Histona , Técnicas de Cocultura/métodos , Meios de Cultura/química , Meios de Cultura/metabolismo , Células-Tronco Embrionárias/metabolismo , Feminino , Camundongos , Técnicas de Cultura de Órgãos/métodos , Células-Tronco Pluripotentes/metabolismo , Isoformas de Proteínas/análise , Isoformas de Proteínas/biossíntese , Retina/metabolismo , Doenças Retinianas/terapia , Transativadores/deficiência , Fatores de Transcrição/análise , Fatores de Transcrição/biossínteseRESUMO
In contrast to the high degree of evolutionary conservation of the Vsx2/Chx10 gene family, vertebrate orthologues of Vsx1 display more divergent sequences and spatio-temporal expression patterns. Here, we report the cloning and expression pattern of Xenopus laevis Vsx1. Differently from the mouse and zebrafish orthologues, Xvsx1 transcription is activated at early neurula both in the evaginating eye vesicles and in the presumptive spinal cord. Compared to other retinal homeobox genes, such as Xrx1, Xsix3 and Xpax6, Xvsx1 is activated at a later stage; in addition, its anterior expression appears to be more specifically restricted to the retina. At tail bud stage, Xvsx1 expression in retinal progenitors persists, and its neural tube expression, which in the spinal cord corresponds to interneurons, progressively expands anteriorly reaching the midbrain-hindbrain boundary. During retinal neurogenesis, Xvsx1 expression is maintained in retinal progenitors and in a peripheral region of the ciliary marginal zone, while in the central retina, it becomes restricted to differentiated bipolar cells.