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1.
Stem Cells Dev ; 33(9-10): 201-213, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38390839

RESUMEN

Because derivation of retinal organoids (ROs) and transplantation are frequently split between geographically distant locations, we developed a special shipping device and protocol capable of the organoids' delivery to any location. Human embryonic stem cell (hESC)-derived ROs were differentiated from the hESC line H1 (WA01), shipped overnight to another location, and then transplanted into the subretinal space of blind immunodeficient retinal degeneration (RD) rats. Development of transplants was monitored by spectral-domain optical coherence tomography. Visual function was accessed by optokinetic tests and superior colliculus (SC) electrophysiology. Cryostat sections through transplants were stained with hematoxylin and eosin; or processed for immunohistochemistry to label human donor cells, retinal cell types, and synaptic markers. After transplantation, ROs integrated into the host RD retina, formed functional photoreceptors, and improved vision in rats with advanced RD. The survival and vision improvement are comparable with our previous results of hESC-ROs without a long-distance delivery. Furthermore, for the first time in the stem cell transplantation field, we demonstrated that the response heatmap on the SC showed a similar shape to the location of the transplant in the host retina, which suggested the point-to-point projection of the transplant from the retina to SC. In conclusion, our results showed that using our special device and protocol, the hESC-derived ROs can be shipped over long distance and are capable of survival and visual improvement after transplantation into the RD rats. Our data provide a proof-of-concept for stem cell replacement as a therapy for RD patients.


Asunto(s)
Células Madre Embrionarias Humanas , Organoides , Retina , Degeneración Retiniana , Animales , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/trasplante , Degeneración Retiniana/terapia , Degeneración Retiniana/patología , Humanos , Organoides/citología , Organoides/trasplante , Ratas , Retina/citología , Retina/patología , Diferenciación Celular , Trasplante de Células Madre/métodos , Supervivencia Celular , Tomografía de Coherencia Óptica
2.
J Vis Exp ; (174)2021 08 05.
Artículo en Inglés | MEDLINE | ID: mdl-34424232

RESUMEN

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and Leber Congenital Amaurosis (LCA) cause progressive and debilitating vision loss. There is an unmet need for therapies that can restore vision once photoreceptors have been lost. Transplantation of human pluripotent stem cell (hPSC)-derived retinal tissue (organoids) into the subretinal space of an eye with advanced RD brings retinal tissue sheets with thousands of healthy mutation-free photoreceptors and has a potential to treat most/all blinding diseases associated with photoreceptor degeneration with one approved protocol. Transplantation of fetal retinal tissue into the subretinal space of animal models and people with advanced RD has been developed successfully but cannot be used as a routine therapy due to ethical concerns and limited tissue supply. Large eye inherited retinal degeneration (IRD) animal models are valuable for developing vision restoration therapies utilizing advanced surgical approaches to transplant retinal cells/tissue into the subretinal space. The similarities in globe size, and photoreceptor distribution (e.g., presence of macula-like region area centralis) and availability of IRD models closely recapitulating human IRD would facilitate rapid translation of a promising therapy to the clinic. Presented here is a surgical technique of transplanting hPSC-derived retinal tissue into the subretinal space of a large animal model allowing assessment of this promising approach in animal models.


Asunto(s)
Células Madre Embrionarias Humanas , Células Madre Pluripotentes , Degeneración Retiniana , Animales , Gatos , Modelos Animales de Enfermedad , Humanos , Retina , Trasplante de Células Madre
3.
Stem Cells Dev ; 30(8): 399-417, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33677999

RESUMEN

Progressive vision loss, caused by retinal degenerative (RD) diseases such as age-related macular degeneration, retinitis pigmentosa, and Leber congenital amaurosis, severely impacts quality of life and affects millions of people. Finding efficient treatment for blinding diseases is among the greatest unmet clinical needs. The evagination of optic vesicles from developing pluripotent stem cell-derived neuroepithelium and self-organization, lamination, and differentiation of retinal tissue in a dish generated considerable optimism for developing innovative approaches for treating RD diseases, which previously were not feasible. Retinal organoids may be a limitless source of multipotential retinal progenitors, photoreceptors (PRs), and the whole retinal tissue, which are productive approaches for developing RD disease therapies. In this study we compared the distribution and expression level of molecular markers (genetic and epigenetic) in human fetal retina (age 8-16 weeks) and human embryonic stem cell (hESC)-derived retinal tissue (organoids) by immunohistochemistry, RNA-seq, flow cytometry, and mass-spectrometry (to measure methylated and hydroxymethylated cytosine level), with a focus on PRs to evaluate the clinical application of hESC-retinal tissue for vision restoration. Our results revealed high correlation in gene expression profiles and histological profiles between human fetal retina (age 8-13 weeks) and hESC-derived retinal tissue (10-12 weeks). The transcriptome signature of hESC-derived retinal tissue from retinal organoids maintained for 24 weeks in culture resembled the transcriptome of human fetal retina of more advanced developmental stages. The histological profiles of 24 week-old hESC-derived retinal tissue displayed mature PR immunophenotypes and presence of developing inner and outer segments. Collectively, our work highlights the similarity of hESC-derived retinal tissue at early stages of development (10 weeks), and human fetal retina (age 8-13 weeks) and it supports the development of regenerative medicine therapies aimed at using tissue from hESC-derived retinal organoids (hESC-retinal implants) for mitigating vision loss.


Asunto(s)
Diferenciación Celular/genética , Células Madre Embrionarias Humanas/metabolismo , Organoides/metabolismo , Células Madre Pluripotentes/metabolismo , Retina/metabolismo , Transcriptoma/genética , Línea Celular , Metilación de ADN , Proteínas de Homeodominio/metabolismo , Células Madre Embrionarias Humanas/citología , Humanos , Inmunohistoquímica , Antígeno Ki-67/metabolismo , Microscopía Confocal , Microscopía Electrónica de Transmisión , Organoides/citología , Organoides/ultraestructura , Factor de Transcripción PAX6/metabolismo , Células Madre Pluripotentes/citología , RNA-Seq/métodos , Retina/citología , Retina/embriología , Factores de Tiempo , Factores de Transcripción/metabolismo
4.
J Ocul Pharmacol Ther ; 37(1): 60-71, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33449859

RESUMEN

Purpose: To determine the long-term biocompatibility of HyStem® hydrogel in the rabbit eye for use as a carrier for cell or drug delivery into the ocular space. Methods: HyStem hydrogel formulation solidifies ∼20 min after reconstitution, thus can potentially form a solid deposit after injection in situ. To study the ocular disposition of fluorescein-labeled HyStem, we delivered 50 µL/eye over 1 min into the vitreous space of the rabbit. We used 3 Dutch-Belted and 3 New Zealand-pigmented rabbits, all females, delivered the gel into the right eyes, and injected 50 µL BSS Plus into the left eyes as a control. Retinal morphology was assessed by optical coherence tomography (OCT) and white light fundus photography. Fluorescence fundus photography enabled measurement of the clearance of the labeled hydrogel from the posterior chamber. Visual function was evaluated using flash and flicker electroretinography (ERG) pre- and postinjection and at weekly intervals thereafter for 6 weeks. Retinal immunohistochemistry for microglial inflammatory markers was carried out with antiglial fibrillary acidic protein (GFAP) antibody, isolectin B4 (IB4), and 4',6-diamidino-2-phenylindole (DAPI). Results: The gel was successfully delivered into the vitreous space without the formation of a discrete retinal deposit. Fundus imaging, OCT measurements of retinal thickness, and immunohistochemical data indicated an absence of retinal inflammation, and ERG indicated no impact on retinal function. The half-time of HyStem clearance calculated from the loss of fundus fluorescence was 3.9 days. Conclusions: HyStem hydrogel appears to be biocompatible in the ocular space of a large eye and safe for long-term intraocular application.


Asunto(s)
Materiales Biocompatibles/administración & dosificación , Ojo/efectos de los fármacos , Hidrogeles/administración & dosificación , Animales , Tolerancia a Medicamentos , Femenino , Inyecciones Intraoculares , Conejos
5.
J Ocul Pharmacol Ther ; 37(3): 147-156, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33052761

RESUMEN

Blindness, associated with death of retinal cells at the back of the eye, is caused by a number of conditions with high prevalence such as glaucoma, age-related macular degeneration, and diabetic retinopathy. In addition, a large number of orphan inherited (mostly monogenic) conditions, such as retinitis pigmentosa and Leber Congenital Amaurosis, add to the overall number of patients with blinding retinal degenerative diseases. Blindness caused by deterioration and loss of retina is so far incurable. Modern biomedical research leveraging molecular and regenerative medicine approaches had a number of groundbreaking discoveries and proof-of-principle treatments of blindness in animals. However, these methods are slow to be standardized and commercialized as therapies to benefit people losing their eyesight due to retinal degenerative conditions. In this review, we will outline major regenerative medicine approaches, which are emerging as promising for preserving or/and restoring vision. We discuss the potential of each of these approaches to reach commercialization step and be converted to treatments, which could at least ameliorate blindness caused by retinal cell death.


Asunto(s)
Ceguera/terapia , Organoides/citología , Células Madre Pluripotentes/citología , Degeneración Retiniana/terapia , Trasplante de Células Madre , Animales , Humanos
6.
Front Cell Neurosci ; 14: 179, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33132839

RESUMEN

The self-formation of retinal tissue from pluripotent stem cells generated a tremendous promise for developing new therapies of retinal degenerative diseases, which previously seemed unattainable. Together with use of induced pluripotent stem cells or/and CRISPR-based recombineering the retinal organoid technology provided an avenue for developing models of human retinal degenerative diseases "in a dish" for studying the pathology, delineating the mechanisms and also establishing a platform for large-scale drug screening. At the same time, retinal organoids, highly resembling developing human fetal retinal tissue, are viewed as source of multipotential retinal progenitors, young photoreceptors and just the whole retinal tissue, which may be transplanted into the subretinal space with a goal of replacing patient's degenerated retina with a new retinal "patch." Both approaches (transplantation and modeling/drug screening) were projected when Yoshiki Sasai demonstrated the feasibility of deriving mammalian retinal tissue from pluripotent stem cells, and generated a lot of excitement. With further work and testing of both approaches in vitro and in vivo, a major implicit limitation has become apparent pretty quickly: the absence of the uniform layer of Retinal Pigment Epithelium (RPE) cells, which is normally present in mammalian retina, surrounds photoreceptor layer and develops and matures first. The RPE layer polarize into apical and basal sides during development and establish microvilli on the apical side, interacting with photoreceptors, nurturing photoreceptor outer segments and participating in the visual cycle by recycling 11-trans retinal (bleached pigment) back to 11-cis retinal. Retinal organoids, however, either do not have RPE layer or carry patches of RPE mostly on one side, thus directly exposing most photoreceptors in the developing organoids to neural medium. Recreation of the critical retinal niche between the apical RPE and photoreceptors, where many retinal disease mechanisms originate, is so far unattainable, imposes clear limitations on both modeling/drug screening and transplantation approaches and is a focus of investigation in many labs. Here we dissect different retinal degenerative diseases and analyze how and where retinal organoid technology can contribute the most to developing therapies even with a current limitation and absence of long and functional outer segments, supported by RPE.

7.
J Tissue Eng Regen Med ; 14(2): 388-394, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31908157

RESUMEN

Retinal organoid technology enables generation of an inexhaustible supply of three-dimensional retinal tissue from human pluripotent stem cells (hPSCs) for regenerative medicine applications. The high similarity of organoid-derived retinal tissue and transplantable human fetal retina provides an opportunity for evaluating and modeling retinal tissue replacement strategies in relevant animal models in the effort to develop a functional retinal patch to restore vision in patients with profound blindness caused by retinal degeneration. Because of the complexity of this very promising approach requiring specialized stem cell and grafting techniques, the tasks of retinal tissue derivation and transplantation are frequently split between geographically distant teams. Delivery of delicate and perishable neural tissue such as retina to the surgical sites requires a reliable shipping protocol and also controlled temperature conditions with damage-reporting mechanisms in place to prevent transplantation of tissue damaged in transit into expensive animal models. We have developed a robust overnight tissue shipping protocol providing reliable temperature control, live monitoring of the shipment conditions and physical location of the package, and damage reporting at the time of delivery. This allows for shipping of viable (transplantation-competent) hPSC-derived retinal tissue over large distances, thus enabling stem cell and surgical teams from different parts of the country to work together and maximize successful engraftment of organoid-derived retinal tissue. Although this protocol was developed for preclinical in vivo studies in animal models, it is potentially translatable for clinical transplantation in the future and will contribute to developing clinical protocols for restoring vision in patients with retinal degeneration.


Asunto(s)
Organoides/citología , Células Madre Pluripotentes/citología , Medicina Regenerativa/métodos , Retina/patología , Ingeniería de Tejidos/métodos , Ceguera , Diferenciación Celular , Humanos , Células Madre Pluripotentes Inducidas/trasplante , Organoides/metabolismo , Retina/embriología , Degeneración Retiniana , Trasplante de Células Madre/métodos , Células Madre/citología , Temperatura , Andamios del Tejido
8.
Stem Cells Dev ; 28(17): 1151-1166, 2019 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-31210100

RESUMEN

To develop biological approaches to restore vision, we developed a method of transplanting stem cell-derived retinal tissue into the subretinal space of a large-eye animal model (cat). Human embryonic stem cells (hESC) were differentiated to retinal organoids in a dish. hESC-derived retinal tissue was introduced into the subretinal space of wild-type cats following a pars plana vitrectomy. The cats were systemically immunosuppressed with either prednisolone or prednisolone plus cyclosporine A. The eyes were examined by fundoscopy and spectral-domain optical coherence tomography imaging for adverse effects due to the presence of the subretinal grafts. Immunohistochemistry was done with antibodies to retinal and human markers to delineate graft survival, differentiation, and integration into cat retina. We successfully delivered hESC-derived retinal tissue into the subretinal space of the cat eye. We observed strong infiltration of immune cells in the graft and surrounding tissue in the cats treated with prednisolone. In contrast, we showed better survival and low immune response to the graft in cats treated with prednisolone plus cyclosporine A. Immunohistochemistry with antibodies (STEM121, CALB2, DCX, and SMI-312) revealed large number of graft-derived fibers connecting the graft and the host. We also show presence of human-specific synaptophysin puncta in the cat retina. This work demonstrates feasibility of engrafting hESC-derived retinal tissue into the subretinal space of large-eye animal models. Transplanting retinal tissue in degenerating cat retina will enable rapid development of preclinical in vivo work focused on vision restoration.


Asunto(s)
Técnicas de Reprogramación Celular/métodos , Células Madre Embrionarias Humanas/trasplante , Retina/trasplante , Trasplante de Células Madre/métodos , Animales , Calbindina 2/genética , Calbindina 2/metabolismo , Gatos , Diferenciación Celular , Línea Celular , Células Cultivadas , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Supervivencia de Injerto , Células Madre Embrionarias Humanas/citología , Humanos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Neuropéptidos/genética , Neuropéptidos/metabolismo , Retina/citología , Retina/metabolismo , Sinaptofisina/genética , Sinaptofisina/metabolismo
9.
J Vis Exp ; (138)2018 08 29.
Artículo en Inglés | MEDLINE | ID: mdl-30222161

RESUMEN

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a variety of human retinal degenerative diseases and pinpoint new treatment approaches. The nuclear architecture of mouse retina is organized in two different patterns: conventional and inverted. Conventional pattern is universal where heterochromatin is localized to the periphery of the nucleus, while active euchromatin resides in the nuclear interior. In contrast, inverted nuclear pattern is unique to the adult rod photoreceptor cell nuclei where heterochromatin localizes to the nuclear center, and euchromatin resides in the nuclear periphery. DNA methylation is predominantly observed in chromocenters. DNA methylation is a dynamic covalent modification on the cytosine residues (5-methylcytosine, 5mC) of CpG dinucleotides that are enriched in the promoter regions of many genes. Three DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) participate in methylation of DNA during development. Detecting 5mC with immunohistochemical techniques is very challenging, contributing to variability in results, as all DNA bases including 5mC modified bases are hidden within the double-stranded DNA helix. However, detailed delineation of 5mC distribution during development is very informative. Here, we describe a reproducible technique for robust immunohistochemical detection of 5mC and another epigenetic DNA marker 5-hydroxymethylcytosine (5hmC), which colocalizes with the "open", transcriptionally active chromatin in developing and postmitotic mouse retina.


Asunto(s)
5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Inmunohistoquímica/métodos , Retina/metabolismo , Animales , Humanos , Ratones
10.
Hum Mol Genet ; 27(2): 283-294, 2018 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-29126234

RESUMEN

Bardet-Biedl syndrome (BBS) is an autosomal recessive ciliopathy characterized by developmental abnormalities and vision loss. To date, mutations in 21 genes have been linked to BBS. The products of eight of these BBS genes form a stable octameric complex termed the BBSome. Mutations in BBS8, a component of the BBSome, cause early vision loss, but the role of BBS8 in supporting vision is not known. To understand the mechanisms by which BBS8 supports rod and cone photoreceptor function, we generated animal models lacking BBS8. The loss of BBS8 protein led to concomitant decrease in the levels of BBSome subunits, BBS2 and BBS5 and increase in the levels of the BBS1 and BBS4 subunits. BBS8 ablation was associated with severe reduction of rod and cone photoreceptor function and progressive degeneration of each photoreceptor subtype. We observed disorganized and shortened photoreceptor outer segments (OS) at post-natal day 10 as the OS elaborates. Interestingly, loss of BBS8 led to changes in the distribution of photoreceptor axonemal proteins and hyper-acetylation of ciliary microtubules. In contrast to properly localized phototransduction machinery, we observed OS accumulation of syntaxin3, a protein normally found in the cytoplasm and the synaptic termini. In conclusion, our studies demonstrate the requirement for BBS8 in early development and elaboration of ciliated photoreceptor OS, explaining the need for BBS8 in normal vision. The findings from our study also imply that early targeting of both rods and cones in BBS8 patients is crucial for successful restoration of vision.


Asunto(s)
Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Células Fotorreceptoras/metabolismo , Animales , Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/metabolismo , Síndrome de Bardet-Biedl/patología , Cilios/metabolismo , Proteínas del Citoesqueleto , Modelos Animales de Enfermedad , Ratones , Ratones Noqueados , Neuronas/metabolismo , Neuronas/patología , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Fotorreceptoras Retinianas Conos/patología , Células Fotorreceptoras Retinianas Bastones/metabolismo
11.
Can J Physiol Pharmacol ; 95(9): 1046-1057, 2017 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-28666094

RESUMEN

We investigated whether North American ginseng (Panax quinquefolius) could reduce development of the metabolic syndrome phenotype in a mouse model (ETKO) of the disease. Young ETKO mice have no disease but similar to humans start to develop the fatty liver, hypertriglyceridemia, obesity, and insulin resistance at 25-30 weeks of age, and the disease continues to progress with ageing. ETKO mice were orally given an ethanol extract of ginseng roots at 4 and 32 weeks of age. Treatments with ginseng eliminated the ETKO fatty liver, reduced hepatic and intestinal lipoprotein secretion, and reduced the level of circulating lipids. Improvements by ginseng treatments were manifested as a reduction in the expression of genes involved in the regulation of fatty acid and triglyceride (fat) synthesis and secretion by the lipoproteins on one hand, and the stimulation of fatty acid oxidation and triglyceride degradation by lipolysis on the other hand. These processes altogether improved glucose, fatty acid, and triglyceride metabolism, reduced liver fat load, and reversed the progression of metabolic syndrome. These data confirm that treatments with North American ginseng could alleviate metabolic syndrome through the maintenance of a better balance between glucose and fatty acid metabolism, lipoprotein secretion, and energy homeostasis in disease-prone states.


Asunto(s)
Dislipidemias/tratamiento farmacológico , Etanol/química , Hígado Graso/tratamiento farmacológico , Síndrome Metabólico/complicaciones , Panax/química , Extractos Vegetales/farmacología , Animales , Modelos Animales de Enfermedad , Dislipidemias/complicaciones , Ácidos Grasos/metabolismo , Hígado Graso/complicaciones , Lipoproteínas/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Ratones , Tamaño de los Órganos/efectos de los fármacos , Oxidación-Reducción/efectos de los fármacos , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/uso terapéutico , Periodo Posprandial/efectos de los fármacos , Triglicéridos/sangre , Triglicéridos/metabolismo
12.
Exp Eye Res ; 159: 132-146, 2017 06.
Artículo en Inglés | MEDLINE | ID: mdl-27865785

RESUMEN

Characterizing the role of epigenetic regulation in the mammalian retina is critical for understanding fundamental mechanisms of retinal development and disease. DNA methylation, an epigenetic modifier of genomic DNA, plays an important role in modulating networks of tissue and cell-specific gene expression. However, the impact of DNA methylation on retinal development and homeostasis of retinal neurons remains unclear. Here, we have created a tissue-specific DNA methyltransferase (Dnmt) triple mutant mouse in an effort to characterize the impact of DNA methylation on retinal development and homeostasis. An Rx-Cre transgene was used to drive targeted mutation of all three murine Dnmt genes in the mouse retina encoding major DNA methylation enzymes DNMT1, DNMT3A and DNMT3B. The triple mutant mice represent a hypomorph model since Dnmt1 catalytic activity was still present and excision of Dnmt3a and Dnmt3b had only about 90% efficiency. Mutation of all three Dnmts resulted in global genomic hypomethylation and dramatic reorganization of the photoreceptor and synaptic layers within retina. Transcriptome and proteomic analyses demonstrated enrichment of dysregulated phototransduction and synaptic genes. The 5 mC signal in triple mutant retina was confined to the central heterochromatin but reduced in the peripheral heterochromatin region of photoreceptor nuclei. In addition, we found a reduction of the 5 mC signal in ganglion cell nuclei. Collectively, this data suggests cooperation of all three Dnmts in the formation and homeostasis of photoreceptors and other retinal neurons within the mammalian retina, and highlight the relevance of epigenetic regulation to sensory retinal disorders and vision loss.


Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/genética , ADN/genética , Mutación , Células Fotorreceptoras de Vertebrados/metabolismo , Animales , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , ADN Metiltransferasa 3A , Análisis Mutacional de ADN , Immunoblotting , Inmunohistoquímica , Ratones , Ratones Mutantes , Microscopía Electrónica , Modelos Animales , Células Fotorreceptoras de Vertebrados/ultraestructura , Reacción en Cadena en Tiempo Real de la Polimerasa , Neuronas Retinianas/metabolismo , Neuronas Retinianas/ultraestructura , ADN Metiltransferasa 3B
13.
Hum Mol Genet ; 25(10): 2031-2044, 2016 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-26936825

RESUMEN

The small GTPase, ADP-ribosylation factor-like 3 (ARL3), has been proposed to participate in the transport of proteins in photoreceptor cells. Moreover, it has been implicated in the pathogenesis associated with X-linked retinitis pigmentosa (XLRP) resulting from mutations in the ARL3 GTPase activating protein, retinitis pigmentosa 2 (RP2). To determine the importance of ARL3 in rod photoreceptor cells, we generated transgenic mice expressing a dominant active form of ARL3 (ARL3-Q71L) under a rod-specific promoter. ARL3-Q71L animals exhibited extensive rod cell death after post-natal day 30 (PN30) and degeneration was complete by PN70. Prior to the onset of cell death, rod photoresponse was significantly reduced along with a robust decrease in rod phosphodiesterase 6 (PDE6) and G-protein receptor kinase-1 (GRK1) levels. Furthermore, assembled phosphodiesterase-6 (PDE6) subunits, rod transducin and G-protein receptor kinase-1 (GRK1) accumulated on large punctate structures within the inner segment in ARL3-Q71L retina. Defective trafficking of prenylated proteins is likely due to sequestration of prenyl binding protein δ (PrBPδ) by ARL3-Q71L as we demonstrate a specific interaction between these proteins in the retina. Unexpectedly, our studies also revealed a novel role for ARL3 in the migration of photoreceptor nuclei. In conclusion, this study identifies ARL3 as a key player in prenylated protein trafficking in rod photoreceptor cells and establishes the potential role for ARL3 dysregulation in the pathogenesis of RP2-related forms of XLRP.


Asunto(s)
Factores de Ribosilacion-ADP/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Pirofosfatasas/genética , Retinitis Pigmentosa/genética , Factores de Ribosilacion-ADP/biosíntesis , Animales , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/biosíntesis , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/genética , Proteínas del Ojo/biosíntesis , Proteínas del Ojo/genética , Quinasa 1 del Receptor Acoplado a Proteína-G/biosíntesis , Quinasa 1 del Receptor Acoplado a Proteína-G/genética , Proteínas de Unión al GTP , Regulación de la Expresión Génica , Enfermedades Genéticas Ligadas al Cromosoma X/patología , Humanos , Proteínas de la Membrana , Ratones , Ratones Transgénicos , Prenilación de Proteína/genética , Retina/metabolismo , Retina/patología , Células Fotorreceptoras Retinianas Bastones/metabolismo , Células Fotorreceptoras Retinianas Bastones/patología , Retinitis Pigmentosa/patología , Segmento Externo de la Célula en Bastón/metabolismo , Segmento Externo de la Célula en Bastón/patología
14.
Stem Cells Dev ; 24(23): 2778-95, 2015 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-26283078

RESUMEN

Stem cell-based therapy of retinal degenerative conditions is a promising modality to treat blindness, but requires new strategies to improve the number of functionally integrating cells. Grafting semidifferentiated retinal tissue rather than progenitors allows preservation of tissue structure and connectivity in retinal grafts, mandatory for vision restoration. Using human embryonic stem cells (hESCs), we derived retinal tissue growing in adherent conditions consisting of conjoined neural retina and retinal pigment epithelial (RPE) cells and evaluated cell fate determination and maturation in this tissue. We found that deriving such tissue in adherent conditions robustly induces all eye field genes (RX, PAX6, LHX2, SIX3, SIX6) and produces four layers of pure populations of retinal cells: RPE (expressing NHERF1, EZRIN, RPE65, DCT, TYR, TYRP, MITF, PMEL), early photoreceptors (PRs) (coexpressing CRX and RCVRN), inner nuclear layer neurons (expressing CALB2), and retinal ganglion cells [RGCs, expressing BRN3B and Neurofilament (NF) 200]. Furthermore, we found that retinal progenitors divide at the apical side of the hESC-derived retinal tissue (next to the RPE layer) and then migrate toward the basal side, similar to that found during embryonic retinogenesis. We detected synaptogenesis in hESC-derived retinal tissue, and found neurons containing many synaptophysin-positive boutons within the RGC and PR layers. We also observed long NF200-positive axons projected by RGCs toward the apical side. Whole-cell recordings demonstrated that putative amacrine and/or ganglion cells exhibited electrophysiological responses reminiscent of those in normal retinal neurons. These responses included voltage-gated Na(+) and K(+) currents, depolarization-induced spiking, and responses to neurotransmitter receptor agonists. Differentiation in adherent conditions allows generation of long and flexible pieces of 3D retinal tissue suitable for isolating transplantable slices of tissue for retinal replacement therapies.


Asunto(s)
Células Madre Embrionarias/citología , Neuronas Retinianas/citología , Epitelio Pigmentado de la Retina/citología , Ingeniería de Tejidos , Potenciales de Acción , Células Cultivadas , Células Madre Embrionarias/metabolismo , Humanos , Neurogénesis , Potasio/metabolismo , Neuronas Retinianas/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Sodio/metabolismo , Sinapsis/metabolismo , Sinapsis/fisiología , Sinaptofisina/genética , Sinaptofisina/metabolismo
15.
Mol Cell Biol ; 35(15): 2641-57, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-25986609

RESUMEN

Phosphatidylethanolamine (PE) is the most abundant inner membrane phospholipid. PE synthesis from ethanolamine and diacylglycerol is regulated primarily by CTP:phosphoethanolamine cytidylyltransferase (Pcyt2). Pcyt2(+/-) mice have reduced PE synthesis and, as a consequence, perturbed glucose and fatty acid metabolism, which gradually leads to the development of hyperlipidemia, obesity, and insulin resistance. Glucose and fatty acid uptake and the corresponding transporters Glut4 and Cd36 are similarly impaired in male and female Pcyt2(+/-) hearts. These mice also have similarly reduced phosphatidylinositol 3-kinase (PI3K)/Akt1 signaling and increased reactive oxygen species (ROS) production in the heart. However, only Pcyt2(+/-) males develop hypertension and cardiac hypertrophy. Pcyt2(+/-) males have upregulated heart AceI expression, heart phospholipids enriched in arachidonic acid and other n-6 polyunsaturated fatty acids, and dramatically increased ROS production in the aorta. In contrast, Pcyt2(+/-) females have unmodified heart phospholipids but have reduced heart triglyceride levels and altered expression of the structural genes Acta (low) and Myh7 (high). These changes together protect Pcyt2(+/-) females from cardiac dysfunction under conditions of reduced glucose and fatty acid uptake and heart insulin resistance. Our data identify Pcyt2 and membrane PE biogenesis as important determinants of gender-specific differences in cardiac lipids and heart function.


Asunto(s)
Cardiomegalia/genética , Hipertensión/genética , Fosfatidiletanolaminas/biosíntesis , ARN Nucleotidiltransferasas/genética , Actinas/biosíntesis , Actinas/genética , Animales , Aorta/patología , Ácido Araquidónico/biosíntesis , Antígenos CD36/genética , Ácidos Grasos/metabolismo , Femenino , Glucosa/metabolismo , Transportador de Glucosa de Tipo 4/genética , Corazón/fisiología , Hiperlipidemias/metabolismo , Resistencia a la Insulina , Peroxidación de Lípido/fisiología , Masculino , Ratones , Ratones Transgénicos , Cadenas Pesadas de Miosina/biosíntesis , Cadenas Pesadas de Miosina/genética , Obesidad/metabolismo , Peptidil-Dipeptidasa A/biosíntesis , Fosfatidilinositol 3-Quinasa/genética , Fosfatidilinositol 3-Quinasa/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factores Sexuales , Transducción de Señal
16.
FASEB J ; 29(5): 1663-75, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25466896

RESUMEN

Fibroblasts from a patient with postural orthostatic tachycardia syndrome (POTS), who presented with low plasma choline and betaine, were studied to determine the metabolic characteristics of the choline deficiency. Choline is required for the synthesis of the phospholipid phosphatidylcholine (PC) and for betaine, an important osmoregulator. Here, choline transport, lipid homeostasis, and mitochondria function were analyzed in skin fibroblasts from POTS and compared with control cells. The choline transporter-like protein 1/solute carrier 44A1 (CTL1/SLC44A1) and mRNA expression were 2-3 times lower in POTS fibroblasts, and choline uptake was reduced 60% (P < 0.05). Disturbances of membrane homeostasis were observed by reduced ratios between PC:phosphatidylethanolamine and sphingomyelin:cholesterol, as well as by modified phospholipid fatty acid composition. Choline deficiency also impaired mitochondria function, which was observed by a reduction in oxygen consumption, mitochondrial potential, and glycolytic activity. When POTS cells were treated with choline, transporter was up-regulated, and uptake of choline increased, offering an option for patient treatment. The characteristics of the POTS fibroblasts described here represent a first model of choline and CTL1/SLC44A1 deficiency, in which choline transport, membrane homeostasis, and mitochondrial function are impaired.


Asunto(s)
Membrana Celular/patología , Deficiencia de Colina/etiología , Colina/farmacología , Fibroblastos/patología , Mitocondrias/patología , Síndrome de Taquicardia Postural Ortostática/complicaciones , Piel/patología , Transporte Biológico , Western Blotting , Estudios de Casos y Controles , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Deficiencia de Colina/metabolismo , Deficiencia de Colina/patología , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas de Transporte de Catión Orgánico/genética , Proteínas de Transporte de Catión Orgánico/metabolismo , Consumo de Oxígeno/efectos de los fármacos , Fosfatidilcolinas/metabolismo , Fosfolípidos/metabolismo , Síndrome de Taquicardia Postural Ortostática/fisiopatología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/efectos de los fármacos , Piel/metabolismo
17.
Hum Mol Genet ; 23(4): 1002-12, 2014 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-24108108

RESUMEN

Defects in the photoreceptor-specific gene encoding aryl hydrocarbon receptor interacting protein like-1 (AIPL1) are linked to blinding diseases, including Leber congenital amaurosis (LCA) and cone dystrophy. While it is apparent that AIPL1 is needed for rod and cone function, the role of AIPL1 in cones is not clear. In this study, using an all-cone animal model lacking Aipl1, we show a light-independent degeneration of M- and S-opsin containing cones that proceeds in a ventral-to-dorsal gradient. Aipl1 is needed for stability, assembly and membrane association of cone PDE6, an enzyme crucial for photoreceptor function and survival. Furthermore, RetGC1, a protein linked to LCA that is needed for cGMP synthesis, was dramatically reduced in cones lacking Aipl1. A defect in RetGC1 is supported by our finding that cones lacking Aipl1 exhibited reduced levels of cGMP. These findings are in contrast to the role of Aipl1 in rods, where destabilization of rod PDE6 results in an increase in cGMP levels, which drives rapid rod degeneration. Our results illustrate mechanistic differences behind the death of rods and cones in retinal degenerative disease caused by deficiencies in AIPL1.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Ceguera/enzimología , GMP Cíclico/metabolismo , Amaurosis Congénita de Leber/enzimología , Células Fotorreceptoras Retinianas Conos/enzimología , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Animales , Dominio Catalítico , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Estabilidad de Enzimas , Guanilato Ciclasa/metabolismo , Humanos , Ratones , Ratones Noqueados , Transporte de Proteínas , Receptores de Superficie Celular/metabolismo , Retina/patología
18.
Ann Surg Oncol ; 20 Suppl 3: S424-32, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-23117476

RESUMEN

BACKGROUND: MCPH1 is a proximal regulator of DNA damage response pathway that is involved in recruitment of phosphorylated ATM to double-stranded DNA breaks. METHODS: To understand the importance of MCPH1 and ATM in deregulation of DNA damage response pathway in breast carcinoma, we studied m-RNA expression and genetic/epigenetic alterations of these genes in primary breast carcinoma samples. RESULTS: Our study revealed reduced expression (mRNA/protein) and high alterations (deletion/methylation) (96 %, 121 of 126) of MCPH1 and ATM. Mutation was, however, rare in inactivation of MCPH1. In immunohistochemical analysis, reduced protein expression of MCPH1, ATM and p-ATM were concordant with their molecular alterations (P = 0.03-0.01). Alterations of MCPH1 and deletion of ATM were significantly high in estrogen/progesterone receptor-negative than estrogen/progesterone receptor-positive breast carcinoma samples compared to early or late age of onset tumors, indicating differences in pathogenesis of the molecular subtypes (P = 0.004-0.01). These genes also showed differential association with tumor stage, grade and lymph node status in different subtypes of breast carcinoma (P = 0.00001-0.01). Their coalterations showed significant association with tumor progression and prognosis (P = 0.003-0.05). Interestingly, patients with alterations of these genes or MCPH1 alone had poor outcome after treatment with DNA-interacting drugs and/or radiation (P = 0.01-0.05). CONCLUSIONS: Inactivation of MCPH1-ATM-associated DNA damage response pathway might have an important role in the development of breast carcinoma with diagnostic, prognostic and therapeutic implications.


Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/genética , Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Proteínas del Tejido Nervioso/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/patología , Proteínas de Ciclo Celular , Proteínas del Citoesqueleto , Metilación de ADN , Femenino , Estudios de Seguimiento , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica , Humanos , Técnicas para Inmunoenzimas , Estadificación de Neoplasias , Proteínas del Tejido Nervioso/metabolismo , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
19.
Mol Oncol ; 5(5): 454-64, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-21803008

RESUMEN

To understand the importance of frequent deletions at chromosome 11q24.1-24.2 region in breast carcinoma, alterations (deletion/methylation) of the candidate genes LOH11CR2A, ROBO3, ROBO4, HEPACAM, PIG8 and CHEK1 located in this region were analyzed in 106 breast carcinoma samples. Among these genes, LOH11CR2A showed highest frequency of deletion (56%), followed by PIG8 (35%), CHEK1 (31%) and ROBO3/ROBO4/HEPACAM loci (28%). Comparable frequency of promoter methylation (26-35%) was observed for LOH11CR2A, CHEK1 and PIG8. Overall alterations (deletion/methylation) of these genes were in the following order: LOH11CR2A (60%) > PIG8 (46%) > CHEK1 (41%) and showed significant association with each other. Breast carcinoma samples that were estrogen/progesterone receptor negative showed significantly high deletion and overall alterations than estrogen/progesterone receptor positive samples for LOH11CR2A, CHEK1 and PIG8. The methylation and overall alteration of LOH11CR2A were significantly associated with tumor stages in breast carcinoma. However, in early/late onset and estrogen/progesterone receptor positive/negative breast carcinoma, the overall alterations of LOH11CR2A, PIG8 and CHEK1 were differentially associated with advanced stages, tumor grade and lymph node metastasis. Alterations of PIG8 and CHEK1 were significantly associated with poor prognosis in patients with early age of onset of the disease indicating significant prognostic importance. Quantitative mRNA expression analysis detected reduced expression of the genes in the order LOH11CR2A > CHEK1 > PIG8. Immunohistochemical analysis showed reduced protein expression of PIG8 and CHEK1 that was concordant with their molecular alterations. Thus, our study suggests that LOH11CR2A, PIG8 and CHEK1 are candidate tumor suppressor genes associated with breast carcinoma and have significant clinical as well as prognostic importance.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Neoplasias de la Mama/genética , Cromosomas Humanos Par 11/genética , Genes Relacionados con las Neoplasias/genética , Mutación/genética , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteínas Quinasas/genética , Adulto , Anciano , Proteínas Reguladoras de la Apoptosis/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama Masculina/genética , Neoplasias de la Mama Masculina/patología , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Deleción Cromosómica , Metilación de ADN/genética , Femenino , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Polimorfismo de Nucleótido Simple/genética , Pronóstico , Regiones Promotoras Genéticas , Proteínas Quinasas/metabolismo , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Adulto Joven
20.
Genes Chromosomes Cancer ; 49(2): 155-70, 2010 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19885927

RESUMEN

To understand the importance of frequent deletion of 3p22.3 in cervical carcinogenesis, alterations (deletion/methylation/expression) of the candidate genes STAC, MLH1, ITGA9, and RBSP3, located in the region, were analyzed in 24 cervical intraepithelial neoplasia (CIN) and 137 uterine cervical carcinoma (CACX) samples. In CIN, RBSP3 deletion (48%) and methylation (26%) were high compared with the other genes (4-9%). In CACX, alterations of these genes were as follows: deletion: STAC (54%) > MLH1 (46%) > RBSP3 (45%) > ITGA9 (41%), methylation: RBSP3 (25%) > ITGA9 (24%) > STAC (19%) > MLH1 (13%). Overall, alterations of RBSP3 showed association with CIN, whereas for STAC and MLH1, this frequency increased significantly from CIN --> Stage I/II and for ITGA9 from CIN --> Stage I/II and also from Stage I/II --> Stage III/IV. Quantitative mRNA expression analysis showed differential reduced expression of these genes in CACX concordant to their molecular alterations. The more active RBSP3B splice variant was underexpressed in CACX. RB1 was infrequently deleted in CACX. Concordance was seen between (i) inactivation of RBSP3 and intense p-RB1 nuclear immunostaining and (ii) low/absence of MLH1 expression and its molecular alterations in CACX. In normal cervical epithelium, p-RB1 immunostaining was low in differentiated cells, whereas MLH1 staining was seen in both nucleus and cytoplasm irrespective of differentiation stage. Alterations of the genes were significantly associated with poor prognosis. High parity (>or=5)/early sexual debut (

Asunto(s)
Cromosomas Humanos Par 3 , Regulación Neoplásica de la Expresión Génica , Lesiones Precancerosas/genética , Proteínas Supresoras de Tumor/genética , Neoplasias del Cuello Uterino/genética , Adulto , Carcinoma in Situ/genética , Carcinoma in Situ/patología , ADN de Neoplasias/genética , ADN de Neoplasias/aislamiento & purificación , Femenino , Eliminación de Gen , Humanos , Repeticiones de Microsatélite , Persona de Mediana Edad , Estadificación de Neoplasias , Lesiones Precancerosas/patología , Pronóstico , ARN Mensajero/genética , ARN Neoplásico/genética , Neoplasias del Cuello Uterino/patología
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