20 research outputs found
Comparative study of human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) as a treatment for retinal dystrophies
Retinal dystrophies (RD) are major causes of familial blindness and are characterized by progressive dysfunction of photoreceptor and/or retinal pigment epithelium (RPE) cells. In this study, we aimed to evaluate and compare the therapeutic effects of two pluripotent stem cell (PSC)-based therapies. We differentiated RPE from human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (hiPSCs) and transplanted them into the subretinal space of the Royal College of Surgeons (RCS) rat. Once differentiated, cells from either source of PSC resembled mature RPE in their morphology and gene expression profile. Following transplantation, both hESC- and hiPSC-derived cells maintained the expression of specific RPE markers, lost their proliferative capacity, established tight junctions, and were able to perform phagocytosis of photoreceptor outer segments. Remarkably, grafted areas showed increased numbers of photoreceptor nuclei and outer segment disk membranes. Regardless of the cell source, human transplants protected retina from cell apoptosis, glial stress and accumulation of autofluorescence, and responded better to light stimuli. Altogether, our results show that hESC- and hiPSC-derived cells survived, migrated, integrated, and functioned as RPE in the RCS rat retina, providing preclinical evidence that either PSC source could be of potential benefit for treating RD
Chronic Glaucoma Using Biodegradable Microspheres to Induce Intraocular Pressure Elevation. Six-Month Follow-Up
Altres ajuts: Rio Hortega Research Grant M17/00213, Research Group UCM 920415, UCM-Santander fellowship (CT17/17-CT17-18).Background: To compare two prolonged animal models of glaucoma over 24 weeks of follow-up. A novel pre-trabecular model of chronic glaucoma was achieved by injection of biodegradable poly lactic-co-glycolic acid (PLGA) microspheres (10-20 µm) (Ms20/10) into the ocular anterior chamber to progressively increase ocular hypertension (OHT). Methods: Rat right eyes were injected to induce OHT: 50% received a suspension of Ms20/10 in the anterior chamber at 0, 2, 4, 8, 12, 16 and 20 weeks, and the other 50% received a sclerosing episcleral vein injection biweekly (EPIm). Ophthalmological clinical signs, intraocular pressure (IOP), neuroretinal functionality measured by electroretinography (ERG), and structural analysis of the retina, retinal nerve fiber layer (RNFL) and ganglion cell layer (GCL) protocols using optical coherence tomography (OCT) and histological exams were performed. Results: Both models showed progressive neuroretinal degeneration (p < 0.05), and contralateral eye affectation. The Ms20/10 model showed a more progressive increase in IOP and better preservation of ocular surface. Although no statistical differences were found between models, the EPIm showed a tendency to produce thicker retinal and thinner GCL thicknesses, slower latency and smaller amplitude as measured using ERG, and more aggressive disturbances in retinal histology. In both models, while the GCL showed the greatest percentage loss of thickness, the RNFL showed the greatest and earliest rate of thickness loss. Conclusions: The intracameral model with biodegradable microspheres resulted more like the conditions observed in humans. It was obtained by a less-aggressive mechanism, which allows for adequate study of the pathology over longer periods
FGF21 gene therapy as treatment for obesity and insulin resistance
Prevalence of type 2 diabetes (T2D) and obesity is increasing worldwide. Currently available therapies are not suited for all patients in the heterogeneous obese/T2D population, hence the need for novel treatments. Fibroblast growth factor 21 (FGF21) is considered a promising therapeutic agent for T2D/obesity. Native FGF21 has, however, poor pharmacokinetic properties, making gene therapy an attractive strategy to achieve sustained circulating levels of this protein. Here, adeno-associated viral vectors (AAV) were used to genetically engineer liver, adipose tissue, or skeletal muscle to secrete FGF21. Treatment of animals under long-term high-fat diet feeding or of ob/ob mice resulted in marked reductions in body weight, adipose tissue hypertrophy and inflammation, hepatic steatosis, inflammation and fibrosis, and insulin resistance for >Â 1Â year. This therapeutic effect was achieved in the absence of side effects despite continuously elevated serum FGF21. Furthermore, FGF21 overproduction in healthy animals fed a standard diet prevented the increase in weight and insulin resistance associated with aging. Our study underscores the potential of FGF21 gene therapy to treat obesity, insulin resistance, and T2D.This work was supported by grants from Ministerio de EconomÃa y Competi-
tividad (MINECO) and FEDER, Plan Nacional I+D+I (SAF2014-54866R), andGeneralitat de Catalunya (2014SGR1669and ICREA Academia Award to F.B.), Spain, from the European Commission (MYOCURE, PHC-14-2015 667751) and the European Foundation for the Study of Diabetes (EFSD/MSD
European Research Programme on Novel Therapies for Type
2 Diabetes,2013). V.J. was recipient of a post-doctoral research fellowship from EFSD/
Lilly. E.C., V.S., and C.M. received a predoctoral fellowship from Ministerio de
Educación, Cultura y Deporte, and J.R. from Ministerio de EconomÃa y
Competitividad, Spain. The authors thank Marta Moya and Maria Molas for
technical assistance.S
Long-term retinal PEDF overexpression prevents neovascularization in a murine adult model of retinopathy
Neovascularization associated with diabetic retinopathy (DR) and other ocular disorders is a leading cause of visual impairment and adult-onset blindness. Currently available treatments are merely palliative and offer temporary solutions. Here, we tested the efficacy of antiangiogenic gene transfer in an animal model that mimics the chronic progression of human DR. Adeno-associated viral (AAV) vectors of serotype 2 coding for antiangiogenic Pigment Epithelium Derived Factor (PEDF) were injected in the vitreous of a 1.5 month-old transgenic model of retinopathy that develops progressive neovascularization. A single intravitreal injection led to long-term production of PEDF and to a striking inhibition of intravitreal neovascularization, normalization of retinal capillary density, and prevention of retinal detachment. This was parallel to a reduction in the intraocular levels of Vascular Endothelial Growth Factor (VEGF). Normalization of VEGF was consistent with a downregulation of downstream effectors of angiogenesis, such as the activity of Matrix Metalloproteinases (MMP) 2 and 9 and the content of Connective Tissue Growth Factor (CTGF). These results demonstrate long-term efficacy of AAV-mediated PEDF overexpression in counteracting retinal neovascularization in a relevant animal model, and provides evidence towards the use of this strategy to treat angiogenesis in DR and other chronic proliferative retinal disorders
Long-Term Efficacy and Safety of Insulin and Glucokinase Gene Therapy for Diabetes : 8-Year Follow-Up in Dogs
Diabetes is a complex metabolic disease that exposes patients to the deleterious effects of hyperglycemia on various organs. Achievement of normoglycemia with exogenous insulin treatment requires the use of high doses of hormone, which increases the risk of life-threatening hypoglycemic episodes. We developed a gene therapy approach to control diabetic hyperglycemia based on co-expression of the insulin and glucokinase genes in skeletal muscle. Previous studies proved the feasibility of gene delivery to large diabetic animals with adeno-associated viral (AAV) vectors. Here, we report the long-term (∼8 years) follow-up after a single administration of therapeutic vectors to diabetic dogs. Successful, multi-year control of glycemia was achieved without the need of supplementation with exogenous insulin. Metabolic correction was demonstrated through normalization of serum levels of fructosamine, triglycerides, and cholesterol and remarkable improvement in the response to an oral glucose challenge. The persistence of vector genomes and therapeutic transgene expression years after vector delivery was documented in multiple samples from treated muscles, which showed normal morphology. Thus, this study demonstrates the long-term efficacy and safety of insulin and glucokinase gene transfer in large animals and especially the ability of the system to respond to the changes in metabolic needs as animals grow older
3-Dimensional histological reconstruction and imaging of the murine pancreas.
Visualization of important disease-driving tissues in their native morphological state, such as the pancreas, given its importance in glucose homeostasis and diabetes, provides critical insight into the etiology and progression of disease and our understanding of how cellular changes impact disease severity. Numerous challenges to maintaining tissue morphology exist when one attempts to preserve or to recreate such tissues for histological evaluation. We have overcome many of these challenges and have developed new methods for visualizing the whole murine pancreas and single islets of Langerhans in an effort to gain a better understanding of how islet cell volume, spatial distribution, and vascularization are altered as diabetes progresses. These methods are readily adaptable without requirement for costly specialized equipment, such as magnetic resonance imaging, positron emission tomography, or computed tomography, and can be used to provide additional robust analysis of diabetes susceptibility in mouse models of Type 1 and Type II diabetes. Mamm Genome 2014 Oct; 25(9-10):549-48
Specific cell types transduced by intravitreal AAV2 in TgIGF-I retinas.
<p>(A) Retinal flat-mount immunostained for GFP showing transduction of cells located on the surface of the retina. Arrows indicate GFP signal in axons, demonstrating transduction of ganglion cells. (B) GFP detection in paraffin-embedded eye sections. Besides transduction of cells in the ganglion cell layer (GCL), there was, to a lesser extend, transduction of cells of the Inner Nuclear layer (INL) <i>(inset, arrows)</i>. (C) Double immunostaining for GFP and calretinin (amacrine neurons) showed that amacrine cells located both in the surface of the retina as well as in the INL were transduced by AAV2-GFP. Bipolar cells, stained with an antibody against PKC-α, do not show colocalization with GFP nor in the cell bodies localized in the INL <i>(right inset)</i> nor in the neuronal projections ending at the GCL <i>(left inset)</i>. Immunohistochemistry for calbindin, expressed in horizontal cells of the INL outmost layer <i>(left inset)</i> and in a subtype of amacrine cells located in the INL and GCL <i>(right inset)</i>, showing that both cell types are transduced by AAV2-GFP. (D) Double immunostaining for GFP and Glutamine synthetase (Müller cells). No expression of GFP was observed in the Müller cells' end feet processes at the retinal surface <i>(right panel)</i> or in the cell bodies in the INL <i>(left panel)</i>. Astrocytes (S100+), localized in the most superficial retinal layer and around blood vessels, were not transduced by AAV2-GFP. Original magnification: 10× (A), 2× (B). Scale bar: 33 µm (C, <i>left and central panel</i>); 74 µm (C, <i>right panel</i>); 74 µm (D, <i>left panel</i>); 18 µm (D, <i>right panel</i>).</p
Inhibition of intraretinal neovascularization by AAV2-hPEDF treatment.
<p>(A) Representative images used for the quantitative assessment of intrarretinal capillary density. Original magnification: 40×. (B) Twenty high-magnification images of the capillary network that excluded major vessels were taken per retinal angiography with fluorescein-conjugated dextran, and the area corresponding to the capillary area was calculated using software tools as percentage of green area over total retinal area. Digital image quantification showed a significant increase in the retinal capillary density in transgenic animals compared with Wt. Six months after a single treatment with AAV2-hPEDF transgenic retinas showed a capillary density similar to that of Wt healthy retinas. N.I., non-injected. Values are expressed as the mean ±SEM of 7 animals/group. *<i>P</i><0.05.</p
Effects of AAV2-hPEDF treatment on VEGF levels in a HIF-1α independent manner.
<p>(A) Detection by ELISA of increased intraocular VEGF levels in both TgIGF-I and AAV2-null injected TgIGF-I mice. Levels normalized 6 months after AAV2-hPEDF treatment. (B) Time-course of retinal HIF-1α expression in TgIGF-I mice. HIF-1α protein was increased in retinas of 7.5 months-old TgIGF-I, remaining unaltered in younger animals. (C) AAV2-PEDF treatment did not affect HIF-1α expression 6 months after injection of the vector. N.I., non-injected. Values are expressed as the mean ±SEM of 4–10 animals/group. ***<i>P</i><0.001, **<i>P</i><0.01, *<i>P</i><0.05.</p
Intravitreal neovessels in non-treated and AAV-null-treated 7.5 month-old TgIGF-I mice.
<p>(A) Representative image of PAS-stained sections of transgenic eyes showing intravitreal vessels. Note the presence of fibrotic tissue associated to the vessel attached to the inner limiting membrane of the retina, in an area where the retinal morphology appears altered. Wild-type eyes did not present vascular structures nor fibrosis in the vitreous cavity. Original magnification: 20× (B) ColIV (basal membrane marker) and CD105 (EC marker) inmunostaining in the same intravitreal neovessel depicted in (A) corresponding to a transgenic mouse aged 7.5 months. A clear CD105+ ring-shaped signal, indicating the presence of a lumen, was surrounded by Collagen IV+ staining. Arrows indicate completely formed intravitreal neovessels. Arrowheads indicate a blood vessel within the retina. Scale bar: 25 µm. (C) Representative image of the CD105+/Col-IV+ vascular structures <i>(asterisks)</i> present in the vitreous cavity of AAV2null-injected eyes 6 months after delivery of the vectors. No such structures were observed in the contralateral PEDF-treated eyes <i>(data not shown)</i>. The dashed line indicates the limit between the retina and the vitreous. Scale bar: 25 µm (<i>left pannel</i>), 7.5 µm (<i>right pannel</i>). <i>GCL</i>, ganglion cell layer; <i>L</i>, lenses, <i>V</i>, vitreous cavity, <i>R</i>, retina.</p