RESUMEN
The scarcity of donors and need for immunosuppression limit pancreatic islet transplantation to a few patients with labile type 1 diabetes. Transplantation of encapsulated stem cell-derived islets (SC islets) might extend the applicability of islet transplantation to a larger cohort of patients. Transplantation of conformal-coated islets into a confined well-vascularized site allows long-term diabetes reversal in fully MHC-mismatched diabetic mice without immunosuppression. Here, we demonstrated that human SC islets reaggregated from cryopreserved cells display glucose-stimulated insulin secretion in vitro. Importantly, we showed that conformally coated SC islets displayed comparable in vitro function with unencapsulated SC islets, with conformal coating permitting physiological insulin secretion. Transplantation of SC islets into the gonadal fat pad of diabetic NOD-scid mice revealed that both unencapsulated and conformal-coated SC islets could reverse diabetes and maintain human-level euglycemia for more than 80 days. Overall, these results provide support for further evaluation of safety and efficacy of conformal-coated SC islets in larger species.
Asunto(s)
Diferenciación Celular , Diabetes Mellitus Tipo 1/terapia , Células Secretoras de Insulina/citología , Trasplante de Islotes Pancreáticos , Células Madre/citología , Animales , Células Cultivadas , Criopreservación/métodos , Modelos Animales de Enfermedad , Femenino , Humanos , Células Secretoras de Insulina/metabolismo , Masculino , Ratones , Células Madre/metabolismo , Trasplante HeterólogoRESUMEN
Methods: Human ARPE-19 cells engineered to secrete high levels of the glial cell line-derived neurotrophic factor (GDNF) were encapsulated into hollow fiber membranes. The devices were implanted into the rat striatum 1 week prior to striatal quinolinic acid injections. Animals were evaluated using a battery of validated motor tests, and histology was performed to determine the extent of GDNF diffusion and associated prevention of neuronal cell loss and behavioral deficits. Results: Encapsulated cell-based delivery of GDNF produced widespread distribution of GDNF throughout the entire implanted striatum. Stereological estimates of striatal neuron number and volume of lesion size revealed that GDNF delivery resulted in near complete neuroprotection. Conclusions: Delivery of neurotrophic molecules such as GDNF using encapsulated cells has reached a technological point where clinical evaluation is justified. Because GDNF has been effective in animal models of Parkinson's disease, stroke, epilepsy, and Huntington's disease, among other debilitating neurodegenerative diseases, encapsulated cell-based delivery of GDNF might represent one innovative means of slowing the neural degeneration seen in a myriad of currently untreatable neurological diseases.
Asunto(s)
Cuerpo Estriado/efectos de los fármacos , Factor Neurotrófico Derivado de la Línea Celular Glial/administración & dosificación , Fármacos Neuroprotectores/administración & dosificación , Ácido Quinolínico/toxicidad , Animales , Encapsulación Celular , Línea Celular , Sistemas de Liberación de Medicamentos , Humanos , Células LLC-PK1 , Masculino , Enfermedades Neurodegenerativas/tratamiento farmacológico , Neuronas/efectos de los fármacos , Ratas Sprague-Dawley , PorcinosRESUMEN
The choroid plexus (CP) has been explored as a cellular therapeutic due to its broad-ranging secretome and demonstrated longevity in a variety of encapsulation modalities. While the CP organ is normally involved in disease repair processes in the brain, the range of indications that could potentially be ameliorated with exogenous CP therapy is widespread, including diseases of the central nervous system, hearing loss, chronic wounds, and others. The CP can be isolated from animal sources and digested into a highly purified epithelial culture that can withstand encapsulation and transplantation. Its epithelium can adapt to different microenvironments, and depending on culture conditions, can be manipulated into various three-dimensional configurations with distinct gene expression profiles. The cocktail of proteins secreted by the CP can be harvested in culture, and purified forms of these extracts have been evaluated in topical applications to treat poorly healing wounds. When encapsulated, the epithelial clusters can be maintained for extended durations in vitro with minimal impact on potency. A treatment for Parkinson's disease utilizing encapsulated porcine CP has been developed and is currently being evaluated in a Phase I clinical trial. The current chapter serves to summarize recent experience with CP factor delivery, and provides a description of the relevant materials and methods employed in these studies.
Asunto(s)
Células Inmovilizadas/citología , Células Inmovilizadas/trasplante , Plexo Coroideo/citología , Células Epiteliales/citología , Células Epiteliales/trasplante , Alginatos/química , Animales , Cápsulas/química , Técnicas de Cultivo de Célula/métodos , Separación Celular/métodos , Tratamiento Basado en Trasplante de Células y Tejidos , Células Cultivadas , Células Inmovilizadas/metabolismo , Plexo Coroideo/metabolismo , Cuerpo Estriado/citología , Cuerpo Estriado/cirugía , Medios de Cultivo Condicionados/metabolismo , Células Epiteliales/metabolismo , Ácido Glucurónico/química , Ácidos Hexurónicos/química , Humanos , Neuronas/citología , Neuronas/metabolismo , Enfermedad de Parkinson/terapia , Ratas , PorcinosRESUMEN
BACKGROUND: Coordinated remodeling of epithelium and vasculature is essential for normal postglandular lung development. The value of the human-to-rodent lung xenograft as model of fetal microvascular development remains poorly defined. AIM: The aim of this study was to determine the fate of the endogenous (human-derived) microvasculature in fetal lung xenografts. METHODS: Lung tissues were obtained from spontaneous pregnancy losses (14-22 weeks' gestation) and implanted in the renal subcapsular or dorsal subcutaneous space of SCID-beige mice (T, B, and NK-cell-deficient) and/or nude rats (T-cell-deficient). Informed parental consent was obtained. Lung morphogenesis, microvascular angiogenesis, and epithelial differentiation were assessed at 2 and 4 weeks post-transplantation by light microscopy, immunohistochemical, and gene expression studies. Archival age-matched postmortem lungs served as control. RESULTS: The vascular morphology, density, and proliferation of renal subcapsular grafts in SCID-beige mice were similar to age-matched control lungs, with preservation of the physiologic association between epithelium and vasculature. The microvasculature of subcutaneous grafts in SCID-beige mice was underdeveloped and dysmorphic, associated with significantly lower VEGF, endoglin, and angiopoietin-2 mRNA expression than renal grafts. Grafts at both sites displayed mild airspace dysplasia. Renal subcapsular grafts in nude rats showed frequent infiltration by host lymphocytes and obliterating bronchiolitis-like changes, associated with markedly decreased endogenous angiogenesis. CONCLUSION: This study demonstrates the critical importance of host and site selection to ensure optimal xenograft development. When transplanted to severely immune suppressed, NK-cell-deficient hosts and engrafted in the renal subcapsular site, the human-to-rodent fetal lung xenograft provides a valid model of postglandular microvascular lung remodeling.
Asunto(s)
Células Epiteliales/fisiología , Pulmón , Microvasos/embriología , Neovascularización Fisiológica/fisiología , Animales , Diferenciación Celular/fisiología , Humanos , Pulmón/irrigación sanguínea , Pulmón/citología , Pulmón/embriología , Ratones , Ratones SCID , Modelos Animales , Ratas , Ratas Desnudas , Trasplante HeterólogoRESUMEN
PURPOSE: Encapsulated cell (EC) biodelivery is a promising, clinically relevant technology platform to safely target the delivery of therapeutic proteins to the central nervous system. The purpose of this study was to evaluate EC biodelivery of the novel neurotrophic factor, Meteorin, to the striatum of rats and to investigate its neuroprotective effects against quinolinic acid (QA)-induced excitotoxicity. METHODS: Meteorin-producing ARPE-19 cells were loaded into EC biodelivery devices and implanted into the striatum of rats. Two weeks after implantation, QA was injected into the ipsilateral striatum followed by assessment of neurological performance two and four weeks after QA administration. RESULTS: Implant-delivered Meteorin effectively protected against QA-induced toxicity, as manifested by both near-normal neurological performance and reduction of brain cell death. Morphological analysis of the Meteorin-treated brains showed a markedly reduced striatal lesion size. The EC biodelivery devices produced stable or even increasing levels of Meteorin throughout the study over 6 weeks. CONCLUSIONS: Stereotactically implanted EC biodelivery devices releasing Meteorin could offer a feasible strategy in the treatment of neurological diseases with an excitotoxic component such as Huntington's disease. In a broader sense, the EC biodelivery technology is a promising therapeutic protein delivery platform for the treatment of a wide range of diseases of the central nervous system.
Asunto(s)
Implantes Absorbibles/normas , Citoprotección/efectos de los fármacos , Modelos Animales de Enfermedad , Enfermedad de Huntington/tratamiento farmacológico , Proteínas del Tejido Nervioso/administración & dosificación , Quinolinas/toxicidad , Animales , Trasplante de Tejido Encefálico/métodos , Cápsulas/administración & dosificación , Línea Celular , Citoprotección/fisiología , Humanos , Enfermedad de Huntington/inducido químicamente , Enfermedad de Huntington/genética , Masculino , Ratones , Factores de Crecimiento Nervioso/administración & dosificación , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Meteorin is a newly discovered secreted protein involved in both glial and neuronal cell differentiation, as well as in cerebral angiogenesis during development; but effects in the adult nervous system are unknown. The growth factor-like properties and expression of Meteorin during the development of the nervous system raises the possibility that it might possess important neuroprotective or regenerative capabilities. This report is the first demonstration that Meteorin has potent neuroprotective effects in vivo. Lentiviral-mediated striatal delivery of Meteorin to rats two weeks prior to injections of quinolinic acid (QA) dramatically reduced the loss of striatal neurons. The cellular protection afforded by Meteorin was associated with normalization of neurological performance on spontaneous forelimb placing and cylinder behavioral tests and a complete protection against QA-induced weight loss. These benefits were comparable in magnitude to those obtained with lentiviral-mediated delivery of ciliary neurotrophic factor (CNTF), a protein with known neuroprotective properties in the same model system. In naive animals, endogenous levels of both Meteorin and CNTF were increased in glial cells in response to QA lesion indicating that Meteorin may exert its protective effects as part of the reactive gliosis cascade in the injured brain. In summary, these data demonstrate that Meteorin strongly protects striatal neurons and deserves additional evaluation as a novel therapeutic for the treatment of neurological disorders with an excitotoxic component such as Huntington's Disease.
Asunto(s)
Cuerpo Estriado/metabolismo , Terapia Genética/métodos , Enfermedad de Huntington/terapia , Lentivirus/genética , Trastornos del Movimiento/terapia , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Animales , Conducta Animal/efectos de los fármacos , Conducta Animal/fisiología , Cuerpo Estriado/patología , Modelos Animales de Enfermedad , Vectores Genéticos/genética , Vectores Genéticos/uso terapéutico , Células HEK293 , Humanos , Enfermedad de Huntington/genética , Enfermedad de Huntington/metabolismo , Masculino , Ratones , Trastornos del Movimiento/genética , Trastornos del Movimiento/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/patología , Ratas , Ratas Sprague-DawleyRESUMEN
We used cryogenic transmission electron microscopy (cryo-TEM) to identify differences in macromolecular structures present in the serum from healthy individuals (HI) and prostate cancer (PCa) patients and show that these differences are potential markers for disease. Using a murine orthotopic model of human PCa, we determined that some of these structural markers in serum are associated with the tumour burden. These findings signify the potential of this nanoscale ex vivo imaging technology of body fluids as a platform for discovering early markers of PCa and other diseases.
Asunto(s)
Biomarcadores de Tumor/sangre , Microscopía por Crioelectrón/métodos , Microscopía Electrónica de Transmisión/métodos , Nanoestructuras/ultraestructura , Neoplasias de la Próstata/sangre , Neoplasias de la Próstata/patología , Anciano , Animales , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Persona de Mediana EdadRESUMEN
The choroid plexus (CP) is a transplantable cell source secreting tropic and trophic factors for the treatment of brain and peripheral trauma characterized by cellular loss or dysfunction. Here we characterize the expression and secretion of vascular endothelial growth factor (VEGF) from neonatal porcine CP. Light and electron microscopy revealed that enzymatic digestion of the CP produced a preparation consisting primarily of epithelial cells without notable contaminating cells. Microarray analysis, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay were used to quantify the nuclear, cytoplasmic, and secretory compartmentalization of VEGF. In vitro, the kinetics of VEGF release were orderly, with stepwise increases in secretion over time. The secretory profile of VEGF from CP grown in configurations ranging from a simple monolayer to free-floating 3-dimensional clusters to clusters encapsulated within alginate-polyornithine microcapsules was similar. VEGF output was not affected notably when the cells were maintained in 90% stress medium or in other maintenance media devoid of serum proteins. Secreted VEGF was bioactive, as confirmed by demonstrating its continued ability to proliferate co-cultured human umbilical vascular endothelial cells. The robust ability of these cells to continue to secrete VEGF (and presumably other bioactive proteins) across a variety of dimensional configurations and medium types has implications for their use in clinical indications requiring novel and imaginative use of engineered ectopic transplant sites.
Asunto(s)
Plexo Coroideo/citología , Plexo Coroideo/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Péptidos/química , Ingeniería de Tejidos/métodos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Alginatos/química , Animales , Técnicas de Cultivo de Célula/métodos , Células Cultivadas , Materiales Biocompatibles Revestidos/química , Ácido Glucurónico/química , Ácidos Hexurónicos/química , Ensayo de Materiales , PorcinosRESUMEN
Neurotrophic factors have the ability to protect and initiate growth of neurons. In the central nervous system, neurotrophic factors are neuroprotective in a wide range of disease states. Similarly, disease pathologies of the neurosensory retina respond favourably in animal models of retinitis pigmentosa, macular degeneration, glaucoma and others. With advances in drug delivery and cell therapy, an almost universal increase in efficacy is being realised. Now, repeated injections of neurotrophic factors are being replaced by controlled delivery of cell-mediated factor secretion, reducing the number of potential acute side effects. Tissue engineering strategies in conjunction with gene-modulated protein therapy or gene transfer are creating a unique treatment niche and are quickly gaining acclaim in the clinic. This review surveys the founding and current work on neurotrophic factors such as CNTF, BDNF, GDNF, LEDGF, PEDF and others. Ongoing clinical trials and successful preclinical studies are summarised as well.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Factores de Crecimiento Nervioso/administración & dosificación , Ingeniería de Proteínas/métodos , Enfermedades de la Retina/tratamiento farmacológico , Animales , Sistemas de Liberación de Medicamentos/tendencias , Terapia Genética/métodos , Humanos , Factores de Crecimiento Nervioso/genética , Ingeniería de Proteínas/tendencias , Enfermedades de la Retina/genéticaRESUMEN
This article investigates the effect of particle size and the incorporation of a bioadhesive polymer, poly(fumaric-co-sebacic) anhydride p(FA:SA), on the relative bioavailability of dicumarol. A novel method was used to reduce particle size of the drug, and encapsulated formulations were fabricated using a phase inversion technique to produce nanospheres and microspheres with varying size. Groups of Yorkshire swine were catheterized and gavaged after fasting for 12 h with each formulation in a 50 mg/mL suspension. Blood was collected at different time points, from 0 to 96 h, and pharmacokinetic analysis revealed that formulations incorporating the smaller drug particles showed the highest bioavailability: micronized drug with 7% p(FA:SA) 17:83 polymer had 190% relative bioavailability, and phase inverted p(FA:SA) 17:83 microspheres with 31% (w/w) loading had 198% relative bioavailability to spray dried formulation. Formulations with larger drug particles achieved 71% relative bioavailability. A nonadhesive formulation, fabricated with poly(lactic acid) (PLA), showed 91% relative bioavailability. Both particle size and polymer composition play a role in oral absorption of dicumarol.