RESUMO
The purpose of this study was to identify a membrane-bound complement inhibitor that could be overexpressed on retinal pigment epithelial cells (RPE) providing a potential therapy for age-related macular degeneration (AMD). This type of therapy may allow replacement of damaged RPE with cells that are able to limit complement activation in the retina. Complement Receptor 1 (CR1) is a membrane-bound complement inhibitor commonly found on erythrocytes and immune cells. In this study, QPCR and flow cytometry data demonstrated that CR1 is not well-expressed by RPE, indicating that its overexpression may provide extra protection from complement activation. To screen CR1 for this ability, a stable CR1-expressing ARPE19 line was created using a combination of antibiotic selection and FACS. Cell-based assays were used to demonstrate that addition of CR1 inhibited deposition of complement proteins C3b and C6 on the transfected line. In the end, this study identifies CR1 as a complement inhibitor that may be overexpressed on stem cell-derived RPE to create a potential "enhanced" cell therapy for AMD. A combination cell/complement therapy may create transplantable RPE better suited to avoid complement-mediated lysis and limit chronic inflammation in the retina.
Assuntos
Células Epiteliais/imunologia , Degeneração Macular/imunologia , Receptores de Complemento 3b/imunologia , Retina/imunologia , Epitélio Pigmentado da Retina/imunologia , Pigmentos da Retina/imunologia , Linhagem Celular , Ativação do Complemento/imunologia , Complemento C3b/imunologia , Complemento C6/imunologia , Eritrócitos/imunologia , HumanosRESUMO
Purpose: Age-related macular degeneration (AMD) is a common disease trending towards epidemic proportions and is a leading cause of irreversible vision loss in people over the age of 65. A pathomechanism of AMD is death and/or dysfunction of retinal pigment epithelial (RPE) cells; RPE loss invariably results in photoreceptor atrophy. Treatment options for AMD are very limited, and include vitamin supplements and lifestyle changes. An exciting potential therapy currently being tested in clinical trials is transplantation of stem cell-derived RPE. Methods: We developed a NIH-registered embryonic stem line (CR-4), and in this study set out to determine if CR4-RPE are tolerated in normal mice and in murine models of retinal degeneration by injecting a bolus of CR4-RPE cells in the subretinal space of immunosuppressed wild-type, Mer mutant (Merkd), and Elovl4 deficient mice. Results: Mice with CR-RPE grafts were monitored daily, were examined routinely using OCT, and histology was prepared and examined at terminal end-points. Based on the parameters of the study, none of the animals with CR-RPE grafts (n=36) experienced any obvious adverse reactions. Conclusions: We conclude that transplanted CR-4 hES-derived RPE cells are well tolerated in immunosuppressed healthy and dystrophic murine retinas.
Assuntos
Células-Tronco Embrionárias Humanas/citologia , Degeneração Macular/terapia , Epitélio Pigmentado da Retina/citologia , Animais , Modelos Animais de Doenças , Proteínas do Olho/metabolismo , Humanos , Degeneração Macular/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Camundongos , Camundongos KnockoutRESUMO
Monosomy of chromosome X is associated with high prenatal mortality of female embryos and severe developmental abnormalities of patients born with Turner's syndrome (45,XO). The CDMLe012-A-1 human embryonic stem cell (hESC) line, derived from a day six blastocyst with a normal 46,XX female karyotype spontaneously lost an X-chromosome during cell culture. This 45,XO karyotype was stably maintained for more than 55 passages. Since the CDMLe012-A-1 cells express pluripotent stem cell markers and differentiate into cells derived from the three germ layers, the cell line represents a stable, pluripotent stem cell model of Turner's syndrome.
Assuntos
Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Síndrome de Turner/genética , Blastocisto/citologia , Blastocisto/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Feminino , Humanos , Cariótipo , Cariotipagem , GravidezRESUMO
The CR-4 human embryonic stem cell line was derived from the inner cell mass of a developing blastocyst. This cell line has been adapted to grow in feeder-free conditions and is especially well-suited for differentiation to retinal pigment epithelium. The line demonstrates a normal human 46,XX female karyotype. Pluripotency was assessed through qRT-PCR for expression of NANOG, OCT-4, and SOX-2. A teratoma assay was performed and results were positive for all three germ layers. Testing for Mycoplasma was negative.