RESUMO
Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.
Assuntos
Transplante de Células/métodos , Epitélio Pigmentado da Retina/transplante , Transplante Heterólogo/métodos , Animais , Humanos , Injeções Intraoculares/métodos , Degeneração Macular/terapia , Ratos , Retina/transplante , Epitélio Pigmentado da Retina/citologiaRESUMO
Age-related macular degeneration (AMD) affects the retinal pigment epithelium (RPE), a cell monolayer essential for photoreceptor survival, and is the leading cause of vision loss in the elderly. There are no disease-altering therapies for dry AMD, which is characterized by accumulation of subretinal drusen deposits and complement-driven inflammation. We report the derivation of human-induced pluripotent stem cells (hiPSCs) from patients with diagnosed AMD, including two donors with the rare ARMS2/HTRA1 homozygous genotype. The hiPSC-derived RPE cells produce several AMD/drusen-related proteins, and those from the AMD donors show significantly increased complement and inflammatory factors, which are most exaggerated in the ARMS2/HTRA1 lines. Using a panel of AMD biomarkers and candidate drug screening, combined with transcriptome analysis, we discover that nicotinamide (NAM) ameliorated disease-related phenotypes by inhibiting drusen proteins and inflammatory and complement factors while upregulating nucleosome, ribosome, and chromatin-modifying genes. Thus, targeting NAM-regulated pathways is a promising avenue for developing therapeutics to combat AMD.
Assuntos
Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Degeneração Macular/tratamento farmacológico , Degeneração Macular/metabolismo , Niacinamida/uso terapêutico , Diferenciação Celular/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Genótipo , Humanos , Imuno-Histoquímica , Retina/efeitos dos fármacos , Retina/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) resets their identity back to an embryonic age and, thus, presents a significant hurdle for modeling late-onset disorders. In this study, we describe a strategy for inducing aging-related features in human iPSC-derived lineages and apply it to the modeling of Parkinson's disease (PD). Our approach involves expression of progerin, a truncated form of lamin A associated with premature aging. We found that expression of progerin in iPSC-derived fibroblasts and neurons induces multiple aging-related markers and characteristics, including dopamine-specific phenotypes such as neuromelanin accumulation. Induced aging in PD iPSC-derived dopamine neurons revealed disease phenotypes that require both aging and genetic susceptibility, such as pronounced dendrite degeneration, progressive loss of tyrosine hydroxylase (TH) expression, and enlarged mitochondria or Lewy-body-precursor inclusions. Thus, our study suggests that progerin-induced aging can be used to reveal late-onset age-related disease features in hiPSC-based disease models.
Assuntos
Envelhecimento/patologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Biológicos , Proteínas Nucleares/metabolismo , Precursores de Proteínas/metabolismo , Adulto , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Reprogramação Celular , Senescência Celular , Criança , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Neurônios Dopaminérgicos/transplante , Neurônios Dopaminérgicos/ultraestrutura , Fibroblastos/metabolismo , Humanos , Lamina Tipo A , Mesencéfalo/patologia , Camundongos , Pessoa de Meia-Idade , Doença de Parkinson/patologia , Fenótipo , Doadores de TecidosRESUMO
In congenital mitochondrial DNA (mtDNA) disorders, a mixture of normal and mutated mtDNA (termed heteroplasmy) exists at varying levels in different tissues, which determines the severity and phenotypic expression of disease. Pearson marrow pancreas syndrome (PS) is a congenital bone marrow failure disorder caused by heteroplasmic deletions in mtDNA. The cause of the hematopoietic failure in PS is unknown, and adequate cellular and animal models are lacking. Induced pluripotent stem (iPS) cells are particularly amenable for studying mtDNA disorders, as cytoplasmic genetic material is retained during direct reprogramming. Here, we derive and characterize iPS cells from a patient with PS. Taking advantage of the tendency for heteroplasmy to change with cell passage, we isolated isogenic PS-iPS cells without detectable levels of deleted mtDNA. We found that PS-iPS cells carrying a high burden of deleted mtDNA displayed differences in growth, mitochondrial function, and hematopoietic phenotype when differentiated in vitro, compared to isogenic iPS cells without deleted mtDNA. Our results demonstrate that reprogramming somatic cells from patients with mtDNA disorders can yield pluripotent stem cells with varying burdens of heteroplasmy that might be useful in the study and treatment of mitochondrial diseases.
Assuntos
DNA Mitocondrial/genética , Células-Tronco Pluripotentes Induzidas/fisiologia , Doenças Mitocondriais/genética , Acil-CoA Desidrogenase de Cadeia Longa/deficiência , Acil-CoA Desidrogenase de Cadeia Longa/metabolismo , Anemia Sideroblástica/genética , Anemia Sideroblástica/metabolismo , Anemia Sideroblástica/patologia , Diferenciação Celular/genética , Linhagem Celular , Pré-Escolar , Síndrome Congênita de Insuficiência da Medula Óssea , DNA Mitocondrial/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Erros Inatos do Metabolismo Lipídico/diagnóstico , Erros Inatos do Metabolismo Lipídico/metabolismo , Erros Inatos do Metabolismo Lipídico/patologia , Doenças Mitocondriais/diagnóstico , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , Doenças Musculares/diagnóstico , Doenças Musculares/metabolismo , Doenças Musculares/patologia , Deleção de SequênciaRESUMO
This chapter describes a protocol for deriving induced pluripotent stem cells (iPSCs) from human fibroblasts. Human fibroblasts, cultured in fibroblast medium, are infected with a cocktail of retroviral vectors expressing the transcription factors OCT4, SOX2, KLF4, and MYC. The culture conditions are then switched to conditions that support human embryonic stem cell growth and emerging iPSC colonies that morphologically resemble human embryonic stem cell (hESC) colonies and have silenced the retroviral vectors (as evidenced by downregulation of retroviral GFP expression) that are mechanically isolated and subsequently cultured in identical fashion to hESCs. Putative iPSC lines are validated to be bona fide human iPSC lines by analyzing them for the expression of pluripotency markers and by differentiation in vitro and in vivo.
Assuntos
Técnicas de Cultura de Células/métodos , Fibroblastos/citologia , Fibroblastos/virologia , Técnicas de Transferência de Genes , Células-Tronco Pluripotentes Induzidas/citologia , Retroviridae/genética , Linhagem Celular , Proliferação de Células , Reprogramação Celular/genética , Células Clonais , Ensaio de Unidades Formadoras de Colônias , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 4 Semelhante a Kruppel , Reprodutibilidade dos Testes , Retroviridae/fisiologia , Coloração e Rotulagem , Fatores de Transcrição/metabolismo , Transfecção , Replicação Viral/fisiologiaRESUMO
Patients with dyskeratosis congenita (DC), a disorder of telomere maintenance, suffer degeneration of multiple tissues. Patient-specific induced pluripotent stem (iPS) cells represent invaluable in vitro models for human degenerative disorders like DC. A cardinal feature of iPS cells is acquisition of indefinite self-renewal capacity, which is accompanied by induction of the telomerase reverse transcriptase gene (TERT). We investigated whether defects in telomerase function would limit derivation and maintenance of iPS cells from patients with DC. Here we show that reprogrammed DC cells overcome a critical limitation in telomerase RNA component (TERC) levels to restore telomere maintenance and self-renewal. We discovered that TERC upregulation is a feature of the pluripotent state, that several telomerase components are targeted by pluripotency-associated transcription factors, and that in autosomal dominant DC, transcriptional silencing accompanies a 3' deletion at the TERC locus. Our results demonstrate that reprogramming restores telomere elongation in DC cells despite genetic lesions affecting telomerase, and show that strategies to increase TERC expression may be therapeutically beneficial in DC patients.
Assuntos
Disceratose Congênita/genética , Células-Tronco Pluripotentes , Telômero/genética , Animais , Proteínas de Ciclo Celular/genética , Linhagem Celular , Reprogramação Celular/genética , Disceratose Congênita/enzimologia , Regulação Enzimológica da Expressão Gênica , Humanos , Camundongos , Proteínas Nucleares/genética , Células-Tronco Pluripotentes/enzimologia , RNA/genética , RNA/metabolismo , Deleção de Sequência/genética , Telomerase/genética , Telomerase/metabolismo , Regulação para CimaRESUMO
Fanconi anemia (FA) is a genetically heterogeneous, autosomal recessive disorder characterized by pediatric bone marrow failure and congenital anomalies. The effect of FA gene deficiency on hematopoietic development in utero remains poorly described as mouse models of FA do not develop hematopoietic failure and such studies cannot be performed on patients. We have created a human-specific in vitro system to study early hematopoietic development in FA using a lentiviral RNA interference (RNAi) strategy in human embryonic stem cells (hESCs). We show that knockdown of FANCA and FANCD2 in hESCs leads to a reduction in hematopoietic fates and progenitor numbers that can be rescued by FA gene complementation. Our data indicate that hematopoiesis is impaired in FA from the earliest stages of development, suggesting that deficiencies in embryonic hematopoiesis may underlie the progression to bone marrow failure in FA. This work illustrates how hESCs can provide unique insights into human development and further our understanding of genetic disease.
Assuntos
Células-Tronco Embrionárias/metabolismo , Proteína do Grupo de Complementação A da Anemia de Fanconi , Proteína do Grupo de Complementação D2 da Anemia de Fanconi , Anemia de Fanconi/metabolismo , Técnicas de Silenciamento de Genes , Medula Óssea/metabolismo , Medula Óssea/patologia , Linhagem Celular , Células-Tronco Embrionárias/patologia , Anemia de Fanconi/genética , Anemia de Fanconi/patologia , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Modelos Biológicos , Interferência de RNARESUMO
Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by enforced expression of transcription factors. Using serial live imaging of human fibroblasts undergoing reprogramming, we identified distinct colony types that morphologically resemble embryonic stem (ES) cells yet differ in molecular phenotype and differentiation potential. By analyzing expression of pluripotency markers, methylation at the OCT4 and NANOG promoters and differentiation into teratomas, we determined that only one colony type represents true iPS cells, whereas the others represent reprogramming intermediates. Proviral silencing and expression of TRA-1-60, DNMT3B and REX1 can be used to distinguish the fully reprogrammed state, whereas alkaline phosphatase, SSEA-4, GDF3, hTERT and NANOG are insufficient as markers. We also show that reprogramming using chemically defined medium favors formation of fully reprogrammed over partially reprogrammed colonies. Our data define molecular markers of the fully reprogrammed state and highlight the need for rigorous characterization and standardization of putative iPS cells.
Assuntos
Reprogramação Celular/genética , Imageamento Tridimensional/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Diferenciação Celular , Linhagem Celular , Forma Celular , Sobrevivência Celular , Ensaio de Unidades Formadoras de Colônias , Células-Tronco Embrionárias/citologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Citometria de Fluxo , Imunofluorescência , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Teratoma/patologia , Fatores de TempoRESUMO
Human dermal fibroblasts obtained by skin biopsy can be reprogrammed directly to pluripotency by the ectopic expression of defined transcription factors. Here, we describe the derivation of induced pluripotent stem cells from CD34+ mobilized human peripheral blood cells using retroviral transduction of OCT4/SOX2/KLF4/MYC. Blood-derived human induced pluripotent stem cells are indistinguishable from human embryonic stem cells with respect to morphology, expression of surface antigens, and pluripotency-associated transcription factors, DNA methylation status at pluripotent cell-specific genes, and the capacity to differentiate in vitro and in teratomas. The ability to reprogram cells from human blood will allow the generation of patient-specific stem cells for diseases in which the disease-causing somatic mutations are restricted to cells of the hematopoietic lineage.
Assuntos
Células Sanguíneas/citologia , Desdiferenciação Celular , Proliferação de Células , Células-Tronco Pluripotentes/citologia , Adulto , Antígenos CD34/metabolismo , Células Sanguíneas/metabolismo , Técnicas de Cultura de Células , Desdiferenciação Celular/fisiologia , Células Cultivadas , Humanos , Fator 4 Semelhante a Kruppel , Masculino , Modelos Biológicos , Células-Tronco Pluripotentes/metabolismoRESUMO
Human embryonic stem (hES) cells are self-renewing, pluripotent cells that are valuable research tools and hold promise for use in regenerative medicine. Most hES cell lines are derived from cryopreserved human embryos that were created during in vitro fertilization (IVF) and are in excess of clinical need. Embryos that are discarded during the IVF procedure because of poor morphology and a low likelihood for generating viable pregnancies or surviving the cryopreservation process are also a viable source of hES cells. In this protocol, we describe how to derive novel hES cells from discarded poor-quality embryos and how to maintain the hES cell lines.