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Stem Cells ; 37(5): 609-622, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30681766


Death of photoreceptors is a common cause of age-related and inherited retinal dystrophies, and thus their replenishment from renewable stem cell sources is a highly desirable therapeutic goal. Human pluripotent stem cells provide a useful cell source in view of their limitless self-renewal capacity and potential to not only differentiate into cells of the retina but also self-organize into tissue with structure akin to the human retina as part of three-dimensional retinal organoids. Photoreceptor precursors have been isolated from differentiating human pluripotent stem cells through application of cell surface markers or fluorescent reporter approaches and shown to have a similar transcriptome to fetal photoreceptors. In this study, we investigated the transcriptional profile of CRX-expressing photoreceptor precursors derived from human pluripotent stem cells and their engraftment capacity in an animal model of retinitis pigmentosa (Pde6brd1), which is characterized by rapid photoreceptor degeneration. Single cell RNA-Seq analysis revealed the presence of a dominant cell cluster comprising 72% of the cells, which displayed the hallmarks of early cone photoreceptor expression. When transplanted subretinally into the Pde6brd1 mice, the CRX+ cells settled next to the inner nuclear layer and made connections with the inner neurons of the host retina, and approximately one-third of them expressed the pan cone marker, Arrestin 3, indicating further maturation upon integration into the host retina. Together, our data provide valuable molecular insights into the transcriptional profile of human pluripotent stem cells-derived CRX+ photoreceptor precursors and indicate their usefulness as a source of transplantable cone photoreceptors. Stem Cells 2019;37:609-622.

Stem Cells ; 37(5): 593-598, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30548510


The rapid improvements in single cell sequencing technologies and analyses afford greater scope for dissecting organoid cultures composed of multiple cell types and create an opportunity to interrogate these models to understand tissue biology, cellular behavior and interactions. To this end, retinal organoids generated from human embryonic stem cells (hESCs) were analyzed by single cell RNA-sequencing (scRNA-Seq) at three time points of differentiation. Combinatorial data from all time points revealed the presence of nine clusters, five of which corresponded to key retinal cell types: retinal pigment epithelium (RPE), retinal ganglion cells (RGCs), cone and rod photoreceptors, and Müller glia. The remaining four clusters expressed genes typical of mitotic cells, extracellular matrix components and those involved in homeostasis. The cell clustering analysis revealed the decreasing presence of mitotic cells and RGCs, formation of a distinct RPE cluster, the emergence of cone and rod photoreceptors from photoreceptor precursors, and an increasing number of Müller glia cells over time. Pseudo-time analysis resembled the order of cell birth during retinal development, with the mitotic cluster commencing the trajectory and the large majority of Müller glia completing the time line. Together, these data demonstrate the feasibility and potential of scRNA-Seq to dissect the inherent complexity of retinal organoids and the orderly birth of key retinal cell types. Stem Cells 2019;37:593-598.

Neurobiol Aging ; 38: 217.e1-217.e6, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26639157


There is a growing body of evidence linking mitochondrial dysfunction, mediated either through inherited mitochondrial DNA (mtDNA) variation or mitochondrial proteomic deficit, to Parkinson's disease (PD). Yet, despite this, the role of somatic mtDNA point mutations and specifically point-mutational burden in PD is poorly understood. Here, we take advantage of recent technical and methodological advances to examine the role of age-related and acquired mtDNA mutation in the largest study of mtDNA in postmortem PD tissue to date. Our data show that PD patients suffer an increase in mtDNA mutational burden in, but no limited to, the substantia nigra pars compacta when compared to matched controls. This mutational burden appears increased in genes encoding cytochrome c oxidase, supportive of previous protein studies of mitochondrial dysfunction in PD. Accepting experimental limitations, our study confirms the important role of age-related mtDNA point mutation in the etiology of PD, moreover, by analyzing 2 distinct brain regions, we are able to show that PD patient brains are more vulnerable to mtDNA mutation overall.

DNA Mitocondrial/genética , Estudos de Associação Genética , Doença de Parkinson/genética , Mutação Puntual/genética , Estudos de Coortes , Complexo IV da Cadeia de Transporte de Elétrons/genética , Humanos
Stem Cells Transl Med ; 3(4): 416-23, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24591732


Hypoplastic left heart syndrome (HLHS) is a serious congenital cardiovascular malformation resulting in hypoplasia or atresia of the left ventricle, ascending aorta, and aortic and mitral valves. Diminished flow through the left side of the heart is clearly a key contributor to the condition, but any myocardial susceptibility component is as yet undefined. Using recent advances in the field of induced pluripotent stem cells (iPSCs), we have been able to generate an iPSC model of HLHS malformation and characterize the properties of cardiac myocytes (CMs) differentiated from these and control-iPSC lines. Differentiation of HLHS-iPSCs to cardiac lineages revealed changes in the expression of key cardiac markers and a lower ability to give rise to beating clusters when compared with control-iPSCs and human embryonic stem cells (hESCs). HLHS-iPSC-derived CMs show a lower level of myofibrillar organization, persistence of a fetal gene expression pattern, and changes in commitment to ventricular versus atrial lineages, and they display different calcium transient patterns and electrophysiological responses to caffeine and ß-adrenergic antagonists when compared with hESC- and control-iPSC-derived CMs, suggesting that alternative mechanisms to release calcium from intracellular stores such as the inositol trisphosphate receptor may exist in HLHS in addition to the ryanodine receptor thought to function in control-iPSC-derived CMs. Together our findings demonstrate that CMs derived from an HLHS patient demonstrate a number of marker expression and functional differences to hESC/control iPSC-derived CMs, thus providing some evidence that cardiomyocyte-specific factors may influence the risk of HLHS.

Regulação da Expressão Gênica , Síndrome do Coração Esquerdo Hipoplásico/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Biológicos , Proteínas Musculares/biossíntese , Miócitos Cardíacos/metabolismo , Células Cultivadas , Humanos , Síndrome do Coração Esquerdo Hipoplásico/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Recém-Nascido , Masculino , Miócitos Cardíacos/patologia
Blood ; 118(10): 2656-8, 2011 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-21765025


The human syndrome of dendritic cell, monocyte, B and natural killer lymphoid deficiency presents as a sporadic or autosomal dominant trait causing susceptibility to mycobacterial and other infections, predisposition to myelodysplasia and leukemia, and, in some cases, pulmonary alveolar proteinosis. Seeking a genetic cause, we sequenced the exomes of 4 unrelated persons, 3 with sporadic disease, looking for novel, heterozygous, and probably deleterious variants. A number of genes harbored novel variants in person, but only one gene, GATA2, was mutated in all 4 persons. Each person harbored a different mutation, but all were predicted to be highly deleterious and to cause loss or mutation of the C-terminal zinc finger domain. Because GATA2 is the only common mutated gene in 4 unrelated persons, it is highly probable to be the cause of dendritic cell, monocyte, B, and natural killer lymphoid deficiency. This disorder therefore constitutes a new genetic form of heritable immunodeficiency and leukemic transformation.

Linfócitos B/patologia , Células Dendríticas/patologia , Suscetibilidade a Doenças/etiologia , Éxons/genética , Fator de Transcrição GATA2/genética , Células Matadoras Naturais/patologia , Tecido Linfoide/patologia , Monócitos/patologia , Mutação/genética , Fator de Transcrição GATA2/química , Humanos , Conformação Proteica
Neurosci Lett ; 317(1): 13-6, 2002 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-11750985


Genetic studies in Alzheimer's disease (AD), have indicated that the apolipoprotein E locus (APO E) is a major susceptibility factor, but that it can only account for approximately 50% of AD cases. Several other studies have attempted to identify additional genetic loci associated with disease development, often based on a candidate gene approach. As several lines of evidence indicate that oxidative stress and free radical damage occur in AD, the transferrin gene (TF) has been suggested as a candidate locus for AD since it is the major transport protein for iron, which itself is a major factor in free radical generation. Previous studies have shown elevated TF C2 allele frequencies in AD, this being specifically associated with carriers of the APO E varepsilon4 allele. We have therefore determined the influence of the common polymorphisms in TF, C1 and C2, in dementia. The frequency of the C2 allele was not significantly associated with AD. Stratification of cases according to the APO E varepsilon4 allele showed a highly significant excess of the C2 allele in AD cases without the varepsilon4 allele, contrasting with previous studies. Given the contrasting findings between reports of altered TF C2 allele frequencies, the TF locus would not appear to be a strong risk factor for AD in this population.

Doença de Alzheimer/genética , Radicais Livres/metabolismo , Ferro/metabolismo , Doença por Corpos de Lewy/genética , Estresse Oxidativo/genética , Polimorfismo Genético/genética , Transferrina/genética , Idoso , Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Apolipoproteína E4 , Apolipoproteínas E/genética , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Análise Mutacional de DNA , Frequência do Gene , Predisposição Genética para Doença/genética , Testes Genéticos , Genótipo , Humanos , Doença por Corpos de Lewy/metabolismo , Doença por Corpos de Lewy/fisiopatologia , Pessoa de Meia-Idade , Neurônios/metabolismo , Neurônios/patologia , Transferrina/metabolismo