RESUMO
Down syndrome (trisomy 21) is the most common genetic cause of intellectual disability, but the precise molecular mechanisms underlying impaired cognition remain unclear. Elucidation of these mechanisms has been hindered by the lack of a model system that contains full trisomy of chromosome 21 (Ts21) in a human genome that enables normal gene regulation. To overcome this limitation, we created Ts21-induced pluripotent stem cells (iPSCs) from two sets of Ts21 human fibroblasts. One of the fibroblast lines had low level mosaicism for Ts21 and yielded Ts21 iPSCs and an isogenic control that is disomic for human chromosome 21 (HSA21). Differentiation of all Ts21 iPSCs yielded similar numbers of neurons expressing markers characteristic of dorsal forebrain neurons that were functionally similar to controls. Expression profiling of Ts21 iPSCs and their neuronal derivatives revealed changes in HSA21 genes consistent with the presence of 50% more genetic material as well as changes in non-HSA21 genes that suggested compensatory responses to oxidative stress. Ts21 neurons displayed reduced synaptic activity, affecting excitatory and inhibitory synapses equally. Thus, Ts21 iPSCs and neurons display unique developmental defects that are consistent with cognitive deficits in individuals with Down syndrome and may enable discovery of the underlying causes of and treatments for this disorder.
Assuntos
Síndrome de Down/genética , Fibroblastos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Cromossomos Humanos Par 21/genética , Fibroblastos/citologia , Perfilação da Expressão Gênica , Genótipo , Humanos , Hibridização in Situ Fluorescente , Células-Tronco Pluripotentes Induzidas/citologia , Mosaicismo , Neurônios/citologia , Neurônios/fisiologia , Estresse Oxidativo , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Potenciais Sinápticos/genéticaRESUMO
Best disease (BD) is an inherited degenerative disease of the human macula that results in progressive and irreversible central vision loss. It is caused by mutations in the retinal pigment epithelium (RPE) gene BESTROPHIN1 (BEST1), which, through mechanism(s) that remain unclear, lead to the accumulation of subretinal fluid and autofluorescent waste products from shed photoreceptor outer segments (POSs). We employed human iPS cell (hiPSC) technology to generate RPE from BD patients and unaffected siblings in order to examine the cellular and molecular processes underlying this disease. Consistent with the clinical phenotype of BD, RPE from mutant hiPSCs displayed disrupted fluid flux and increased accrual of autofluorescent material after long-term POS feeding when compared with hiPSC-RPE from unaffected siblings. On a molecular level, RHODOPSIN degradation after POS feeding was delayed in BD hiPSC-RPE relative to unaffected sibling hiPSC-RPE, directly implicating impaired POS handling in the pathophysiology of the disease. In addition, stimulated calcium responses differed between BD and normal sibling hiPSC-RPE, as did oxidative stress levels after chronic POS feeding. Subcellular localization, fractionation and co-immunoprecipitation experiments in hiPSC-RPE and human prenatal RPE further linked BEST1 to the regulation and release of endoplasmic reticulum calcium stores. Since calcium signaling and oxidative stress are critical regulators of fluid flow and protein degradation, these findings likely contribute to the clinical picture of BD. In a larger context, this report demonstrates the potential to use patient-specific hiPSCs to model and study maculopathies, an important class of blinding disorders in humans.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Distrofia Macular Viteliforme/genética , Distrofia Macular Viteliforme/fisiopatologia , Animais , Bestrofinas , Cálcio/metabolismo , Bovinos , Diferenciação Celular , Linhagem Celular , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica , Homeostase , Humanos , Imuno-Histoquímica , Imunoprecipitação , Macula Lutea/patologia , Microscopia Eletrônica de Transmissão , Estresse Oxidativo , Fagocitose , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/patologia , Distrofia Macular Viteliforme/metabolismoRESUMO
Seven human induced pluripotent stem cell (iPSC) lines were generated from fibroblasts from three neonatal individuals using non-integrative reprogramming. Most control iPSCs are derived from adults, so these iPSCs meet the need for control iPSCs from young individuals. Donors were from different ethnicities and these lines provide unique genetic profiles. All iPSCs have normal karyotypes, express stem cell markers, and exhibit pluripotency, as assessed by capacity to differentiate into three germ layers. These lines are valuable to study human development, as age-matched controls for disorder-specific iPSCs, and as platforms for gene editing to control for age and ethnicity.
Assuntos
Técnicas de Cultura de Células/métodos , Etnicidade , Animais , Linhagem Celular , Feminino , Humanos , Recém-Nascido , Masculino , CamundongosRESUMO
Amyotrophic lateral sclerosis (ALS) presents motoneuron (MN)-selective protein inclusions and axonal degeneration but the underlying mechanisms of such are unknown. Using induced pluripotent cells (iPSCs) from patients with mutation in the Cu/Zn superoxide dismutase (SOD1) gene, we show that spinal MNs, but rarely non-MNs, exhibited neurofilament (NF) aggregation followed by neurite degeneration when glia were not present. These changes were associated with decreased stability of NF-L mRNA and binding of its 3' UTR by mutant SOD1 and thus altered protein proportion of NF subunits. Such MN-selective changes were mimicked by expression of a single copy of the mutant SOD1 in human embryonic stem cells and were prevented by genetic correction of the SOD1 mutation in patient's iPSCs. Importantly, conditional expression of NF-L in the SOD1 iPSC-derived MNs corrected the NF subunit proportion, mitigating NF aggregation and neurite degeneration. Thus, NF misregulation underlies mutant SOD1-mediated NF aggregation and axonal degeneration in ALS MNs.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Biológicos , Neurônios Motores/metabolismo , Proteínas Mutantes/metabolismo , Superóxido Dismutase/metabolismo , Esclerose Lateral Amiotrófica/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Proteínas Mutantes/genética , Mutação , Superóxido Dismutase/genética , Superóxido Dismutase-1RESUMO
The generation of induced pluripotent stem cells (iPSCs) opens up the possibility for personalized cell therapy. Here, we show that transplanted autologous rhesus monkey iPSC-derived neural progenitors survive for up to 6 months and differentiate into neurons, astrocytes, and myelinating oligodendrocytes in the brains of MPTP-induced hemiparkinsonian rhesus monkeys with a minimal presence of inflammatory cells and reactive glia. This finding represents a significant step toward personalized regenerative therapies.
Assuntos
Encéfalo/citologia , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/transplante , Células-Tronco Neurais/transplante , Animais , Astrócitos/citologia , Encéfalo/patologia , Feminino , Células-Tronco Pluripotentes Induzidas/citologia , Intoxicação por MPTP/terapia , Macaca mulatta , Células-Tronco Neurais/citologia , Neuroglia/citologia , Neurônios/citologia , Oligodendroglia/citologiaRESUMO
The BCL6 transcriptional repressor is the most commonly involved oncogene in diffuse large B-cell lymphomas (DLBCLs). Constitutive expression of BCL6 mediates lymphomagenesis through aberrant proliferation, survival, and differentiation blockade. Binding of BCL6 to the SMRT/N-CoR corepressors mediates the BCL6 survival effect in DLBCL. Although the basis for differentiation blockade is unknown in DLBCL, recent data suggest that BCL6 binding to the MTA3 corepressor might be involved. We report that BCL6 and MTA3 are coexpressed in normal germinal center B cells and DLBCL. Depletion of MTA3 in DLBCL cells induced a differentiation-related BCL6 target gene (PRDM1), but not target genes involved in survival. Accordingly, MTA3 and PRDM1 expression are mutually exclusive in germinal center B cells. We performed chromatin immunoprecipitation (ChIP)-on-chip mapping of the PRDM1 locus, identifying a novel BCL6 binding site on intron 3 of the PRDM1 gene, and show that BCL6 recruits MTA3 to this site. In DLBCL cells, MTA3 depletion induced plasmacytic differentiation but did not decrease viability of DLBCL cells. However, MTA3 depletion synergized with a specific BCL6 inhibitor that blocks SMRT/N-CoR binding to decrease DLBCL viability. Taken together, these results show that BCL6 regulates distinct transcriptional programs through the SMRT/N-CoR and MTA3 corepressors, respectively, and provides a basis for combinatorial therapeutic targeting of BCL6.