RESUMEN
Most cardiomyocytes (CMs) in the adult mammalian heart are either binucleated or contain a single polyploid nucleus. Recent studies have shown that polyploidy in CMs plays an important role as an adaptive response to physiological demands and environmental stress and correlates with poor cardiac regenerative ability after injury. However, knowledge about the functional properties of polyploid CMs is limited. In this study, we generated tetraploid pluripotent stem cells (PSCs) by fusion of murine embryonic stem cells (ESCs) and somatic cells isolated from bone marrow or spleen and performed a comparative analysis of the electrophysiological properties of tetraploid fusion-derived PSCs and diploid ESC-derived CMs. Fusion-derived PSCs exhibited characteristics of genuine ESCs and contained a near-tetraploid genome. Ploidy features and marker expression were also retained during the differentiation of fusion-derived cells. Fusion-derived PSCs gave rise to CMs, which were similar to their diploid ESC counterparts in terms of their expression of typical cardiospecific markers, sarcomeric organization, action potential parameters, response to pharmacologic stimulation with various drugs, and expression of functional ion channels. These results suggest that the state of ploidy does not significantly affect the structural and electrophysiological properties of murine PSC-derived CMs. These results extend our knowledge of the functional properties of polyploid CMs and contribute to a better understanding of their biological role in the adult heart.
Asunto(s)
Células Madre Pluripotentes Inducidas , Células Madre Pluripotentes , Ratones , Animales , Miocitos Cardíacos/metabolismo , Tetraploidía , Diploidia , Células Madre Embrionarias , Diferenciación Celular/genética , Poliploidía , MamíferosRESUMEN
Mutations in pitrilysin metallopeptidase 1 (PITRM1), a mitochondrial protease involved in mitochondrial precursor processing and degradation, result in a slow-progressing syndrome characterized by cerebellar ataxia, psychotic episodes, and obsessive behavior, as well as cognitive decline. To investigate the pathogenetic mechanisms of mitochondrial presequence processing, we employed cortical neurons and cerebral organoids generated from PITRM1-knockout human induced pluripotent stem cells (iPSCs). PITRM1 deficiency strongly induced mitochondrial unfolded protein response (UPRmt) and enhanced mitochondrial clearance in iPSC-derived neurons. Furthermore, we observed increased levels of amyloid precursor protein and amyloid ß in PITRM1-knockout neurons. However, neither cell death nor protein aggregates were observed in 2D iPSC-derived cortical neuronal cultures. On the other hand, over time, cerebral organoids generated from PITRM1-knockout iPSCs spontaneously developed pathological features of Alzheimer's disease (AD), including the accumulation of protein aggregates, tau pathology, and neuronal cell death. Single-cell RNA sequencing revealed a perturbation of mitochondrial function in all cell types in PITRM1-knockout cerebral organoids, whereas immune transcriptional signatures were substantially dysregulated in astrocytes. Importantly, we provide evidence of a protective role of UPRmt and mitochondrial clearance against impaired mitochondrial presequence processing and proteotoxic stress. Here, we propose a novel concept of PITRM1-linked neurological syndrome whereby defects of mitochondrial presequence processing induce an early activation of UPRmt that, in turn, modulates cytosolic quality control pathways. Thus, our work supports a mechanistic link between mitochondrial function and common neurodegenerative proteinopathies.
Asunto(s)
Enfermedad de Alzheimer , Células Madre Pluripotentes Inducidas , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides , Humanos , Metaloendopeptidasas , Mitocondrias , OrganoidesRESUMEN
Parkinson's disease-associated kinase LRRK2 has been linked to IFN type II (IFN-γ) response in infections and to dopaminergic neuronal loss. However, whether and how LRRK2 synergizes with IFN-γ remains unclear. In this study, we employed dopaminergic neurons and microglia differentiated from patient-derived induced pluripotent stem cells carrying LRRK2 G2019S, the most common Parkinson's disease-associated mutation. We show that IFN-γ enhances the LRRK2 G2019S-dependent negative regulation of AKT phosphorylation and NFAT activation, thereby increasing neuronal vulnerability to immune challenge. Mechanistically, LRRK2 G2019S suppresses NFAT translocation via calcium signaling and possibly through microtubule reorganization. In microglia, LRRK2 modulates cytokine production and the glycolytic switch in response to IFN-γ in an NFAT-independent manner. Activated LRRK2 G2019S microglia cause neurite shortening, indicating that LRRK2-driven immunological changes can be neurotoxic. We propose that synergistic LRRK2/IFN-γ activation serves as a potential link between inflammation and neurodegeneration in Parkinson's disease.
Asunto(s)
Neuronas Dopaminérgicas/inmunología , Interferón gamma/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Microglía/inmunología , Enfermedad de Parkinson/inmunología , Señalización del Calcio/genética , Diferenciación Celular , Citocinas/metabolismo , Neuronas Dopaminérgicas/metabolismo , Técnicas de Inactivación de Genes , Glucólisis/genética , Células HEK293 , Humanos , Células Madre Pluripotentes Inducidas/fisiología , Interferón gamma/inmunología , Microscopía Intravital , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Microglía/metabolismo , Microtúbulos/metabolismo , Mutación , Factores de Transcripción NFATC/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Cultivo Primario de Células , Transducción de Señal/genética , Transducción de Señal/inmunología , Células THP-1RESUMEN
While mitochondrial dysfunction is emerging as key in Parkinson's disease (PD), a central question remains whether mitochondria are actual disease drivers and whether boosting mitochondrial biogenesis and function ameliorates pathology. We address these questions using patient-derived induced pluripotent stem cells and Drosophila models of GBA-related PD (GBA-PD), the most common PD genetic risk. Patient neurons display stress responses, mitochondrial demise, and changes in NAD+ metabolism. NAD+ precursors have been proposed to ameliorate age-related metabolic decline and disease. We report that increasing NAD+ via the NAD+ precursor nicotinamide riboside (NR) significantly ameliorates mitochondrial function in patient neurons. Human neurons require nicotinamide phosphoribosyltransferase (NAMPT) to maintain the NAD+ pool and utilize NRK1 to synthesize NAD+ from NAD+ precursors. Remarkably, NR prevents the age-related dopaminergic neuronal loss and motor decline in fly models of GBA-PD. Our findings suggest NR as a viable clinical avenue for neuroprotection in PD and other neurodegenerative diseases.
Asunto(s)
Drosophila melanogaster/fisiología , Células Madre Pluripotentes Inducidas/patología , Mitocondrias/patología , NAD/metabolismo , Neuronas/metabolismo , Neuronas/patología , Niacinamida/análogos & derivados , Enfermedad de Parkinson/patología , Animales , Autofagia , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Estrés del Retículo Endoplásmico , Glucosilceramidasa/metabolismo , Humanos , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Dinámicas Mitocondriales , Actividad Motora , Niacinamida/metabolismo , Enfermedad de Parkinson/fisiopatología , Compuestos de Piridinio , Respuesta de Proteína DesplegadaRESUMEN
We report here the generation of human iPS cell line UKKi009-A from dermal fibroblasts of a patient carrying heterozygous mutation c.3035-3045delTCCCTCGATGC, p.Leu1012Pro (fs*55) in KCNH2 gene leading to long QT syndrome type 2 (LQT2). We used the Sleeping Beauty transposon-based plasmids expressing OSKM along with microRNAs 307/367 to reprogram the fibroblasts. The iPS cells possess pluripotent stem cell characteristics and differentiate to cell lineages of all three germ layers. This cell line can serve as a source for in vitro modeling of LQT2. This cell line is distributed by the European Collection of Authenticated Cell Cultures (ECACC).
Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Síndrome de QT Prolongado/patología , Adulto , Secuencia de Bases , Diferenciación Celular , Células Cultivadas , Reprogramación Celular , Hibridación Genómica Comparativa , Canal de Potasio ERG1/genética , Femenino , Fibroblastos/citología , Citometría de Flujo , Genotipo , Heterocigoto , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Cariotipo , Síndrome de QT Prolongado/genética , Microscopía Fluorescente , Polimorfismo de Nucleótido Simple , Reacción en Cadena en Tiempo Real de la Polimerasa , Piel/citología , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Reproducible and efficient differentiation of pluripotent stem cells (PSCs) to cardiomyocytes (CMs) is essential for their use in regenerative medicine, drug testing and disease modeling. The aim of this study was to evaluate the effect of some previously reported cardiogenic substances on cardiac differentiation of mouse PSCs. METHODS: Differentiation was performed by embryoid body (EB)-based method using three different murine PSC lines. The differentiation efficiency was monitored by RT-qPCR, immunocytochemistry and flow cytometry, and the effect mechanistically evaluated by transcriptome analysis of treated EBs. RESULTS: Among the five tested compounds (ascorbic acid, dorsomorphin, cyclic adenosine 3',5'-monophosphate, cardiogenol C, cyclosporin A) only ascorbic acid (AA) exerted a strong and reproducible cardiogenic effect in CGR8 cells which was less consistent in other two PSC lines. AA induced only minor changes in transcriptome of CGR8 cells after administration during the initial two days of differentiation. Cardiospecific genes and transcripts involved in angiogenesis, erythropoiesis and hematopoiesis were up-regulated on day 5 but not on days 2 or 3 of differentiation. The cardiac differentiation efficiency was improved when QS11, a small-molecule synergist of Wnt/ß-catenin signaling pathway, was added to cultures after AA-treatment. CONCLUSION: This study demonstrates that only minor transcriptional changes are sufficient for enhancement of cardiogenesis of murine PSCs by AA and that AA and QS11 exhibit synergistic effects and enhance the efficiency of CM differentiation of murine PSCs.