RESUMO
The use of cell-culture models to investigate development and disease of the cerebellum is a recent advance, facilitated by the discovery that patterning of precursors is capable of giving rise to cells with specific neuronal identity. Pluripotent stem cell-derived organoids, which exhibit self-organisational characteristics reminiscent of early cerebellar tissue, present a number of challenges including recapitulation of conditions resembling the mature brain. An understanding of the processes driving fetal and postnatal maturation is required to reproduce these conditions in vitro and advance the capability of the system to model adult-onset disease. A key tool for achieving this is single-cell RNA sequencing, which enables visualisation of key transcriptional features of subpopulations comprising tissues. Here, we explore and compare available single-cell RNA sequencing data derived from the developing human cerebellum and its synthetic, in vitro counterpart (stem cell-derived cerebellar organoids). We focus on performing a qualitative assessment of the expression of key metabolic pathway genes, given recent findings exemplifying tissue-specific metabolic activity, including hypoxia and metabolic shifts associated with neuronal expansion. Signatures indicative of known cell type-specific metabolic differences, such as the astrocyte-neuron lactate shuttle and glutamate-glutamine cycle were evident at a transcriptional level. Cerebellar tissue and cerebellar organoids showed a number of behavioural similarities, including HIF1 signalling, which may serve to drive expansion of granule cell progenitors in both settings. We further highlight numerous differences between cultured organoids and native tissue which may provide clarity on the state of metabolic state following differentiation of organoids, providing the future framework to test and further hypotheses regarding promoting maturation. Overall, this analysis provides insight into understanding the state of in vitro models of the cerebellum, a critical factor required for modelling susceptibility of various cell types to cerebellar disease.
Assuntos
Cerebelo , Organoides , Técnicas de Cultura de Células , Diferenciação Celular , Humanos , Neurônios/metabolismo , Organoides/metabolismoRESUMO
Ataxia oculomotor apraxia type 2 (AOA2) is a rare autosomal recessive cerebellar ataxia. Recent evidence suggests that the protein defective in this syndrome, senataxin (SETX), functions in RNA processing to protect the integrity of the genome. To date, only patient-derived lymphoblastoid cells, fibroblasts and SETX knockdown cells were available to investigate AOA2. Recent disruption of the Setx gene in mice did not lead to neurobehavioral defects or neurodegeneration, making it difficult to study the etiology of AOA2. To develop a more relevant neuronal model to study neurodegeneration in AOA2, we derived neural progenitors from a patient with AOA2 and a control by induced pluripotent stem cell (iPSC) reprogramming of fibroblasts. AOA2 iPSC and neural progenitors exhibit increased levels of oxidative damage, DNA double-strand breaks, increased DNA damage-induced cell death and R-loop accumulation. Genome-wide expression and weighted gene co-expression network analysis in these neural progenitors identified both previously reported and novel affected genes and cellular pathways associated with senataxin dysfunction and the pathophysiology of AOA2, providing further insight into the role of senataxin in regulating gene expression on a genome-wide scale. These data show that iPSCs can be generated from patients with the autosomal recessive ataxia, AOA2, differentiated into neurons, and that both cell types recapitulate the AOA2 cellular phenotype. This represents a novel and appropriate model system to investigate neurodegeneration in this syndrome.
Assuntos
Reprogramação Celular , Modelos Animais de Doenças , Mutação , Células-Tronco Neurais/metabolismo , RNA Helicases/genética , Ataxias Espinocerebelares/congênito , Animais , Apoptose , Quebras de DNA de Cadeia Dupla , DNA Helicases , Feminino , Fibroblastos/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Camundongos , Enzimas Multifuncionais , Neurônios/fisiologia , Estresse Oxidativo , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/metabolismo , Ataxias Espinocerebelares/fisiopatologiaRESUMO
Down syndrome (DS) is the most frequent cause of human congenital mental retardation. Cognitive deficits in DS result from perturbations of normal cellular processes both during development and in adult tissues, but the mechanisms underlying DS etiology remain poorly understood. To assess the ability of induced pluripotent stem cells (iPSCs) to model DS phenotypes, as a prototypical complex human disease, we generated bona fide DS and wild-type (WT) nonviral iPSCs by episomal reprogramming. DS iPSCs selectively overexpressed chromosome 21 genes, consistent with gene dosage, which was associated with deregulation of thousands of genes throughout the genome. DS and WT iPSCs were neurally converted at >95% efficiency and had remarkably similar lineage potency, differentiation kinetics, proliferation, and axon extension at early time points. However, at later time points DS cultures showed a twofold bias toward glial lineages. Moreover, DS neural cultures were up to two times more sensitive to oxidative stress-induced apoptosis, and this could be prevented by the antioxidant N-acetylcysteine. Our results reveal a striking complexity in the genetic alterations caused by trisomy 21 that are likely to underlie DS developmental phenotypes, and indicate a central role for defective early glial development in establishing developmental defects in DS brains. Furthermore, oxidative stress sensitivity is likely to contribute to the accelerated neurodegeneration seen in DS, and we provide proof of concept for screening corrective therapeutics using DS iPSCs and their derivatives. Nonviral DS iPSCs can therefore model features of complex human disease in vitro and provide a renewable and ethically unencumbered discovery platform.
Assuntos
Síndrome de Down/etiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Diferenciação Celular/fisiologia , Síndrome de Down/genética , Síndrome de Down/patologia , Feminino , Dosagem de Genes , Regulação da Expressão Gênica no Desenvolvimento , Predisposição Genética para Doença , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Masculino , Neuritos/patologia , Neuritos/fisiologia , Neurogênese , Neurônios/patologia , Neurônios/fisiologia , TranscriptomaRESUMO
Oncogenic PIK3CA mutations contribute to colorectal tumorigenesis by activating AKT signaling to decrease apoptosis and increase tumor invasion. A synergistic association of PIK3CA mutation with KRAS mutation has been suggested to increase AKT signaling and resistance to antiepidermal growth factor receptor inhibitor therapy for advanced colorectal cancer, although studies have been conflicting. We sought to clarify this by examining PIK3CA mutation frequency in relation to other key molecular features of defined pathways of tumorigenesis. PIK3CA mutation was assessed by high resolution melt analysis in 829 colorectal cancer samples and 426 colorectal polyps. Mutations were independently correlated with clinicopathological features including patient age, sex and tumor location as well as molecular features including microsatellite instability, KRAS and BRAF mutation, MGMT methylation and the CpG Island Methylator Phenotype (CIMP). Mutation of the helical (Exon 9) and catalytic (Exon 20) domain mutation hotspots were also examined independently. Overall, PIK3CA mutation was positively correlated with KRAS mutation (p < 0.001), MGMT methylation (p = 0.007) and CIMP (p < 0.001). Novel, exon-specific associations linked Exon 9 mutations to a subgroup of cancers characterized by KRAS mutation, MGMT methylation and CIMP-Low, whilst Exon 20 mutations were more closely linked to features of serrated pathway tumors including BRAF mutation, microsatellite instability and CIMP-High or Low. PIK3CA mutations were uncommonly, but exclusively, seen in tubulovillous adenomas (4/124, 3.2%) and 1/4 (25.0%) tubulovillous adenomas with a focus of cancer. These data provide insight into the molecular events driving traditional versus serrated pathway tumorigenesis.
Assuntos
Pólipos do Colo/genética , Neoplasias Colorretais/genética , Oncogenes , Fosfatidilinositol 3-Quinases/genética , Idoso , Sequência de Bases , Classe I de Fosfatidilinositol 3-Quinases , Estudos de Coortes , Primers do DNA , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Reprogramming of cells enables generation of pluripotent stem cells and resulting progeny through directed differentiation, making this technology an invaluable tool for the study of human development and disease. Reprogramming occurs with a wide range of efficiency, a culmination of intrinsic and extrinsic factors including the tissue of origin, the passage number and culture history of the target cells. Another major factor affecting reprogramming is the methodology used and the quality of the reprogramming process itself, including for conventional viral-based approaches viral titer and subsequent viral transduction efficiency, including downstream transgene insertion and stoichiometry. Genetic background is an important parameter affecting the efficiency of the reprogramming process with reports that cells from individuals harboring specific mutations are more difficult to reprogram than control counterparts.Ataxia-Telangiectasia (A-T) fibroblasts underwent reprogramming at reduced efficiency in contrast to their controls. To optimize reprogramming of fibroblasts from patients with A-T, we examined the response of A-T cells to various cell culture conditions after lentiviral transduction with reprogramming factors Oc4/Sox2 (pSIN4-EF2-O2S) and Klf4/c-Myc (pSIN4-CMV-K2M). Parameters included media type (KSR or serum-containing DMEM), treatment with a p53 inhibitor (small-molecule cyclic pifithrin-α), and either a low or high concentration of bFGF. Post-transduction, equivalent numbers of cells from heterozygote and homozygote patients were plated and assessed at regular intervals for survival and proliferation. Our findings indicate that A-T cells responded favorably to the addition of FCS and gradual weaning away from their native media into KSR-containing stem cell media that produced suitable conditions for their reprogramming. We examined a range of properties to identify and isolate good quality iPSCs including the expression status of important stem cell transcription factors/surface proteins, methylation levels at stem cell associated regulatory loci, persistence of transgenes, karyotype status, and teratoma-forming ability.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Lentivirus/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Dano ao DNA/genética , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismoRESUMO
Ataxia-telangiectasia (A-T) is a rare genetic disorder caused by loss of function of the ataxia-telangiectasia-mutated kinase and is characterized by a predisposition to cancer, pulmonary disease, immune deficiency and progressive degeneration of the cerebellum. As animal models do not faithfully recapitulate the neurological aspects, it remains unclear whether cerebellar degeneration is a neurodevelopmental or neurodegenerative phenotype. To address the necessity for a human model, we first assessed a previously published protocol for the ability to generate cerebellar neuronal cells, finding it gave rise to a population of precursors highly enriched for markers of the early hindbrain such as EN1 and GBX2, and later more mature cerebellar markers including PTF1α, MATH1, HOXB4, ZIC3, PAX6, and TUJ1. RNA sequencing was used to classify differentiated cerebellar neurons generated from integration-free A-T and control induced pluripotent stem cells. Comparison of RNA sequencing data with datasets from the Allen Brain Atlas reveals in vitro-derived cerebellar neurons are transcriptionally similar to discrete regions of the human cerebellum, and most closely resemble the cerebellum at 22 weeks post-conception. We show that patient-derived cerebellar neurons exhibit disrupted gene regulatory networks associated with synaptic vesicle dynamics and oxidative stress, offering the first molecular insights into early cerebellar pathogenesis of ataxia-telangiectasia.
RESUMO
Despite their abundance, the molecular functions of long non-coding RNAs in mammalian nervous systems remain poorly understood. Here we show that the long non-coding RNA, NEAT1, directly modulates neuronal excitability and is associated with pathological seizure states. Specifically, NEAT1 is dynamically regulated by neuronal activity in vitro and in vivo, binds epilepsy-associated potassium channel-interacting proteins including KCNAB2 and KCNIP1, and induces a neuronal hyper-potentiation phenotype in iPSC-derived human cortical neurons following antisense oligonucleotide knockdown. Next generation sequencing reveals a strong association of NEAT1 with increased ion channel gene expression upon activation of iPSC-derived neurons following NEAT1 knockdown. Furthermore, we show that while NEAT1 is acutely down-regulated in response to neuronal activity, repeated stimulation results in NEAT1 becoming chronically unresponsive in independent in vivo rat model systems relevant to temporal lobe epilepsy. We extended previous studies showing increased NEAT1 expression in resected cortical tissue from high spiking regions of patients suffering from intractable seizures. Our results indicate a role for NEAT1 in modulating human neuronal activity and suggest a novel mechanistic link between an activity-dependent long non-coding RNA and epilepsy.
Assuntos
Encéfalo/fisiologia , Excitabilidade Cortical , Neurônios/fisiologia , RNA Longo não Codificante/metabolismo , Convulsões/patologia , Animais , Células Cultivadas , Humanos , Proteínas Interatuantes com Canais de Kv/metabolismo , Células-Tronco Pluripotentes/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Ligação Proteica , Ratos , Superfamília Shaker de Canais de PotássioRESUMO
Pluripotent stem cells can differentiate into every cell type of the human body. Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) therefore provides an opportunity to gain insight into the molecular and cellular basis of disease. Because the cellular DNA damage response poses a barrier to reprogramming, generation of iPSCs from patients with chromosomal instability syndromes has thus far proven to be difficult. Here we demonstrate that fibroblasts from patients with ataxia-telangiectasia (A-T), a disorder characterized by chromosomal instability, progressive neurodegeneration, high risk of cancer, and immunodeficiency, can be reprogrammed to bona fide iPSCs, albeit at a reduced efficiency. A-T iPSCs display defective radiation-induced signaling, radiosensitivity, and cell cycle checkpoint defects. Bioinformatic analysis of gene expression in the A-T iPSCs identifies abnormalities in DNA damage signaling pathways, as well as changes in mitochondrial and pentose phosphate pathways. A-T iPSCs can be differentiated into functional neurons and thus represent a suitable model system to investigate A-T-associated neurodegeneration. Collectively, our data show that iPSCs can be generated from a chromosomal instability syndrome and that these cells can be used to discover early developmental consequences of ATM deficiency, such as altered mitochondrial function, that may be relevant to A-T pathogenesis and amenable to therapeutic intervention.