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1.
J Cell Physiol ; 2021 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-33393109

RESUMO

Our understanding of signaling pathways regulating the cell fate of human embryonic stem cells (hESCs) is limited. Calcineurin-NFAT signaling is associated with a wide range of biological processes and diseases. However, its role in controlling hESC fate remains unclear. Here, we report that calcineurin A gamma and the NFATc3/SRPX2 axis control the expression of lineage and epithelial-mesenchymal transition (EMT) markers in hESCs. Knockdown of PPP3CC, the gene encoding calcineurin A gamma, or NFATC3, downregulates certain markers both at the self-renewal state and during differentiation of hESCs. Furthermore, NFATc3 interacts with c-JUN and regulates the expression of SRPX2, the gene encoding a secreted glycoprotein known as a ligand of uPAR. We show that SRPX2 is a downstream target of NFATc3. Both SRPX2 and uPAR participate in controlling expression of lineage and EMT markers. Importantly, SRPX2 knockdown diminishes the upregulation of multiple lineage and EMT markers induced by co-overexpression of NFATc3 and c-JUN in hESCs. Together, this study uncovers a previously unknown role of calcineurin A gamma and the NFATc3/SRPX2 axis in modulating the fate determination of hESCs.

2.
Cell Rep ; 31(2): 107500, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32294436

RESUMO

Diffusely infiltrating gliomas are known to cause alterations in cortical function, vascular disruption, and seizures. These neurological complications present major clinical challenges, yet their underlying mechanisms and causal relationships to disease progression are poorly characterized. Here, we follow glioma progression in awake Thy1-GCaMP6f mice using in vivo wide-field optical mapping to monitor alterations in both neuronal activity and functional hemodynamics. The bilateral synchrony of spontaneous neuronal activity gradually decreases in glioma-infiltrated cortical regions, while neurovascular coupling becomes progressively disrupted compared to uninvolved cortex. Over time, mice develop diverse patterns of high amplitude discharges and eventually generalized seizures that appear to originate at the tumors' infiltrative margins. Interictal and seizure events exhibit positive neurovascular coupling in uninfiltrated cortex; however, glioma-infiltrated regions exhibit disrupted hemodynamic responses driving seizure-evoked hypoxia. These results reveal a landscape of complex physiological interactions occurring during glioma progression and present new opportunities for exploring novel biomarkers and therapeutic targets.

5.
PLoS Pathog ; 13(12): e1006773, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29281729

RESUMO

The histone demethylase LSD1 has been known as a key transcriptional coactivator for DNA viruses such as herpes virus. Inhibition of LSD1 was found to block viral genome transcription and lytic replication of DNA viruses. However, RNA virus genomes do not rely on chromatin structure and histone association, and the role of demethylase activity of LSD1 in RNA virus infections is not anticipated. Here, we identify that, contrary to its role in enhancing DNA virus replication, LSD1 limits RNA virus replication by demethylating and activating IFITM3 which is a host restriction factor for many RNA viruses. We have found that LSD1 is recruited to demethylate IFITM3 at position K88 under IFNα treatment. However, infection by either Vesicular Stomatitis Virus (VSV) or Influenza A Virus (IAV) triggers methylation of IFITM3 by promoting its disassociation from LSD1. Accordingly, inhibition of the enzymatic activity of LSD1 by Trans-2-phenylcyclopropylamine hydrochloride (TCP) increases IFITM3 monomethylation which leads to more severe disease outcomes in IAV-infected mice. In summary, our findings highlight the opposite role of LSD1 in fighting RNA viruses comparing to DNA viruses infection. Our data suggest that the demethylation of IFITM3 by LSD1 is beneficial for the host to fight against RNA virus infection.


Assuntos
Histona Desmetilases/metabolismo , Vírus da Influenza A/patogenicidade , Proteínas de Membrana/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Sítios de Ligação , Progressão da Doença , Inibidores Enzimáticos/farmacologia , Feminino , Células HEK293 , Histona Desmetilases/antagonistas & inibidores , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A/fisiologia , Proteínas de Membrana/química , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Metilação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Modelos Biológicos , Infecções por Orthomyxoviridae/etiologia , Infecções por Orthomyxoviridae/metabolismo , Proteínas de Ligação a RNA/química , Tranilcipromina/farmacologia , Vírus da Estomatite Vesicular Indiana/patogenicidade , Vírus da Estomatite Vesicular Indiana/fisiologia , Replicação Viral , Zika virus/patogenicidade , Zika virus/fisiologia
6.
Cell Death Dis ; 8(10): e3149, 2017 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-29072700

RESUMO

Aneuploidy including trisomy results in developmental disabilities and is the leading cause of miscarriages in humans. Unlike trisomy 21, pathogenic mechanisms of trisomy 18 remain unclear. Here, we successfully generated induced pluripotent stem cells (iPSCs) from human amniotic fluid cells (AFCs) with trisomy 18 pregnancies. We found that trisomy 18 iPSCs (18T-iPSCs) were prone to differentiate spontaneously. Intriguingly, 18T-iPSCs lost their extra 18 chromosomes and converted to diploid cells after 10 generations. fluorescence in situ hybridization analysis showed chromosome loss was a random event that might happen in any trisomic cells. Selection undifferentiated cells for passage accelerated the recovery of euploid cells. Overall, our findings indicate the genomic instability of trisomy 18 iPSCs bearing an extra chromosome 18.


Assuntos
Cromossomos/genética , Hibridização in Situ Fluorescente/métodos , Células-Tronco Pluripotentes Induzidas/patologia , Síndrome da Trissomía do Cromossomo 18/genética , Diferenciação Celular , Cromossomos/metabolismo , Humanos , Síndrome da Trissomía do Cromossomo 18/metabolismo
7.
Curr Opin Genet Dev ; 46: 141-148, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28806594

RESUMO

Embryonic stem cells (ESCs) are characterized by their ability of unlimited self-renewal in vitro and pluripotent developmental potential, which endows them with great values in basic research and future clinical application. However, realization of full potential of ESCs is dependent on the elucidation of molecular mechanisms governing ESCs, among which signaling pathways play critical roles. A great deal of efforts has been made in the past decades to understand what and how signaling pathways contribute to the establishment and maintenance of pluripotency. In this review, we discuss signaling networks in both mouse and human ESCs, focusing on signals involved in the control of self-renewal and differentiation. In addition, the modulation of signaling pathways by pluripotency-associated transcription factors is also briefly summarized.


Assuntos
Diferenciação Celular/genética , Autorrenovação Celular/genética , Transdução de Sinais/genética , Fatores de Transcrição/genética , Animais , Células-Tronco Embrionárias , Humanos , Camundongos
9.
Oncotarget ; 8(5): 7900-7913, 2017 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-27926491

RESUMO

Alzheimer's disease (AD) is the most common age-related dementia characterized by progressive neuronal loss. However, the molecular mechanisms for the neuronal loss is still debated. Here, we used induced pluripotent stem cells (iPSCs) derived from somatic cells of familial AD patients carrying PSEN1 mutations to study the early pathogenic event of AD. We found that premature neuronal differentiation with decreased proliferation and increased apoptosis occured in AD-iPSC-derived neural progenitor cells (AD-NPCs) once neuronal differentiation was initiated, together with higher levels of Aß42 and phosphorylated tau. Premature neuronal differentiation in AD-NPCs was caused by PSEN1 mutations and might be correlated to multiple dysregulated processes including but not limited to Wnt-Notch pathway. Our study documented previously unappreciated early NPC dysfunction in AD-NPCs, providing valuable new insights into the early mechanisms underlying AD pathogenesis.


Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Mutação , Células-Tronco Neurais/patologia , Presenilina-1/genética , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Apoptose , Linhagem Celular , Proliferação de Células , Feminino , Marcadores Genéticos , Predisposição Genética para Doença , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Pessoa de Meia-Idade , Células-Tronco Neurais/metabolismo , Neurogênese , Fragmentos de Peptídeos/metabolismo , Fenótipo , Fosforilação , Fatores de Tempo , Proteínas tau/metabolismo
10.
Artigo em Inglês | MEDLINE | ID: mdl-27574312

RESUMO

Although modern techniques such as two-photon microscopy can now provide cellular-level three-dimensional imaging of the intact living brain, the speed and fields of view of these techniques remain limited. Conversely, two-dimensional wide-field optical mapping (WFOM), a simpler technique that uses a camera to observe large areas of the exposed cortex under visible light, can detect changes in both neural activity and haemodynamics at very high speeds. Although WFOM may not provide single-neuron or capillary-level resolution, it is an attractive and accessible approach to imaging large areas of the brain in awake, behaving mammals at speeds fast enough to observe widespread neural firing events, as well as their dynamic coupling to haemodynamics. Although such wide-field optical imaging techniques have a long history, the advent of genetically encoded fluorophores that can report neural activity with high sensitivity, as well as modern technologies such as light emitting diodes and sensitive and high-speed digital cameras have driven renewed interest in WFOM. To facilitate the wider adoption and standardization of WFOM approaches for neuroscience and neurovascular coupling research, we provide here an overview of the basic principles of WFOM, considerations for implementation of wide-field fluorescence imaging of neural activity, spectroscopic analysis and interpretation of results.This article is part of the themed issue 'Interpreting BOLD: a dialogue between cognitive and cellular neuroscience'.


Assuntos
Mapeamento Encefálico/métodos , Encéfalo/fisiologia , Neurônios/fisiologia , Imagem Óptica/métodos , Animais , Encéfalo/irrigação sanguínea , Encéfalo/diagnóstico por imagem , Mapeamento Encefálico/instrumentação , Hemodinâmica , Humanos , Camundongos , Imagem Óptica/instrumentação , Ratos
11.
Stem Cell Res Ther ; 6: 219, 2015 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-26553210

RESUMO

INTRODUCTION: Degenerative retinal diseases like age-related macular degeneration (AMD) are the leading cause of blindness. Cell transplantation showed promising therapeutic effect for such diseases, and embryonic stem cell (ESC) is one of the sources of such donor cells. Here, we aimed to generate retinal progenitor cells (RPCs) from rat ESCs (rESCs) and to test their therapeutic effects in rat model. METHODS: The rESCs (DA8-16) were cultured in N2B27 medium with 2i, and differentiated to two types of RPCs following the SFEBq method with modifications. For rESC-RPC1, the cells were switched to adherent culture at D10, while for rESC-RPC2, the suspension culture was maintained to D14. Both RPCs were harvested at D16. Primary RPCs were obtained from P1 SD rats, and some of them were labeled with EGFP by infection with lentivirus. To generate Rax::EGFP knock-in rESC lines, TALENs were engineered to facilitate homologous recombination in rESCs, which were cotransfected with the targeting vector and TALEN vectors. The differentiated cells were analyzed with live image, immunofluorescence staining, flow cytometric analysis, gene expression microarray, etc. RCS rats were used to mimic the degeneration of retina and test the therapeutic effects of subretinally transplanted donor cells. The structure and function of retina were examined. RESULTS: We established two protocols through which two types of rESC-derived RPCs were obtained and both contained committed retina lineage cells and some neural progenitor cells (NPCs). These rESC-derived RPCs survived in the host retinas of RCS rats and protected the retinal structure and function in early stage following the transplantation. However, the glia enriched rESC-RPC1 obtained through early and longer adherent culture only increased the b-wave amplitude at 4 weeks, while the longer suspension culture gave rise to evidently neuronal differentiation in rESC-RPC2 which significantly improved the visual function of RCS rats. CONCLUSIONS: We have successfully differentiated rESCs to glia enriched RPCs and retinal neuron enriched RPCs in vitro. The retinal neuron enriched rESC-RPC2 protected the structure and function of retina in rats with genetic retinal degeneration and could be a candidate cell source for treating some degenerative retinal diseases in human trials.


Assuntos
Células-Tronco Embrionárias/citologia , Retina/citologia , Degeneração Retiniana/cirurgia , Transplante de Células-Tronco , Animais , Antígenos de Diferenciação/metabolismo , Diferenciação Celular/genética , Movimento Celular , Sobrevivência Celular , Células Cultivadas , Células-Tronco Embrionárias/transplante , Perfilação da Expressão Gênica , Marcação de Genes , Técnicas In Vitro , Neurônios/citologia , Ratos , Ratos Sprague-Dawley , Retina/cirurgia , Células-Tronco/citologia , Células-Tronco/metabolismo , Visão Ocular
12.
Cell Stem Cell ; 17(2): 204-12, 2015 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-26253202

RESUMO

Neuronal conversion from human fibroblasts can be induced by lineage-specific transcription factors; however, the introduction of ectopic genes limits the therapeutic applications of such induced neurons (iNs). Here, we report that human fibroblasts can be directly converted into neuronal cells by a chemical cocktail of seven small molecules, bypassing a neural progenitor stage. These human chemical-induced neuronal cells (hciNs) resembled hiPSC-derived neurons and human iNs (hiNs) with respect to morphology, gene expression profiles, and electrophysiological properties. This approach was further applied to generate hciNs from familial Alzheimer's disease patients. Taken together, our transgene-free and chemical-only approach for direct reprogramming of human fibroblasts into neurons provides an alternative strategy for modeling neurological diseases and for regenerative medicine.


Assuntos
Doença de Alzheimer/patologia , Fibroblastos/patologia , Neurônios/citologia , Bibliotecas de Moléculas Pequenas/farmacologia , Adulto , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Masculino , Neurônios/efeitos dos fármacos
13.
Cell Res ; 23(10): 1187-200, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23856644

RESUMO

Recent success in the derivation of haploid embryonic stem cells (haESCs) from mouse via parthenogenesis and androgenesis has enabled genetic screening in mammalian cells and generation of gene-modified animals. However, whether haESCs can be derived from primates remains unknown. Here, we report the derivation of haESCs from parthenogenetic blastocysts of Macaca fascicularis monkeys. These cells, termed as PG-haESCs, are pluripotent and can differentiate to cells of three embryonic germ layers in vitro or in vivo. Interestingly, the haploidy of one monkey PG-haESC line (MPH1) is more stable compared with that of the other one (MPH2), as shown by the existence of haploid cells for more than 140 days without fluorescence-activated cell sorting (FACS) enrichment of haploid cells. Importantly, transgenic monkey PG-haESC lines can be generated by lentivirus- and piggyBac transposon-mediated gene transfer. Moreover, genetic screening is feasible in monkey PG-haESCs. Our results demonstrate that PG-haESCs can be generated from monkeys, providing an ideal tool for genetic analyses in primates.


Assuntos
Células-Tronco Embrionárias/citologia , Haploidia , Macaca fascicularis/embriologia , Animais , Blastocisto/citologia , Diferenciação Celular , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Macaca fascicularis/genética , Partenogênese
14.
Ann Neurol ; 73(1): 104-19, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23225543

RESUMO

OBJECTIVE: Although amyloid-beta (Aß) peptide deposition into insoluble plaques is a pathological hallmark of Alzheimer disease; soluble oligomeric Aß has been hypothesized to more directly underlie impaired learning and memory in dementia of the Alzheimer type. However, the lack of a sensitive, specific, and quantitative assay for Aß oligomers has hampered rigorous tests of this hypothesis. METHODS: We developed a plate-based single molecule counting fluorescence immunoassay for oligomeric Aß sensitive to low pg/ml concentrations of synthetic Aß dimers using the same Aß-specific monoclonal antibody to both capture and detect Aß. The Aß oligomer assay does not recognize monomeric Aß, amyloid precursor protein, or other non-Aß peptide oligomers. RESULTS: Aß oligomers were detected in aqueous cortical lysates from patients with dementia of the Alzheimer type and nondemented patients with Aß plaque pathology. However, Aß oligomer concentrations in demented patients' lysates were tightly correlated with Aß plaque coverage (r = 0.88), but this relationship was weaker in those from nondemented patients (r = 0.30) despite equivalent Aß plaque pathology. The ratio of Aß oligomer levels to plaque density fully distinguished demented from nondemented patients, with no overlap between groups in this derived variable. Other Aß and plaque measures did not distinguish demented from nondemented patients. Aß oligomers were not detected in cerebrospinal fluid with this assay. INTERPRETATION: The results raise the intriguing hypothesis that the linkage between plaques and oligomers may be a key pathophysiological event underlying dementia of the Alzheimer type. This Aß oligomer assay may be useful for many tests of the oligomer hypothesis.


Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Idoso de 80 Anos ou mais , Doença de Alzheimer/diagnóstico , Córtex Cerebral/química , Feminino , Humanos , Masculino , Multimerização Proteica/fisiologia
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