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1.
Circ Cardiovasc Qual Outcomes ; 16(4): e009524, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-37013814

RESUMO

BACKGROUND: Sudden cardiac death (SCD) in younger individuals is frequently caused by heritable cardiac conditions. The unexpected nature of SCD leaves families with many unanswered questions and an insufficient understanding of the cause of death and their own risk for heritable disease. We explored the experiences of families of young SCD victims upon learning about their relative's cause of death and how they perceive their own risk for heritable cardiac conditions. METHODS: We conducted a qualitative descriptive study, by interviewing families of young (ages 12-45) SCD victims, who died between 2014 and 2018 from a heritable cardiac condition and were investigated by the Office of the Chief Coroner of Ontario, Canada. We used thematic analysis to analyze the transcripts. RESULTS: Between 2018 and 2020, we interviewed 19 family members, of which 10 were males and 9 were females, ages ranging from 21 to 65 (average 46.2±13.1). Four main themes were revealed, each representing a distinct time period that families experience along a trajectory: (1) interactions between bereaved family and others, in particular coroners, shaped their search for answers about their relative's cause of death, with the types, formats, and timing of communication varying by case; (2) searching for answers and processing the cause of death; (3) incidental implications of the SCD event, such as financial strain and lifestyle changes contributed to cumulative stress; (4) receiving answers (or not) and moving forward. CONCLUSIONS: Families rely on communication with others, yet the type, formats, and timing of information received varies, which can influence families' experiences of processing the death (and its cause), their perceived risk and their decision to pursue cascade screening. These results may provide key insights for the interprofessional health care team responsible for the delivery and communication of the cause of death to families of SCD victims.


Assuntos
Atitude Frente a Morte , Morte Súbita Cardíaca , Família , Pesar , Humanos , Morte Súbita Cardíaca/epidemiologia , Ontário/epidemiologia , Masculino , Feminino , Adulto Jovem , Adulto , Pessoa de Meia-Idade , Idoso , Família/psicologia , Entrevista Psicológica
2.
Dev Biol ; 455(2): 382-392, 2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31315026

RESUMO

Estrogen related receptor beta (Esrrb) is an orphan nuclear receptor that is required for self-renewal and pluripotency in mouse embryonic stem (ES) cells. However, in the early post-implantation mouse embryo, Esrrb is specifically expressed in the extraembryonic ectoderm (ExE) and plays a crucial role in trophoblast development. Previous studies showed that Esrrb is also required to maintain trophoblast stem (TS) cells, the in vitro stem cell model of the early trophoblast lineage. In order to identify regulatory targets of Esrrb in vivo, we performed microarray analysis of Esrrb-null versus wild-type post-implantation ExE, and identified 30 genes down-regulated in Esrrb-mutants. Among them is Bmp4, which is produced by the ExE and known to be critical for primordial germ cell (PGC) specification in vivo. We further identified an enhancer region bound by Esrrb at the Bmp4 locus by performing Esrrb ChIP-seq and luciferase reporter assay using TS cells. Finally, we established a knockout mouse line in which the enhancer region was deleted using CRISPR/Cas9 technology. Both Esrrb-null embryos and enhancer knockout embryos expressed lower levels of Bmp4 in the ExE, and had reduced numbers of PGCs. These results suggested that Esrrb functions as an upstream factor of Bmp4 in the ExE, regulating proper PGC development in mice.


Assuntos
Desenvolvimento Embrionário , Células Germinativas , Receptores de Estrogênio/fisiologia , Animais , Proteína Morfogenética Óssea 4/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Ectoderma/embriologia , Elementos Facilitadores Genéticos , Camundongos , Camundongos Knockout , Análise Serial de Proteínas
3.
Sci Rep ; 9(1): 9027, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31227724

RESUMO

Cell lineage conversion of fibroblasts to specialized cell types through transdifferentiation may provide a fast and alternative cell source for regenerative medicine. Here we show that transient transduction of fibroblasts with the four reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) in addition to the early lung transcription factor Nkx2-1 (also known as Ttf1), followed by directed differentiation of the cells, can convert mouse embryonic and human adult dermal fibroblasts into induced lung-like epithelial cells (iLEC). These iLEC differentiate into multiple lung cell types in air liquid interface cultures, repopulate decellularized rat lung scaffolds, and form lung epithelia composed of Ciliated, Goblet, Basal, and Club cells after transplantation into immune-compromised mice. As proof-of-concept, differentiated human iLEC harboring the Cystic Fibrosis mutation dF508 demonstrated pharmacological rescue of CFTR function using the combination of lumacaftor and ivacaftor. Overall, this is a promising alternative approach for generation of patient-specific lung-like progenitors to study lung function, disease and future regeneration strategies.


Assuntos
Transdiferenciação Celular , Reprogramação Celular , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Células Epiteliais/citologia , Fibroblastos/citologia , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Pulmão/citologia , Camundongos , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Medicina Regenerativa/métodos , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Fator Nuclear 1 de Tireoide/genética , Fator Nuclear 1 de Tireoide/metabolismo
4.
Nat Protoc ; 10(3): 363-81, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25654755

RESUMO

Airway epithelial cells are of great interest for research on lung development, regeneration and disease modeling. This protocol describes how to generate cystic fibrosis (CF) transmembrane conductance regulator protein (CFTR)-expressing airway epithelial cells from human pluripotent stem cells (PSCs). The stepwise approach from PSC culture to differentiation into progenitors and then mature epithelia with apical CFTR activity is outlined. Human PSCs that were inefficient at endoderm differentiation using our previous lung differentiation protocol were able to generate substantial lung progenitor cell populations. Augmented CFTR activity can be observed in all cultures as early as at 35 d of differentiation, and full maturation of the cells in air-liquid interface cultures occurs in <5 weeks. This protocol can be used for drug discovery, tissue regeneration or disease modeling.


Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células-Tronco Pluripotentes/citologia , Mucosa Respiratória/citologia , Humanos , Mucosa Respiratória/metabolismo
5.
Curr Biol ; 23(13): 1195-201, 2013 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-23791728

RESUMO

During mammalian development, the first two lineages to be specified are the trophectoderm (TE) and the inner cell mass (ICM). The Hippo pathway kinases Lats 1 and 2 (Lats1/2) and the transcriptional coactivator Yap play important roles in this specification process [1]. In outside cells of the embryo, Yap is nuclear localized and cooperates with Tead4 to induce the TE-specifying transcription factor Cdx2. In inside cells, Lats1/2 phosphorylate Yap and prevent its nuclear localization. The factors acting upstream of Lats1/2 and Yap in this context have not been identified. Here, we demonstrate that the upstream Hippo pathway member Nf2/Merlin is required for Lats1/2-dependent Yap phosphorylation in the preimplantation embryo. Injection of dominant-negative Nf2 mRNA causes Yap mislocalization and ectopic Cdx2 expression, effects that can be rescued by overexpression of Lats2 kinase. Zygotic Nf2 mutant blastocysts have mild defects in Yap localization and Cdx2 expression, but these become much more severe upon removal of both maternal and zygotic Nf2. The inside cells of maternal-zygotic mutants fail to establish a pluripotent ICM and form excess TE, resulting in peri-implantation lethality. Together, these data establish a clear role for Nf2 upstream of Yap in the preimplantation embryo and demonstrate that Hippo signaling is essential to segregate the ICM from the TE.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Regulação da Expressão Gênica no Desenvolvimento , Neurofibromina 2/genética , Fosfoproteínas/genética , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Blastocisto/metabolismo , Fator de Transcrição CDX2 , Proteínas de Ciclo Celular , Via de Sinalização Hippo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Neurofibromina 2/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Reação em Cadeia da Polimerase , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Sinalização YAP
6.
Dev Cell ; 22(4): 887-901, 2012 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-22424930

RESUMO

The advent of reprogramming and its impact on stem cell biology has renewed interest in lineage restriction in mammalian embryos, the source of embryonic (ES), epiblast (EpiSC), trophoblast (TS), and extraembryonic endoderm (XEN) stem cell lineages. Isolation of specific cell types during stem cell differentiation and reprogramming, and also directly from embryos, is a major technical challenge because few cell-surface proteins are known that can distinguish each cell type. We provide a large-scale proteomic resource of cell-surface proteins for the four embryo-derived stem cell lines. We validated 27 antibodies against lineage-specific cell-surface markers, which enabled investigation of specific cell populations during ES-EpiSC reprogramming and ES-to-XEN differentiation. Identified markers also allowed prospective isolation and characterization of viable lineage progenitors from blastocysts by flow cytometry. These results provide a comprehensive stem cell proteomic resource and enable new approaches to interrogate the mechanisms that regulate cell fate specification.


Assuntos
Biomarcadores/metabolismo , Linhagem da Célula , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/metabolismo , Endoderma/metabolismo , Proteômica , Trofoblastos/metabolismo , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Western Blotting , Diferenciação Celular , Células Cultivadas , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Endoderma/citologia , Citometria de Fluxo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Trofoblastos/citologia
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