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1.
Development ; 148(6)2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33722957

RESUMO

The peri-implantation window of mammalian development is the crucial window for primordial germ cell (PGC) specification. Whereas pre-implantation dynamics are relatively conserved between species, the implantation window marks a stage of developmental divergence between key model organisms, and thus potential variance in the cell and molecular mechanisms for PGC specification. In humans, PGC specification is very difficult to study in vivo To address this, the combined use of human and nonhuman primate embryos, and stem cell-based embryo models are essential for determining the origin of PGCs, as are comparative analyses to the equivalent stages of mouse development. Understanding the origin of PGCs in the peri-implantation embryo is crucial not only for accurate modeling of this essential process using stem cells, but also in determining the role of global epigenetic reprogramming upon which sex-specific differentiation into gametes relies.


Assuntos
Células Germinativas/metabolismo , Animais , Diferenciação Celular , Metilação de DNA , Desenvolvimento Embrionário , Células-Tronco Embrionárias/classificação , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células Germinativas/classificação , Células Germinativas/citologia , Humanos , Modelos Biológicos , Cromossomo X/genética , Cromossomo X/metabolismo
3.
Genome Biol ; 17: 29, 2016 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-26887813

RESUMO

Single-cell RNA sequencing (scRNA-seq) has broad applications across biomedical research. One of the key challenges is to ensure that only single, live cells are included in downstream analysis, as the inclusion of compromised cells inevitably affects data interpretation. Here, we present a generic approach for processing scRNA-seq data and detecting low quality cells, using a curated set of over 20 biological and technical features. Our approach improves classification accuracy by over 30 % compared to traditional methods when tested on over 5,000 cells, including CD4+ T cells, bone marrow dendritic cells, and mouse embryonic stem cells.


Assuntos
Sequência de Bases/genética , RNA/genética , Análise de Célula Única , Animais , Células da Medula Óssea/classificação , Linfócitos T CD4-Positivos/classificação , Células Dendríticas/classificação , Células-Tronco Embrionárias/classificação , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos
5.
Nat Biotechnol ; 33(11): 1165-72, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26458175

RESUMO

Chromatin profiling provides a versatile means to investigate functional genomic elements and their regulation. However, current methods yield ensemble profiles that are insensitive to cell-to-cell variation. Here we combine microfluidics, DNA barcoding and sequencing to collect chromatin data at single-cell resolution. We demonstrate the utility of the technology by assaying thousands of individual cells and using the data to deconvolute a mixture of ES cells, fibroblasts and hematopoietic progenitors into high-quality chromatin state maps for each cell type. The data from each single cell are sparse, comprising on the order of 1,000 unique reads. However, by assaying thousands of ES cells, we identify a spectrum of subpopulations defined by differences in chromatin signatures of pluripotency and differentiation priming. We corroborate these findings by comparison to orthogonal single-cell gene expression data. Our method for single-cell analysis reveals aspects of epigenetic heterogeneity not captured by transcriptional analysis alone.


Assuntos
Imunoprecipitação da Cromatina/métodos , Células-Tronco Embrionárias/classificação , Células-Tronco Embrionárias/citologia , Análise de Célula Única/métodos , Animais , Cromatina/genética , Biologia Computacional , Código de Barras de DNA Taxonômico , Humanos , Camundongos , Técnicas Analíticas Microfluídicas , Análise de Sequência de DNA
6.
Mamm Genome ; 26(9-10): 448-55, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26373861

RESUMO

The availability of and access to quality genetically defined, health-status known mouse resources is critical for biomedical research. By ensuring that mice used in research experiments are biologically, genetically, and health-status equivalent, we enable knowledge transfer, hypothesis building based on multiple data streams, and experimental reproducibility based on common mouse resources (reagents). Major repositories for mouse resources have developed over time and each has significant unique resources to offer. Here we (a) describe The International Mouse Strain Resource that offers users a combined catalog of worldwide mouse resources (live, cryopreserved, embryonic stem cells), with direct access to repository sites holding resources of interest and (b) discuss the commitment to nomenclature standards among resources that remain a challenge in unifying mouse resource catalogs.


Assuntos
Pesquisa Biomédica , Linhagem Celular/classificação , Células-Tronco Embrionárias/classificação , Camundongos Endogâmicos/classificação , Animais , Catalogação , Humanos , Internet , Camundongos
7.
Mamm Genome ; 26(9-10): 456-66, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26340938

RESUMO

The International Knockout Mouse Consortium (IKMC; http://www.mousephenotype.org ) has generated mutations in almost every protein-coding mouse gene and is completing the companion Cre driver resource to expand tissue-specific conditional mutagenesis. Accordingly, the IKMC has carried out high-throughput gene trapping and targeting producing conditional mutations in murine embryonic stem cells in more than 18,500 genes, from which at least 4900 mutant mouse lines have been established to date. This resource is currently being upgraded with more powerful tools, such as visualization and manipulation cassettes that can be easily introduced into IKMC alleles for multifaceted functional studies. In addition, we discuss how existing IKMC products can be used in combination with CRISPR technology to accelerate genome engineering projects. All information and materials from this extraordinary biological resource together with coordinated phenotyping efforts can be retrieved at www.mousephenotype.org . The comprehensive IKMC knockout resource in combination with an extensive set of modular gene cassettes will continue to enhance functional gene annotation in the future and solidify its impact on biomedical research.


Assuntos
Células-Tronco Embrionárias/classificação , Camundongos Knockout/classificação , Anotação de Sequência Molecular , Animais , Sistemas CRISPR-Cas/genética , Cooperação Internacional , Camundongos , Mutação
8.
Proc Natl Acad Sci U S A ; 111(34): 12438-43, 2014 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-25114218

RESUMO

The apical domain of embryonic (radial glia) and adult (B1 cells) neural stem cells (NSCs) contains a primary cilium. This organelle has been suggested to function as an antenna for the detection of morphogens or growth factors. In particular, primary cilia are essential for Hedgehog (Hh) signaling, which plays key roles in brain development. Their unique location facing the ventricular lumen suggests that primary cilia in NSCs could play an important role in reception of signals within the cerebrospinal fluid. Surprisingly, ablation of primary cilia using conditional alleles for genes essential for intraflagellar transport [kinesin family member 3A (Kif3a) and intraflagellar transport 88 (Ift88)] and Cre drivers that are activated at early [Nestin; embryonic day 10.5 (E10.5)] and late [human glial fibrillary acidic protein (hGFAP); E13.5] stages of mouse neural development resulted in no apparent developmental defects. Neurogenesis in the ventricular-subventricular zone (V-SVZ) shortly after birth was also largely unaffected, except for a restricted ventral domain previously known to be regulated by Hh signaling. However, Kif3a and Ift88 genetic ablation also disrupts ependymal cilia, resulting in hydrocephalus by postnatal day 4. To directly study the role of B1 cells' primary cilia without the confounding effects of hydrocephalus, we stereotaxically targeted elimination of Kif3a from a subpopulation of radial glia, which resulted in ablation of primary cilia in a subset of B1 cells. Again, this experiment resulted in decreased neurogenesis only in the ventral V-SVZ. Primary cilia ablation led to disruption of Hh signaling in this subdomain. We conclude that primary cilia are required in a specific Hh-regulated subregion of the postnatal V-SVZ.


Assuntos
Cílios/fisiologia , Células-Tronco Neurais/classificação , Células-Tronco Neurais/ultraestrutura , Animais , Animais Recém-Nascidos , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Proliferação de Células , Células-Tronco Embrionárias/classificação , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/ultraestrutura , Feminino , Técnicas de Silenciamento de Genes , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas Hedgehog/fisiologia , Humanos , Cinesinas/antagonistas & inibidores , Cinesinas/genética , Cinesinas/metabolismo , Camundongos , Camundongos Transgênicos , Nestina/genética , Nestina/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese/fisiologia , Gravidez , Transdução de Sinais , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
9.
Biomaterials ; 35(23): 5987-97, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24780170

RESUMO

Human embryonic stem cells (hESCs) are generally induced to differentiate by forming spherical structures termed embryoid bodies (EBs) in the presence of soluble growth factors. hEBs are generated by suspending small clumps of hESC colonies; however, the resulting hEBs are heterogeneous because this method lacks the ability to control the number of cells in individual EBs. This heterogeneity affects factors that influence differentiation such as cell-cell contact and the diffusion of soluble factors, and consequently, the differentiation capacity of each EB varies. Here, we fabricated size-tunable concave microwells to control the physical environment, thereby regulating the size of EBs formed from single hESCs. Defined numbers of single hESCs were forced to aggregate and generate uniformly sized EBs with high fidelity, and the size of the EBs was controlled using concave microwells of different diameters. Differentiation patterns in H9- and CHA15-hESCs were affected by EB size in both the absence and presence of growth factors. By screening EB size in the presence of various BMP4 concentrations, a two-fold increase in endothelial cell differentiation was achieved. Because each hESC line has unique characteristics, the findings of this study demonstrate that concave microwells could be used to screen different EB sizes and growth factor concentrations to optimize differentiation for each hESC line.


Assuntos
Separação Celular/instrumentação , Separação Celular/métodos , Corpos Embrioides/citologia , Corpos Embrioides/fisiologia , Células-Tronco Embrionárias/classificação , Microfluídica/instrumentação , Microfluídica/métodos , Reatores Biológicos , Linhagem Celular , Corpos Embrioides/classificação , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos
10.
DNA Res ; 20(4): 391-402, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23649898

RESUMO

Mouse embryonic stem (ES) cells are prototypical stem cells that remain undifferentiated in culture for long periods, yet maintain the ability to differentiate into essentially all cell types. Previously, we have reported that ES cells oscillate between two distinct states, which can be distinguished by the transient expression of Zscan4 genes originally identified for its specific expression in mouse two-cell stage embryos. Here, we report that the nascent protein synthesis is globally repressed in the Zscan4-positive state of ES cells, which is mediated by the transient expression of newly identified eukaryotic translation initiation factor 1A (Eif1a)-like genes. Eif1a-like genes, clustered on Chromosome 12, show the high sequence similarity to the Eifa1 and consist of 10 genes (Eif1al1-Eif1al10) and 9 pseudogenes (Eif1al-ps1-Eif1al-ps9). The analysis of the expressed sequence tag database showed that Eif1a-like genes are expressed mostly in the two-cell stage mouse embryos. Microarray analyses and quantitative real-time polymerase chain reaction analyses show that Eif1a-like genes are expressed specifically in the Zscan4-positive state of ES cells. These results indicate a novel mechanism to repress protein synthesis by Eif1a-like genes and a unique mode of protein synthesis regulation in ES cells, which undergo a transient and reversible repression of global protein synthesis in the Zscan4-positive state.


Assuntos
Células-Tronco Embrionárias/metabolismo , Fator de Iniciação 1 em Eucariotos/genética , Regulação da Expressão Gênica no Desenvolvimento , Biossíntese de Proteínas , Fatores de Transcrição/genética , Animais , Cromossomos de Mamíferos , Embrião de Mamíferos , Células-Tronco Embrionárias/classificação , Células-Tronco Embrionárias/citologia , Fator de Iniciação 1 em Eucariotos/classificação , Fator de Iniciação 1 em Eucariotos/metabolismo , Etiquetas de Sequências Expressas , Camundongos , Família Multigênica , Isoformas de Proteínas/classificação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fatores de Transcrição/metabolismo
11.
São Paulo; s.n; s.n; 2013. 198 p. tab, graf, ilus.
Tese em Português | LILACS | ID: biblio-846927

RESUMO

Algumas das estratégias utilizadas para entender a biologia de células tronco embrionária (CTE) são baseadas na identificação de cascatas de sinalização que induzem a diferenciação e auto-renovação das CTE através da interferência seletiva de processos específicos. A família das proteínas quinase C (PKC) é conhecida por participar dos processos de auto-renovação e diferenciação celular em CTE, entretanto, o papel específico das diferentes isoenzimas das PKCs ainda precisa ser elucidado. Desta forma investigamos. o papel das PKCs atípicas (aPKCs) em CTE indiferenciadas utilizando um inibidor específico para estas serina/ treonina quinases, o peptídeo pseudossubstrato das aPKCs, e fosfoproteômica. A maioria das proteinas identificadas cuja fosforilação reduziu após o tratamento com o inibidor das aPKC, são proteínas envolvidas com o metabolismo principalmente com a via glicolítica. Além disso, a inibição das aPKCs levou a redução do consumo de glicose, secreção de lactato, acompanhada da redução da atividade da lactato desidrogenase, e aumento da fosforilação oxidativa, sendo analisada através do consumo de oxigênio após o tratamento com oligomicina e FCCP. Verificamos também que as aPKCs são capazes de fosforilar diretamente a piruvato quinase. A glicólise aeróbica parece ser fundamental para a manutenção da indiferenciação das CTE, e demonstramos que as aPKCs participam deste processo auxiliando na auto-renovação das CTE indiferenciadas. Também observamos que as aPKCs assim como a PKCßI modulam a fosforilação da α-tubulina, porém ao passo que as aPKCs interagem com a α-tubulina durante a interfase, a PKCßI interage com a mesma apenas durate a mitose. Estes resultados motivaram a segunda parte da tese, na qual o papel da fosforilação da α-tubulina pela PKCßI foi investigado. O resíduo de treonina 253, conservado em diversas espécies de vertebrados e localizado na interface de polimerização entre a α- e a ß-tubulina foi identificado, como um novo sítio de fosforilação da α-tubulina pela PKCßI. Este sítio não está em um consenso linear para a PKC, entretanto é um consenso formado estruturalmente, onde aminoácidos básicos distantes na sequência linear se tornam justapostos na estrutura terciária da proteína. Estudos de simulação por dinâmica molecular demonstraram que a interação entre a α e ß-tubulina aumenta após esta fosforilação, uma vez que T253 fosforilada passa a interagir com K105, um residuo conservado na ß-tubulina. A fosforilação in vitro de α-tubulina aumenta a taxa de polimerização da tubulina e a inibição da PKCßI em células reduziu a taxa de repolimerização do microtubulo após o tratamento com nocodazol. Além disso, a importância da fosforilação deste sítio foi demonstrada pelo fato de que um mutante fosfomimético GFP-α-tubulina, T253E ser mais incorporado no fuso mitótico ao passo que T253A foi menos incorporado do que a proteína selvagem. Nossos dados suportam a hipótese que os consensos estruturais formados podem ser importantes sítios de reconhecimento pelas quinases e que a fosforilação de T253 da α-tubulina afeta a estabilidade do polímero. Em conclusão, utilizando métodos de fosfoproteômica e interferência seletiva de vias de sinalização, combinados a validações experimentais dos alvos identificados podemos propor a importância funcional das aPKCs e PKCßI em CTE indiferenciadas


Some of the strategies used to understand stem cell biology are based on the identification of signalling cascades that lead to differentiation and self-renewal of embryonic stem cells (ESC) by selective interference of specific signalling processes. The protein kinase C (PKC) family is known to participate in ESC self-renewal and differentiation, however, the specific role of the different PKC isoenzymes in these cells remains to be determined. Therefore, we investigated the role of atypical PKCs (aPKC) in undifferntiated ESC using a specific inhibitor for these serine/ threonine kinases, pseudo-substrate peptide of aPKCs, and phosphoproteomics. The majority of proteins whose phosphorylation decreased upon aPKC inhibition, are proteins involved in metabolism in particular with the glycolytic pathway. Besides that, inhibiton of aPKCs led to a decrease in glucose uptake and lactate secretion, followed by a decrease in lactate dehydrogenase activity, and an increase in mitochondrial activity as measured by oxygen consumption after treatment with olygomycin and a chemical uncoupler. We also verified that aPKCs are able to directly phosphorylated pyruvate kinase. Aerobic glicolysis seems to be fundamental for the maintainance of undifferentiated ESC, and we demonstrated that aPKCs participte in these processes helping to maintain self-renewal of undifferentiated ESC. We also observed that aPKCs as PKCßI modulate the phosphorylation of α-tubulin, however, while aPKCs interact with α-tubulin during interfase PKCßI interacts with α-tubulin only during mitosis. These results lead to the second part of this thesis. We investigated the role of α-tubulina phosphorylation by PKCßI. Indentifying threonine 253, a conserved residue in several vertebrate species, of localized at the polymerization interface between α- and ß-tubulin, as a phosphorylation site of α-tubulin by PKCßI. This site is not in a linear consensus for PKC, however, it is in a structuraly formed consensus, where basic aminoacids distant in the linear sequence are juxtaposed in the three dimentional protein structure. Simulation studies by molecular dynamics show that the interaction between α and ß-tubulin increases upon this phosphorylation, once, phosphorylated T253 interacts with com K105, a conserved residue in ß-tubulin. The in vitro phosphorylation of α-tubulin increased tubulin polymerization rate and inhibiton of PKCßI in cells reduced repolimeration rate of microtubles upon treatment with nocodazole. Besides that, the importance of this phosphorylation site were demonstrated by the fact that a phosphomimetic mutant GFP-α-tubulina, T253E is more incorporated in mitotic fuses while T253A is less than wild type. Our data support the hypothesis that structural consensus may be important sites recognized and that T253 phosphorylation of α-tubulin afects the polymer stability. In conclusion, using phosphoproteomics methods and selective interference of signal transduction pathways combined with experimental validation studies of the identified targets we can propose roles for aPKCs and PKCßI in undifferentiated ESC


Assuntos
Células-Tronco Embrionárias/classificação , Proteína Quinase C beta/análise , Estudo de Validação , Fracionamento Celular/métodos , Metabolismo/genética , Nocodazol/análise , Fosforilação/genética , Proteína Quinase C/análise , Remodelação do Consumo , Tubulinos/crescimento & desenvolvimento , Eletroforese em Gel Diferencial Bidimensional/métodos
12.
São Paulo; s.n; s.n; 2013. 112 p. tab, graf, ilus.
Tese em Português | LILACS | ID: biblio-846936

RESUMO

Fontes alternativas de células ß têm sido estudadas para o tratamento de Diabetes mellitus tipo 1, dentre as quais a mais promissora consiste das células-tronco diferenciadas em células produtoras de insulina (IPCs). Alguns trabalhos demonstram a capacidade de células-tronco embrionárias murinas (mESCs) de formarem estruturas semelhantes a ilhotas pancreáticas, porém, os níveis de produção de insulina são insuficientes para a reversão do diabetes em camundongos diabetizados. Este trabalho visa desenvolver um protocolo adequado para geração de IPCs e contribuir para a identificação e caracterização funcional de novos genes associados à organogênese pancreática. Logo no início da diferenciação das mESCs em IPCs, foi possível verificar o surgimento de células progenitoras, evidenciado pela expressão de marcadores importantes da diferenciação beta-pancreática. Ao final do processo de diferenciação in vitro, ocorreu a formação de agrupamentos (clusters) semelhantes a ilhotas, corando positivamente por ditizona, que é específica para células ß-pancreáticas. Para avaliar seu potencial in vivo, estes clusters foram microencapsulados em Biodritina® e transplantados em camundongos diabetizados. Apesar dos níveis de insulina produzidos não serem suficientes para estabelecer a normoglicemia, os animais tratados com IPCs apresentaram melhores condições, quando comparados ao grupo controle, tendo melhor controle glicêmico, ganho de massa corpórea e melhor aparência da pelagem, na ausência de apatia. Além disso, análise dos clusters transplantados nestes animais indicou aumento da expressão de genes relacionados à maturação das células ß. Porém, quando estes clusters foram microencapsuladas em Bioprotect® e submetidos à maturação in vivo em animais normais, ocorreu um aumento drástico na expressão de todos os genes analisados, indicando sua maturação completa em células beta. O transplante destas células completamente maturadas em animais diabetizados, tornou-os normoglicêmicos e capazes de responder ao teste de tolerância à glicose (OGTT) de forma semelhante aos animais normais. A segunda parte do trabalho visou analisar genes diferencialmente expressos identificados em estudo anterior do nosso grupo, comparando, através de DNA microarray, mESCs indiferenciadas e diferenciadas em IPCs. Um dos genes diferencialmente expressos é aquele que codifica para a Purkinge cell protein 4 (Pcp4), sendo 3.700 vezes mais expresso em IPCs. Para investigar o possível papel do gene Pcp4 em células ß e no processo de diferenciação ß-pancreática, adotou-se o enfoque de genômica funcional, superexpressando e inibindo sua expressão em células MIN-6 e mESCs. Apesar da alteração na expressão de Pcp4 em células MIN-6 não ter interferido de forma expressiva na expressão dos genes analisados, quando inibido, modificou o perfil da curva de crescimento celular, aumentando seu tempo de dobramento de forma significativa e diminuindo da viabilidade celular em ensaios de indução de apoptose. Já na diferenciação de mESCs em IPCs, a superexpressão de Pcp4 interferiu de forma positiva apresentando uma tendência a aumentar a expressão dos genes relacionado à diferenciaçãoß-pancreática. Concluindo, desenvolvemos um novo protocolo de diferenciação de mESCs em IPCs as quais foram capazes de reverter o diabetes em camundongos diabetizados e descrevemos, pela primeira vez, o gene Pcp4 como sendo expresso em células ß-pancreáticas, podendo estar relacionado à manutenção da viabilidade celular e maturação destas células


New cellular sources for type 1 Diabetes mellitus treatment have been previously investigated, the most promising of which seems to be the insulin producing cells (IPCs), obtained by stem cells differentiation. Some reports show that murine embryonic stem cells (mESCs) are able to form islet-like structures, however, their insulin production is insufficient to render diabetic mice normoglycemic. This work aims at developing an adequate protocol for generation of IPCs and searching for new genes which could be involved in the pancreatic organogenesis process. Early on during mESCs differentiation into IPCs, we observed the presence of progenitor cells, which were able to express pancreatic ß-cell markers. At the end of the differentiation process, the islet-like clusters positively stained for the insulin-specific dithizone. These clusters were microencapsulated in Biodritin® microcapsules, and then transplanted into diabetized mice. Although the levels of insulin production were insufficient for the animals to achieve normoglycemia, those which received IPCs displayed improved conditions, when compared to the control group, as judged by a better glycemic control, body weight gain and healthy fur appearance, in the absence of apathy. In addition, when these transplantated clusters were retrieved, high levels of expression of the genes related to ß-cell maturation were detected. IPCs were also microencapsulated in Bioprotect® and subjected to in vivo maturation in normal animals. A dramatic increase of the analyzed genes expression was observed, indicating complete maturation of the differentiated cells. When these cells were transplanted into diabetized mice, these animals achieved normoglycemia and were able to display glucose tolerance test (OGTT) response very similar to that of normal mice. In the second part of this work, we analyzed upregulated genes described in previous work from our group, comparing undifferentiated mESCs to IPCs using a microarray platform. One of these genes is that coding for the Purkinje cell protein 4 (Pcp4), which is 3,700 more expressed than in undifferentiated mESC cells. We adopted a functional genomics approach to investigate the role played by the Pcp4 gene in ß-cells and in ß-cell differentiation, by inducing overexpression and knocking down this gene in MIN-6 and mESC cells. Although the differential expression of Pcp4 in MIN-6 was not able to interfere with the expression of the genes analyzed, we observed different cell growth rates, with increased doubling time and decreased cell viability when its expression was knocked down. In addition, overexpression of Pcp4 in mESCs subjected to differentiation into IPCs apparently increases the expression of genes related to ß-cell differentiation. In conclusion, we developed a new protocol for ESCs differentiation into IPCs, which is able to revert diabetes in diabetized mice, and we also describe here, for the first time, the Pcp4 gene as being expressed in pancreatic ß-cells and possibly being related to maintenance of cell viability and ß-cell maturation


Assuntos
Camundongos , Genes , Insulina/fisiologia , Diabetes Mellitus Tipo 1/prevenção & controle , Células-Tronco Embrionárias/classificação , Expressão Gênica , Ilhotas Pancreáticas , Biologia Molecular , Células-Tronco Embrionárias Murinas/metabolismo , Organogênese , Pâncreas , Células de Purkinje/classificação
13.
Stem Cells ; 30(12): 2732-45, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23034951

RESUMO

Embryonic (ES) and epiblast (EpiSC) stem cells are pluripotent but committed to an embryonic lineage fate. Conversely, trophoblast (TS) and extraembryonic endoderm (XEN) stem cells contribute predominantly to tissues of the placenta and yolk sac, respectively. Here we show that each of these four stem cell types is defined by a unique DNA methylation profile. Despite their distinct developmental origin, TS and XEN cells share key epigenomic hallmarks, chiefly characterized by robust DNA methylation of embryo-specific developmental regulators, as well as a subordinate role of 5-hydroxymethylation. We also observe a substantial methylation reinforcement of pre-existing epigenetic repressive marks that specifically occurs in extraembryonic stem cells compared to in vivo tissue, presumably due to continued high Dnmt3b expression levels. These differences establish a major epigenetic barrier between the embryonic and extraembryonic stem cell types. In addition, epigenetic lineage boundaries also separate the two extraembryonic stem cell types by mutual repression of key lineage-specific transcription factors. Thus, global DNA methylation patterns are a defining feature of each stem cell type that underpin lineage commitment and differentiative potency of early embryo-derived stem cells. Our detailed methylation profiles identify a cohort of developmentally regulated sequence elements, such as orphan CpG islands, that will be most valuable to uncover novel transcriptional regulators and pivotal "gatekeeper" genes in pluripotency and lineage differentiation.


Assuntos
Metilação de DNA , Células-Tronco Embrionárias/classificação , Células-Tronco Pluripotentes/classificação , Animais , Diferenciação Celular/fisiologia , Linhagem da Célula , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Endoderma/citologia , Feminino , Impressão Genômica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , Gravidez , Trofoblastos/citologia , Inativação do Cromossomo X
14.
Cancer Gene Ther ; 19(8): 517-22, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22653384

RESUMO

As stem cells are capable of self-renewal and can generate differentiated progenies for organ development, they are considered as potential source for regenerative medicine and tissue replacement after injury or disease. Along with this capacity, stem cells have the therapeutic potential for treating human diseases including cancers. According to the origins, stem cells are broadly classified into two types: embryonic stem cells (ESCs) and adult stem cells. In terms of differentiation potential, ESCs are pluripotent and adult stem cells are multipotent. Amnion, which is a membranous sac that contains the fetus and amniotic fluid and functions in protecting the developing embryo during gestation, is another stem cell source. Amnion-derived stem cells are classified as human amniotic membrane-derived epithelial stem cells, human amniotic membrane-derived mesenchymal stem cells and human amniotic fluid-derived stem cells. They are in an intermediate stage between pluripotent ESCs and lineage-restricted adult stem cells, non-tumorigenic, and contribute to low immunogenicity and anti-inflammation. Furthermore, they are easily available and do not cause any controversial issues in their recovery and applications. Not only are amnion-derived stem cells applicable in regenerative medicine, they have anticancer capacity. In non-engineered stem cells transplantation strategies, amnion-derived stem cells effectively target the tumor and suppressed the tumor growth by expressing cytotoxic cytokines. Additionally, they also have a potential as novel delivery vehicles transferring therapeutic genes to the cancer formation sites in gene-directed enzyme/prodrug combination therapy. Owing to their own advantageous properties, amnion-derived stem cells are emerging as a new candidate in anticancer therapy.


Assuntos
Células-Tronco Adultas/citologia , Âmnio/citologia , Líquido Amniótico/citologia , Células-Tronco Embrionárias/citologia , Neoplasias/terapia , Células-Tronco Adultas/classificação , Células-Tronco Adultas/transplante , Diferenciação Celular , Linhagem da Célula , Células-Tronco Embrionárias/classificação , Células-Tronco Embrionárias/transplante , Humanos , Células-Tronco Mesenquimais/citologia
15.
Stem Cells ; 30(3): 580-4, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22162332

RESUMO

'In this report, we use single cell gene expression to identify transcriptional patterns emerging during the differentiation of human embryonic stem cells (hESCs) into the endodermal lineage. Endoderm-specific transcripts are highly variable between individual CXCR4(+) endodermal cells, suggesting that either the cells generated from in vitro differentiation are distinct or that these embryonic cells tolerate a high degree of transcript variability. Housekeeping transcripts, on the other hand, are far more consistently expressed within the same cellular population. However, when we compare the levels of housekeeping transcripts between hESCs and derived endoderm, patterns emerge that can be used to clearly separate the two embryonic cell types. We further compared four additional human cell types, including 293T, induced pluripotent stem cell (iPSC), HepG2, and endoderm-derived iPSC. In each case, the relative levels of housekeeping transcripts defined a particular cell fate. Interestingly, we find that three transcripts, LDHA, NONO, and ACTB, contribute the most to this diversity and together serve to segregate all six cell types. Overall, this suggests that levels of housekeeping transcripts, which are expressed within all cells, can be leveraged to distinguish between human cell types and thus may serve as important biomarkers for stem cell biology and other disciplines.


Assuntos
Genes Essenciais , Antropologia Cultural , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Diferenciação Celular , Células-Tronco Embrionárias/classificação , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/fisiologia , Endoderma/citologia , Endoderma/metabolismo , Perfilação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/classificação , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Análise de Célula Única , Transcrição Gênica
16.
PLoS Comput Biol ; 7(10): e1002225, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22028635

RESUMO

Many types of epigenetic profiling have been used to classify stem cells, stages of cellular differentiation, and cancer subtypes. Existing methods focus on local chromatin features such as DNA methylation and histone modifications that require extensive analysis for genome-wide coverage. Replication timing has emerged as a highly stable cell type-specific epigenetic feature that is regulated at the megabase-level and is easily and comprehensively analyzed genome-wide. Here, we describe a cell classification method using 67 individual replication profiles from 34 mouse and human cell lines and stem cell-derived tissues, including new data for mesendoderm, definitive endoderm, mesoderm and smooth muscle. Using a Monte-Carlo approach for selecting features of replication profiles conserved in each cell type, we identify "replication timing fingerprints" unique to each cell type and apply a k nearest neighbor approach to predict known and unknown cell types. Our method correctly classifies 67/67 independent replication-timing profiles, including those derived from closely related intermediate stages. We also apply this method to derive fingerprints for pluripotency in human and mouse cells. Interestingly, the mouse pluripotency fingerprint overlaps almost completely with previously identified genomic segments that switch from early to late replication as pluripotency is lost. Thereafter, replication timing and transcription within these regions become difficult to reprogram back to pluripotency, suggesting these regions highlight an epigenetic barrier to reprogramming. In addition, the major histone cluster Hist1 consistently becomes later replicating in committed cell types, and several histone H1 genes in this cluster are downregulated during differentiation, suggesting a possible instrument for the chromatin compaction observed during differentiation. Finally, we demonstrate that unknown samples can be classified independently using site-specific PCR against fingerprint regions. In sum, replication fingerprints provide a comprehensive means for cell characterization and are a promising tool for identifying regions with cell type-specific organization.


Assuntos
Impressões Digitais de DNA/métodos , Período de Replicação do DNA/fisiologia , Células-Tronco Embrionárias/classificação , Células-Tronco Pluripotentes/classificação , Animais , Linhagem Celular , Cromatina/metabolismo , Metilação de DNA , Endoderma/citologia , Epigenômica , Regulação da Expressão Gênica no Desenvolvimento , Histonas/genética , Histonas/metabolismo , Humanos , Mesoderma/citologia , Camundongos , Método de Monte Carlo , Músculo Liso/citologia
17.
Cytotherapy ; 13(5): 582-93, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21231803

RESUMO

BACKGROUND AIMS: Fetal membrane from human placenta tissue has been described as a potential source of stem cells. Despite abundant literature on amnion stem cells, there are limited studies on the stem cell properties of chorion-derived stem cells. METHODS: The main aim was to determine the stemness properties of serial-passaged human chorion-derived stem cells (hCDSC). Quantitative polymerase chain reaction (PCR) was performed to reveal the following stemness gene expression in serial-passaged hCDSC: Oct-4, Sox-2, FGF-4, Rex-1, TERT, Nanog (3), Nestin, FZD-9, ABCG-2 and BST-1. Cell growth rate was evaluated from passage (P) 1 until P5. The colony-forming unit-fibroblast (CFU-F) frequency of P3 and P5 cells and multilineage differentiation potential of P5 cells were determined. The immunophenotype of hCDSC was compared using the surface markers CD9, CD31, CD34, CD44, CD45, CD73, CD90, CD117, HLA-ABC and HLA-DR, -DP and -DQ. Immunostaining for trophoblast markers was done on P0, P1, P3 and P5 cells to detect the contamination of trophoblasts in culture, while chromosomal abnormality was screened by cytogenetic analysis of P5 cells. RESULTS: The surface markers for mesenchymal lineage in hCDSC were more highly expressed at P5 compared with P3 and P0, indicating the increased purity of these stem cells after serial passage. Indeed, all the stemness genes except TERT were expressed at P1, P3 and P5 hCDSC. Furthermore, human chorion contained high clonogenic precursors with a 1:30 CFU-F frequency. Successful adipogenic, chondrogenic and osteogenic differentiation demonstrated the multilineage potential of hCDSC. The karyotyping analysis showed hCDSC maintained chromosomal stability after serial passage. CONCLUSIONS: hCDSC retain multipotent potential even at later passages, hence are a promising source for cell therapy in the future.


Assuntos
Adipogenia , Condrogênese , Córion/citologia , Células-Tronco Embrionárias/citologia , Células-Tronco Multipotentes/citologia , Osteogênese , Placenta/citologia , Antígenos de Superfície/análise , Proliferação de Células , Células Cultivadas , Células-Tronco Embrionárias/classificação , Células-Tronco Embrionárias/metabolismo , Feminino , Expressão Gênica/genética , Humanos , Imunofenotipagem , Células-Tronco Multipotentes/classificação , Células-Tronco Multipotentes/metabolismo , Gravidez
18.
Stem Cell Rev Rep ; 7(2): 471-7, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21188651

RESUMO

Prolonged in vitro culture of human embryonic stem (hES) cells can result in chromosomal abnormalities believed to confer a selective advantage. This potential occurrence has crucial implications for the appropriate use of hES cells for research and therapeutic purposes. In view of this, time-point karyotypic evaluation to assess genetic stability is recommended as a necessary control test to be carried out during extensive 'passaging'. Standard techniques currently used for the cytogenetic assessment of ES cells include G-banding and/or Fluorescence in situ Hybridization (FISH)-based protocols for karyotype analysis, including M-FISH and SKY. Critical for both banding and FISH techniques are the number and quality of metaphase spreads available for analysis at the microscope. Protocols for chromosome preparation from hES and human induced pluripotent stem (hiPS) cells published so far appear to differ considerably from one laboratory to another. Here we present an optimized technique, in which both the number and the quality of chromosome metaphase spreads were substantially improved when compared to current standard techniques for chromosome preparations. We believe our protocol represents a significant advancement in this line of work, and has the required attributes of simplicity and consistency to be widely accepted as a reference method for high quality, fast chromosomal analysis of human ES and iPS cells.


Assuntos
Células-Tronco Embrionárias/classificação , Células-Tronco Pluripotentes Induzidas/classificação , Cariotipagem/métodos , Técnicas de Cultura de Células , Cromossomos Humanos , Demecolcina/química , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Humanos , Hibridização in Situ Fluorescente , Indicadores e Reagentes/química , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Nocodazol/química , Coloração e Rotulagem/métodos
19.
J Neurosci ; 30(44): 14635-48, 2010 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-21048121

RESUMO

Sox2 is expressed by neural stem and progenitor cells, and a sox2 enhancer identifies these cells in the forebrains of both fetal and adult transgenic mouse reporters. We found that an adenovirus encoding EGFP placed under the regulatory control of a 0.4 kb sox2 core enhancer selectively identified multipotential and self-renewing neural progenitor cells in dissociates of human fetal forebrain. Upon EGFP-based fluorescence-activated cell sorting (FACS), the E/sox2:EGFP(+) isolates were propagable for up to 1 year in vitro, and remained multilineage competent throughout. E/sox2:EGFP(+) cells expressed more telomerase enzymatic activity than matched E/sox2:EGFP-depleted populations, and maintained their telomeric lengths with successive passage. Gene expression analysis of E/sox2:EGFP-sorted neural progenitor cells, normalized to the unsorted forebrain dissociates from which they derived, revealed marked overexpression of genes within the notch and wnt pathways, and identified multiple elements of each pathway that appear selective to human neural progenitors. Sox2 enhancer-based FACS thus permits the prospective identification and direct isolation of a telomerase-active population of neural stem cells from the human fetal forebrain, and the elucidation of both the transcriptome and dominant signaling pathways of these critically important cells.


Assuntos
Células-Tronco Embrionárias/citologia , Elementos Facilitadores Genéticos/genética , Citometria de Fluxo/métodos , Células-Tronco Neurais/citologia , Fatores de Transcrição SOXB1/genética , Telomerase/biossíntese , Linhagem da Célula/genética , Separação Celular/métodos , Células Cultivadas , Células-Tronco Embrionárias/classificação , Células-Tronco Embrionárias/enzimologia , Feto , Humanos , Células-Tronco Neurais/classificação , Células-Tronco Neurais/enzimologia , Estudos Prospectivos , Telomerase/genética
20.
Cell Transplant ; 19(11): 1383-95, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20587141

RESUMO

For human embryonic stem cells (hESCs) to be used clinically, it is imperative that immune responses evoked by hESCs and their derivates after transplantation should be prevented. Human leukocyte antigens (HLA) and ABO blood group antigens are important histocompatibility factors in graft rejection. HLA matching between recipient and unrelated donors, in particular, is important in improving outcomes in hematopoietic cell transplantation (HCT). We have established and successfully maintained 29 hESC lines and analyzed the HLA and ABO genotypes of these lines. HLA-A, -B, -C and -DR (DRB1) genotyping was performed by polymerase chain reaction (PCR) sequence-based typing and ABO genotyping was carried out by PCR restriction fragment length polymorphism methods. To determine what proportion of the Korean population would be covered by these cell lines in organ transplantation, 27 cell lines with HLA-A, -B, and -DR data were evaluated for HCT (cord blood) donors and 28 cell lines with HLA-DR and ABO data were evaluated for solid organ (kidney) transplantation donors, and then compared the data with those from 6,740 donated cord bloods. When 2 HLA mismatches are allowed for HCT, as currently accepted for cord blood transplantation, it was estimated that about 16% and 25% of the possible recipients can find one or more donor cell lines with ≤2 mismatches at A, B, DRB1 allele level and at A, B antigen/DRB1 allele level, respectively. When HLA-DR antigen level matching and ABO compatibility was considered for solid organ (kidney) transplantation, it was estimated that about 29% and 96% of the possible recipients can find one or more ABO-compatible donor cell lines with 0 and 1 DR mismatches, respectively. We provided the first report on the HLA and ABO genotypes of hESC lines, and estimated the degree of HLA and ABO matching in organ transplantation for the Korean population.


Assuntos
Sistema ABO de Grupos Sanguíneos/genética , Células-Tronco Embrionárias/transplante , Antígenos HLA/genética , Alelos , Tipagem e Reações Cruzadas Sanguíneas/estatística & dados numéricos , Linhagem Celular , Células-Tronco Embrionárias/classificação , Células-Tronco Embrionárias/imunologia , Sangue Fetal/química , Sangue Fetal/metabolismo , Genótipo , Transplante de Células-Tronco Hematopoéticas , Teste de Histocompatibilidade , Humanos , Polimorfismo de Fragmento de Restrição , Análise de Sequência de DNA , Doadores de Tecidos
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