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1.
Cell Stem Cell ; 17(4): 435-47, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26387754

RESUMO

ELABELA (ELA) is a peptide hormone required for heart development that signals via the Apelin Receptor (APLNR, APJ). ELA is also abundantly secreted by human embryonic stem cells (hESCs), which do not express APLNR. Here we show that ELA signals in a paracrine fashion in hESCs to maintain self-renewal. ELA inhibition by CRISPR/Cas9-mediated deletion, shRNA, or neutralizing antibodies causes reduced hESC growth, cell death, and loss of pluripotency. Global phosphoproteomic and transcriptomic analyses of ELA-pulsed hESCs show that it activates PI3K/AKT/mTORC1 signaling required for cell survival. ELA promotes hESC cell-cycle progression and protein translation and blocks stress-induced apoptosis. INSULIN and ELA have partially overlapping functions in hESC medium, but only ELA can potentiate the TGFß pathway to prime hESCs toward the endoderm lineage. We propose that ELA, acting through an alternate cell-surface receptor, is an endogenous secreted growth factor in human embryos and hESCs that promotes growth and pluripotency.


Assuntos
Células-Tronco Embrionárias Humanas/metabolismo , Hormônios Peptídicos/metabolismo , Transdução de Sinais , Anticorpos Neutralizantes , Receptores de Apelina , Diferenciação Celular , Linhagem Celular , Autorrenovação Celular , Endoderma/citologia , Endoderma/metabolismo , Perfilação da Expressão Gênica , Células-Tronco Embrionárias Humanas/citologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Comunicação Parácrina , Fosfatidilinositol 3-Quinases/metabolismo , Proteômica , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno , Receptores Acoplados a Proteínas G/metabolismo
2.
Stem Cell Reports ; 1(5): 379-86, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24286026

RESUMO

Human embryonic stem cells (hESCs) regularly acquire nonrandom genomic aberrations during culture, raising concerns about their safe therapeutic application. The International Stem Cell Initiative identified a copy number variant (CNV) amplification of chromosome 20q11.21 in 25% of hESC lines displaying a normal karyotype. By comparing four cell lines paired for the presence or absence of this CNV, we show that those containing this amplicon have higher population doubling rates, attributable to enhanced cell survival through resistance to apoptosis. Of the three genes encoded within the minimal amplicon and expressed in hESCs, only overexpression of BCL2L1 (BCL-XL isoform) provides control cells with growth characteristics similar to those of CNV-containing cells, whereas inhibition of BCL-XL suppresses the growth advantage of CNV cells, establishing BCL2L1 as a driver mutation. Amplification of the 20q11.21 region is also detectable in human embryonal carcinoma cell lines and some teratocarcinomas, linking this mutation with malignant transformation.


Assuntos
Cromossomos Humanos Par 20/genética , Variações do Número de Cópias de DNA , Células-Tronco Embrionárias/metabolismo , Seleção Genética , Proteína bcl-X/metabolismo , Linhagem Celular , Amplificação de Genes , Loci Gênicos , Humanos , Mutação , Proteína bcl-X/genética
3.
Nat Biotechnol ; 29(12): 1132-44, 2011 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-22119741

RESUMO

The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups. Most lines remained karyotypically normal, but there was a progressive tendency to acquire changes on prolonged culture, commonly affecting chromosomes 1, 12, 17 and 20. DNA methylation patterns changed haphazardly with no link to time in culture. Structural variants, determined from the SNP arrays, also appeared sporadically. No common variants related to culture were observed on chromosomes 1, 12 and 17, but a minimal amplicon in chromosome 20q11.21, including three genes expressed in human ES cells, ID1, BCL2L1 and HM13, occurred in >20% of the lines. Of these genes, BCL2L1 is a strong candidate for driving culture adaptation of ES cells.


Assuntos
Células-Tronco Embrionárias/citologia , Crescimento/genética , Células-Tronco Pluripotentes Induzidas/citologia , Proteínas de Ligação a RNA/metabolismo , Proteína bcl-X/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Cromossomos Humanos Par 20/genética , Evolução Clonal/genética , Metilação de DNA , Etnicidade/genética , Regulação da Expressão Gênica no Desenvolvimento , Variação Genética , Genótipo , Humanos , Proteína 1 Inibidora de Diferenciação/genética , Proteína 1 Inibidora de Diferenciação/metabolismo , Polimorfismo de Nucleotídeo Único , Proteínas de Ligação a RNA/genética , Seleção Genética/genética , Proteína bcl-X/genética
4.
Curr Protoc Stem Cell Biol ; Chapter 5: Unit5C.1, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21913171

RESUMO

This unit describes the generation of tetracycline-inducible short hairpin RNA interference (shRNAi) human embryonic stem cell (hESC) lines. Using this vector-based approach enables stable and long-term expression of target hairpins under the control of doxycycline/tetracycline. Target degradation can be controlled in both a dose- and time-dependent manner that can even be switched off, depending upon the particular requirements of the study.


Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Embrionárias/citologia , Interferência de RNA , RNA Interferente Pequeno/genética , Animais , Soluções Tampão , Linhagem Celular , Clonagem Molecular/métodos , Regulação para Baixo , Doxiciclina/farmacologia , Vetores Genéticos , Humanos , Camundongos , Oligonucleotídeos/genética , RNA Interferente Pequeno/metabolismo , Transfecção/métodos
5.
Nat Genet ; 43(4): 365-9, 2011 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-21358634

RESUMO

Multiple self-healing squamous epithelioma (MSSE), also known as Ferguson-Smith disease (FSD), is an autosomal-dominant skin cancer condition characterized by multiple squamous-carcinoma-like locally invasive skin tumors that grow rapidly for a few weeks before spontaneously regressing, leaving scars. High-throughput genomic sequencing of a conservative estimate (24.2 Mb) of the disease locus on chromosome 9 using exon array capture identified independent mutations in TGFBR1 in three unrelated families. Subsequent dideoxy sequencing of TGFBR1 identified 11 distinct monoallelic mutations in 18 affected families, firmly establishing TGFBR1 as the causative gene. The nature of the sequence variants, which include mutations in the extracellular ligand-binding domain and a series of truncating mutations in the kinase domain, indicates a clear genotype-phenotype correlation between loss-of-function TGFBR1 mutations and MSSE. This distinguishes MSSE from the Marfan syndrome-related disorders in which missense mutations in TGFBR1 lead to developmental defects with vascular involvement but no reported predisposition to cancer.


Assuntos
Mutação , Proteínas Serina-Treonina Quinases/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Neoplasias Cutâneas/genética , Sequência de Aminoácidos , Sequência de Bases , Carcinoma/genética , Carcinoma/metabolismo , Códon sem Sentido , Sequência Conservada , Primers do DNA/genética , Feminino , Mutação da Fase de Leitura , Estudos de Associação Genética , Haplótipos , Humanos , Ceratoacantoma/genética , Ceratoacantoma/metabolismo , Masculino , Síndrome de Marfan/genética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/química , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Homologia de Sequência de Aminoácidos , Neoplasias Cutâneas/metabolismo
6.
Stem Cells ; 28(5): 863-73, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20235236

RESUMO

Transforming growth factor (TGF)-beta superfamily proteins play a key role in the regulation of human embryonic stem cells (hESCs). Those of the TGFbeta/activin/nodal branch seem to support self-renewal and pluripotency, whereas those of the bone morphogenic protein (BMP) branch induce differentiation. In contrast to this generalization, we found that hESC remained undifferentiated after knockdown of SMAD4 with inducible short hairpin RNA interference, although the knockdown inhibited TGFbeta signaling and rendered the cells nonresponsive to BMP-induced differentiation. Moreover, the rapid differentiation of hESC after pharmacological inhibition of TGFbeta/activin/nodal receptor signaling was restricted after SMAD4 knockdown. These results suggest that TGFbeta/activin/nodal signaling supports the undifferentiated phenotype of hESC by suppressing BMP activity. During long-term culture, SMAD4 knockdown cell populations became less stable and more permissive to neural induction, a situation that was rescued by re-establishment of SMAD4 expression. These results suggest that SMAD4 is not required for maintenance of the undifferentiated state of hESC, but rather to stabilize that state.


Assuntos
Divisão Celular/genética , Linhagem da Célula/genética , Células-Tronco Embrionárias/metabolismo , Proteína Smad4/genética , Ativinas/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Regulação para Baixo/fisiologia , Células-Tronco Embrionárias/citologia , Humanos , Proteína Nodal/metabolismo , Interferência de RNA/fisiologia , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/metabolismo
7.
Stem Cells ; 27(4): 776-82, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19350677

RESUMO

Manipulation of gene function in embryonic stem cells by either over expression or downregulation is critical for understanding their subsequent cell fate. We have developed a tetracycline-inducible short hairpin RNA interference (shRNAi) for human embryonic stem cells (hESCs) and demonstrated doxycycline dose-dependent knockdown of the transcription factor OCT4 and the cell surface antigen beta2-microglobulin. The induced knockdown of OCT4 resulted in rapid differentiation of hESCs with a significant increase in transcription of genes associated with trophoblast and endoderm lineages, the extent of which was controlled by the degree of induction. Transgene toxicity, which may occur in conditional over-expression strategies with hESCs, was not observed with wild-type Tet repressor protein. The system allows efficient, reversible, and long-term downregulation of target genes in hESCs and enables the generation of stable transfectants for the knockdown of genes essential for cell survival and self-renewal, not necessarily possible by nonconditional shRNAi methods. STEM CELLS 2009;27:776-782.


Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Técnicas de Silenciamento de Genes/métodos , Fator 3 de Transcrição de Octâmero/genética , Interferência de RNA/fisiologia , Diferenciação Celular , Linhagem Celular , Linhagem da Célula/genética , Citometria de Fluxo , Humanos , Immunoblotting , Imuno-Histoquímica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Transfecção
8.
Stem Cells Dev ; 17(6): 1195-205, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18393631

RESUMO

Human embryonic stem cells (hESCs) replicate in vitro by the process of self-renewal, whilst maintaining their pluripotency. Understanding the pathways involved in the regulation of this process will assist in developing fully-defined conditions for the robust proliferation of hESCs necessary for therapeutic applications. We previously demonstrated that sphingosine-1-phosphate (S1P) plays an important role in survival and proliferation of hESCs. and here the key signaling pathways and downstream targets of S1P were investigated in a representative cell line (Shef 4). A significant rise in ERK1/2 activation with S1P treatment was witnessed in hESCs maintained on murine embryonic fibroblasts (MEFs) exhibiting significantly higher levels of active ERK1/2 than those grown on Matrigel. RT-PCR and microarray analysis of micro-dissected, non-differentiated hESC revealed 1049 differentially expressed genes in S1P treated preparations compared with controls (n = 3). S1P regulated apoptosis through several BCL-2 family members, including BAX and BID, with increased expression of cell cycle progression genes associated with proliferation of hESC cultures. S1P treatment also increased expression of cell adhesion genes specifically cadherins and integrins. However, gene expression associated with pluripotency was decreased with S1P treatment indicating that an increased rate of hESC turnover (higher proliferation and lower apoptosis) may be balanced by an increased susceptibility to differentiate.


Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Lisofosfolipídeos/farmacologia , Células-Tronco Pluripotentes/metabolismo , Esfingosina/análogos & derivados , Transcrição Gênica/efeitos dos fármacos , Animais , Apoptose/fisiologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/biossíntese , Proteínas de Ciclo Celular/biossíntese , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Células-Tronco Embrionárias/citologia , Ativação Enzimática/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/metabolismo , Perfilação da Expressão Gênica/métodos , Humanos , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Células-Tronco Pluripotentes/citologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Esfingosina/farmacologia , Transcrição Gênica/fisiologia , Proteína X Associada a bcl-2/biossíntese
9.
Stem Cells Dev ; 15(5): 729-40, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17105408

RESUMO

Human embryonic stem (hES) cells have the ability to self-renew while maintaining their pluripotency. The ability of stem cells to self-renew expansively is essential in both development and maintenance of adult tissues. ES cell lines were first generated from mouse blastocysts, these lines provided much needed information regarding ES cell propagation, growth factor dependence, and marker expression. However, the application potential of murine models is restricted in value because many differences between mouse and human ES cells have since been identified. The process of hES cells self-renewal appears to be regulated by many different pathways; however, the molecular mechanisms enabling this process are not fully characterized. Further defining these mechanisms will enable growth of hES cells under defined conditions and aid controlled differentiation of cells into specified lineages, in turn providing cells suitable for therapeutic applications. This review provides a summary of the mechanisms known to control self-renewal and pluripotency in hES cells.


Assuntos
Células-Tronco Embrionárias/citologia , Proliferação de Células , Humanos , Transdução de Sinais , Fatores de Transcrição/metabolismo
11.
J Interferon Cytokine Res ; 25(8): 467-84, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16108730

RESUMO

As most mechanisms of adaptive immunity evolved during the divergence of vertebrates, the immune systems of extant vertebrates represent different successful variations on the themes initiated in their earliest common ancestors. The genes involved in elaborating these mechanisms have been subject to exceptional selective pressures in an arms race with highly adaptable pathogens, resulting in highly divergent sequences of orthologous genes and the gain and loss of members of gene families as different species find different solutions to the challenge of infection. Consequently, it has been difficult to transfer to the chicken detailed knowledge of the molecular mechanisms of the mammalian immune system and, thus, to enhance the already significant contribution of chickens toward understanding the evolution of immunity. The availability of the chicken genome sequence provides the opportunity to resolve outstanding questions concerning which molecular components of the immune system are shared between mammals and birds and which represent their unique evolutionary solutions. We have integrated genome data with existing knowledge to make a new comparative census of members of cytokine and chemokine gene families, distinguishing the core set of molecules likely to be common to all higher vertebrates from those particular to these 300 million-year-old lineages. Some differences can be explained by the different architectures of the mammalian and avian immune systems. Chickens lack lymph nodes and also the genes for the lymphotoxins and lymphotoxin receptors. The lack of functional eosinophils correlates with the absence of the eotaxin genes and our previously reported observation that interleukin- 5 (IL-5) is a pseudogene. To summarize, in the chicken genome, we can identify the genes for 23 ILs, 8 type I interferons (IFNs), IFN-gamma, 1 colony-stimulating factor (GM-CSF), 2 of the 3 known transforming growth factors (TGFs), 24 chemokines (1 XCL, 14 CCL, 8 CXCL, and 1 CX3CL), and 10 tumor necrosis factor superfamily (TNFSF) members. Receptor genes present in the genome suggest the likely presence of 2 other ILs, 1 other CSF, and 2 other TNFSF members.


Assuntos
Quimiocinas/genética , Galinhas/genética , Citocinas/genética , Genômica , Sequência de Aminoácidos , Animais , Citocinas/química , Humanos , Inflamação/genética , Dados de Sequência Molecular , Filogenia , Receptores de Quimiocinas/genética , Alinhamento de Sequência
12.
Dev Comp Immunol ; 28(5): 375-94, 2004 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-15062639

RESUMO

As the adaptive immune system arose in jawed vertebrates, a reasonable working hypothesis is that cytokines involved exclusively in controlling the adaptive immune system, e.g. T1 and T2 cytokines, will only be found in jawed vertebrates whilst those with a role in innate responses, e.g. type I IFNs or pro-inflammatory cytokines, will be universal, or have functional equivalents in 'lower' animals. Progress to date in cloning cytokines, including interleukins, in vertebrates outside of mammals is limited and non-existent in invertebrates. T1 and T2 interleukins have only been cloned in birds. Receptors for T1 and T2 interleukins have been cloned in fish, however, suggesting the presence of the corresponding interleukins. Pro-inflammatory interleukins have been cloned in birds, fish and amphibia, but not reptiles. Cross-reactive bioassays, polyclonal antisera and mAbs suggest that an IL-6-like factor exists in starfish, as yet the only evidence for innate immune response cytokines in 'lower' animals.


Assuntos
Evolução Molecular , Interleucinas/genética , Sequência de Aminoácidos , Animais , Humanos , Mediadores da Inflamação/imunologia , Interleucina-12/genética , Dados de Sequência Molecular , Família Multigênica , Homologia de Sequência de Aminoácidos , Células Th1/imunologia , Células Th2/imunologia
13.
J Interferon Cytokine Res ; 24(10): 600-10, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15626157

RESUMO

A genomics approach based on the conservation of synteny was used to develop a bacterial artificial chromosome (BAC) contig across the chicken T2 cytokine gene cluster. Sequencing of representative BACs showed that the chicken genome encodes genes for the homologs of mammalian interleukin-3 (IL-3), IL-4, IL-5, IL-13, and granulocyte-macrophage colony-stimulating factor (GM-CSF). These sequences represent the first T2 cytokines found outside of mammals, and their location demonstrates that the T2 cluster is ancient (at least 300 million years old). Four of these genes (IL-3, IL-4, IL-13, and GM-CSF) are expressed at the mRNA level and can be expressed as recombinant protein. In contrast to the other four genes, the chicken IL-5 (ChIL-5) gene we sequenced lacks a recognizable promoter and regulatory sequences in the predicted 3'-untranslated region (3'-UTR). Further, there is no evidence for its expression at the mRNA level. We, therefore, hypothesize that it is a pseudogene. Genomic analysis revealed that a recently characterized cytokinelike transcript, KK34, not identified in our initial analysis of the BAC sequence, is also encoded in this cluster. This gene may represent a duplication of an ancestral IL-5 gene and may encode the functional homolog of IL-5 in the chicken.


Assuntos
Galinhas/genética , Galinhas/imunologia , Citocinas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromossomos Artificiais Bacterianos/genética , DNA Complementar/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Humanos , Interleucina-13/genética , Interleucina-3/genética , Interleucina-4/genética , Interleucina-5/genética , Camundongos , Dados de Sequência Molecular , Família Multigênica , Regiões Promotoras Genéticas , Pseudogenes , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie
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