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1.
Nat Methods ; 8(12): 1037-40, 2011 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-22020065

RESUMO

NKX2-5 is expressed in the heart throughout life. We targeted eGFP sequences to the NKX2-5 locus of human embryonic stem cells (hESCs); NKX2-5(eGFP/w) hESCs facilitate quantification of cardiac differentiation, purification of hESC-derived committed cardiac progenitor cells (hESC-CPCs) and cardiomyocytes (hESC-CMs) and the standardization of differentiation protocols. We used NKX2-5 eGFP(+) cells to identify VCAM1 and SIRPA as cell-surface markers expressed in cardiac lineages.


Assuntos
Separação Celular/métodos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Homeodomínio/metabolismo , Mioblastos Cardíacos/citologia , Miócitos Cardíacos/citologia , Fatores de Transcrição/metabolismo , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Biomarcadores/análise , Diferenciação Celular , Perfilação da Expressão Gênica , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/genética , Humanos , Mioblastos Cardíacos/metabolismo , Miócitos Cardíacos/metabolismo , Reação em Cadeia da Polimerase , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Fatores de Transcrição/genética , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo
2.
Stem Cells ; 30(9): 1999-2009, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22761039

RESUMO

Monoclonal antibodies against cell surface markers are powerful tools in the study of tissue regeneration, repair, and neoplasia, but there is a paucity of specific reagents to identify stem and progenitor cells in tissues of endodermal origin. The epitope defined by the GCTM-5 monoclonal antibody is a putative marker of hepatic progenitors. We sought to analyze further the distribution of the GCTM-5 antigen in normal tissues and disease states and to characterize the antigen biochemically. The GCTM-5 epitope was specifically expressed on tissues derived from the definitive endoderm, in particular the fetal gut, liver, and pancreas. Antibody reactivity was detected in subpopulations of normal adult biliary and pancreatic duct cells, and GCTM-5-positive cells isolated from the nonparenchymal fraction of adult liver expressed markers of progenitor cells. The GCTM-5-positive cell populations in liver and pancreas expanded greatly in numbers in disease states such as biliary atresia, cirrhosis, and pancreatitis. Neoplasms arising in these tissues also expressed the GCTM-5 antigen, with pancreatic adenocarcinoma in particular showing strong and consistent reactivity. The GCTM-5 epitope was also strongly displayed on cells undergoing intestinal metaplasia in Barrett's esophagus, a precursor to esophageal carcinoma. Biochemical, mass spectrometry, and immunochemical studies revealed that the GCTM-5 epitope is associated with the mucin-like glycoprotein FCGBP. The GCTM-5 epitope on the mucin-like glycoprotein FCGBP is a cell surface marker for the study of normal differentiation lineages, regeneration, and disease progression in tissues of endodermal origin.


Assuntos
Moléculas de Adesão Celular/imunologia , Epitopos/biossíntese , Glicoproteínas/imunologia , Fígado/citologia , Células-Tronco/imunologia , Diferenciação Celular/imunologia , Endoderma/citologia , Endoderma/imunologia , Epitopos/imunologia , Humanos , Fígado/imunologia , Células-Tronco/citologia
3.
BMC Dev Biol ; 7: 12, 2007 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-17335568

RESUMO

BACKGROUND: The mechanisms responsible for the maintenance of pluripotency in human embryonic stem cells, and those that drive their commitment into particular differentiation lineages, are poorly understood. In fact, even our knowledge of the phenotype of hESC is limited, because the immunological and molecular criteria presently used to define this phenotype describe the properties of a heterogeneous population of cells. RESULTS: We used a novel approach combining immunological and transcriptional analysis (immunotranscriptional profiling) to compare gene expression in hESC populations at very early stages of differentiation. Immunotranscriptional profiling enabled us to identify novel markers of stem cells and their differentiated progeny, as well as novel potential regulators of hESC commitment and differentiation. The data show clearly that genes associated with the pluripotent state are downregulated in a coordinated fashion, and that they are co-expressed with lineage specific transcription factors in a continuum during the early stages of stem cell differentiation. CONCLUSION: These findings, that show that maintenance of pluripotency and lineage commitment are dynamic, interactive processes in hESC cultures, have important practical implications for propagation and directed differentiation of these cells, and for the interpretation of mechanistic studies of hESC renewal and commitment. Since embryonic stem cells at defined stages of commitment can be isolated in large numbers by immunological means, they provide a powerful model for studying molecular genetics of stem cell commitment in the embryo.


Assuntos
Células-Tronco Embrionárias/citologia , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Células-Tronco Pluripotentes/citologia , Animais , Técnicas de Cultura de Células , Linhagem Celular , Linhagem da Célula , Técnica Indireta de Fluorescência para Anticorpo , Regulação da Expressão Gênica no Desenvolvimento , Marcadores Genéticos , Humanos , Camundongos , Proteínas Recombinantes/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sialoglicoproteínas/genética
4.
PLoS One ; 9(11): e112909, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25412507

RESUMO

Medulloblastoma is the most common form of malignant paediatric brain tumour and is the leading cause of childhood cancer related mortality. The four molecular subgroups of medulloblastoma that have been identified - WNT, SHH, Group 3 and Group 4 - have molecular and topographical characteristics suggestive of different cells of origin. Definitive identification of the cell(s) of origin of the medulloblastoma subgroups, particularly the poorer prognosis Group 3 and Group 4 medulloblastoma, is critical to understand the pathogenesis of the disease, and ultimately for the development of more effective treatment options. To address this issue, the gene expression profiles of normal human neural tissues and cell types representing a broad neuro-developmental continuum, were compared to those of two independent cohorts of primary human medulloblastoma specimens. Clustering, co-expression network, and gene expression analyses revealed that WNT and SHH medulloblastoma may be derived from distinct neural stem cell populations during early embryonic development, while the transcriptional profiles of Group 3 and Group 4 medulloblastoma resemble cerebellar granule neuron precursors at weeks 10-15 and 20-30 of embryogenesis, respectively. Our data indicate that Group 3 medulloblastoma may arise through abnormal neuronal differentiation, whereas deregulation of synaptic pruning-associated apoptosis may be driving Group 4 tumorigenesis. Overall, these data provide significant new insight into the spatio-temporal relationships and molecular pathogenesis of the human medulloblastoma subgroups, and provide an important framework for the development of more refined model systems, and ultimately improved therapeutic strategies.


Assuntos
Neoplasias Cerebelares/patologia , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Meduloblastoma/patologia , Neurogênese , Células Cultivadas , Neoplasias Cerebelares/genética , Criança , Pré-Escolar , Regulação Neoplásica da Expressão Gênica , Humanos , Meduloblastoma/genética , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Neurônios/metabolismo
5.
Stem Cell Rev Rep ; 6(3): 351-66, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20625855

RESUMO

Stem cells have great potential for understanding early development, treating human disease, tissue trauma and early phase drug discovery. The factors that control the regulation of stem cell survival, proliferation, migration and differentiation are still emerging. Some evidence now exists demonstrating the potent effects of various G-protein coupled receptor (GPCR) ligands on the biology of stem cells. This review aims to give an overview of the current knowledge of the regulation of embryonic and somatic stem cell maintenance and differentiation by GPCR ligands.


Assuntos
Diferenciação Celular , Proliferação de Células , Receptores Acoplados a Proteínas G/fisiologia , Células-Tronco/fisiologia , Animais , Diferenciação Celular/genética , Humanos , Ligantes , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Células-Tronco/metabolismo
6.
Stem Cells Dev ; 19(4): 569-78, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19594361

RESUMO

Human embryonic stem cell-derived neural stem cells (hESC-NSCs) are an attractive cell type for studying aspects of brain development and pathology. To develop the full potential of this model system, it is important to establish a reliable methodology for the manipulation of gene expression in hNSCs. To address this issue, we used an adenoviral vector with a CMV promoter-driven green fluorescent protein (GFP) reporter gene (Ad5-GFP). We optimized conditions for Ad5-GFP infection and assessed the efficiency of infection of whole and dissociated embryonic stem cell (ESC)-derived neurospheres as well as the effect of adenoviral vectors on cell surface marker expression, proliferation, and differentiation potential. Our results demonstrate that most neurosphere cells ( approximately 70%) express the coxsackie and adenovirus receptor and can be infected with Ad5. More specifically, the CD133+ hESC-NSC population could be infected more efficiently than the CD133 population and both populations expressed GFP at high levels. At low multiplicity of infection (MOI < 25), the virus had no significant effect on stem cell marker expression (CD133 and Nestin), cell survival, cell proliferation rate, or differentiation potential. This model system provides a practical new approach to study human NSC function in the context of neurodegenerative and neoplastic disorders.


Assuntos
Adenoviridae/genética , Células-Tronco Embrionárias , Técnicas de Transferência de Genes , Vetores Genéticos , Neurônios , Transdução Genética/métodos , Antígeno AC133 , Antígenos CD/metabolismo , Biomarcadores , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Glicoproteínas/metabolismo , Humanos , Proteínas de Filamentos Intermediários/metabolismo , Modelos Biológicos , Proteínas do Tecido Nervoso/metabolismo , Nestina , Neurônios/citologia , Neurônios/metabolismo , Peptídeos/metabolismo
8.
Stem Cells Dev ; 18(2): 269-82, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18651819

RESUMO

The ability to generate purified neural progenitors is critical to the development of embryonic stem cell-based therapies to alleviate human neurological disorders. While many cell culture protocols for directed differentiation of human embryonic stem (hES) cells into neural cells have been described, most yield mixed populations, some containing cells of different embryonic germ layer lineages, or even undifferentiated embryonic stem cells. In this study, we describe a method for single-cell dissociation, isolation by flow cytometry, and subsequent culture of neural progenitors from hES cells. As reported earlier, hES cells treated with the bone morphogenetic protein (BMP) antagonist noggin gave rise to neurospheres at a relatively high frequency. However, these noggin-treated embryonic stem cell cultures were heterogeneous, with cells expressing embryonic stem markers still detectable even following 14 days of differentiation. In order to isolate pure human neural progenitors, we fractionated noggin-treated ES cells on the basis of their expression of the putative neural stem cell marker, CD133, and the GCTM-2, and SSEA-1 antigens, which mark pluripotent stem cells and differentiated cells respectively from hES cell culture. CD133(+) cells formed larger spheres compared to CD133(-) cells. CD133(+)SSEA1(+) cells and CD133(+)SSEA-1(-) cells expressed similar levels of neural genes and formed neurospheres at similar frequencies. By contrast, CD133(+)GCTM-2(+) cells expressed high levels of OCT4 but not neural lineage genes and failed to form neurospheres. CD133(+)GCTM-2(-) cells formed neurospheres at the relative highest frequency. Thus, negative selection with GCTM-2 may be useful for the purification of specific cell types differentiated from hES cells.


Assuntos
Antígenos CD/metabolismo , Separação Celular/métodos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Glicoproteínas/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Peptídeos/metabolismo , Antígeno AC133 , Animais , Proteínas de Transporte/farmacologia , Contagem de Células , Ensaio de Unidades Formadoras de Colônias , Citometria de Fluxo , Imunofluorescência , Humanos , Camundongos
9.
Stem Cells ; 23(1): 103-12, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15625127

RESUMO

The nature of the cells that contribute to the repopulation of the liver after hepatic necrosis or cirrhosis remains uncertain, in part because we lack specific markers to facilitate identification and prospective isolation of progenitor cells. The monoclonal antibody GCTM-5 reacts with a minority subpopulation of cells in spontaneously differentiating cultures of pluripotent human embryonal carcinoma or embryonic stem cells. The epitope recognized by GCTM-5 is found on a 50-kDa protein present on the surface of these cells. In tissue sections of first-trimester human embryos, GCTM-5 specifically stained hepatoblasts and no other cell type examined. In normal pediatric or adult liver, GCTM-5 reacted with a minority population of luminal bile duct cells. In diseased livers, the numbers of GCTM-5-positive cells were increased compared with normal liver; antibody staining was restricted to a subpopulation of ductular reactive cells, and among this subpopulation we observed GCTM-5-positive cells that did not express cytokeratin 19 or N-CAM, classical makers of ductular reactive cells. Live GCTM-5-positive cells could be isolated from diseased livers by immunomagnetic sorting. These results suggest that GCTM-5 will be a useful reagent for defining cell lineage relationships between putative progenitor populations in embryonic liver and in the biliary epithelium during tissue repair.


Assuntos
Antígenos de Superfície/análise , Ductos Biliares/citologia , Biomarcadores Tumorais/análise , Células Epiteliais/citologia , Fígado/citologia , Fígado/embriologia , Células-Tronco/citologia , Adulto , Animais , Anticorpos Monoclonais/metabolismo , Antígenos de Diferenciação/análise , Antígenos de Superfície/genética , Linhagem Celular Tumoral , Embrião de Mamíferos , Células Epiteliais/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Fígado/ultraestrutura , Camundongos , Células-Tronco/metabolismo , Células-Tronco/ultraestrutura
10.
Nat Methods ; 2(4): 259-60, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15782217

RESUMO

Human embryonic stem cells (hESCs) have been advanced as a potential source of cells for use in cell replacement therapies. The ability to identify hESCs and their differentiated progeny readily in transplantation experiments will facilitate the analysis of hESC potential and function in vivo. We have generated a hESC line designated 'Envy', in which robust levels of green fluorescent protein (GFP) are expressed in stem cells and all differentiated progeny.


Assuntos
Diferenciação Celular/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células-Tronco/citologia , Células-Tronco/fisiologia , Sequência de Bases , Linhagem Celular , Humanos , Dados de Sequência Molecular
11.
Hum Reprod ; 17(4): 850-2, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11925371

RESUMO

Ooplasm donation, wherein ooplasm is transferred from a donor oocyte to a recipient oocyte in an effort to increase embryo viability, has been applied in the human, with resulting pregnancies and births. Neither the safety nor efficacy of this method has been adequately investigated. Mitochondrial heteroplasmy in the blood of children conceived using ooplasm donation has recently been described. A follow-up study of children born following the use of this technique primarily focused on the presence of mitochondria from the donor oocyte highlighting possible problems due to mitochondrial heteroplasmy. Other effects related to epigenetic events may also arise, but have not been addressed. Studies using inbred mouse strains reveal that genetically diverse ooplasms can impose diverse epigenetic modifications on parental genomes. Incompatibilities produced by combining maternal genome and ooplasm from different genotypes leads to defects in gene expression and development. Such defects can be heritable and observed in the next generation. Given the potential for epigenetic modifications to arise following ooplasm donation, the safety and efficacy of this method need to be evaluated in a suitable animal model.


Assuntos
Citoplasma/transplante , Oócitos/fisiologia , Animais , Expressão Gênica , Doenças Genéticas Inatas/etiologia , Genoma , Humanos , Camundongos , Doenças Mitocondriais/etiologia , Doação de Oócitos
12.
Mol Reprod Dev ; 61(3): 281-7, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11835573

RESUMO

The Bex1/Rex3 gene was recently identified as an X-linked gene that is differentially expressed between parthenogenetic and normal fertilized, preimplantation stage mouse embryos. The Bex1/Rex3 gene appears to be expressed preferentially from the maternal X chromosome in blastocysts, but from either X chromosome in later stage embryonic tissues and adult tissues. To investigate whether differential expression of the Bex1/Rex3 gene between normal and parthenogenetic blastocyst stage embryos reflects genomic imprinting at the Bex1/Rex3 locus itself, or instead is the result of preferential inactivation of the paternal X chromosome or differences in timing of cellular differentiation, we examined in detail the expression pattern of the Bex1/Rex3 mRNA in normal preimplantation stage embryos, and compared its expression between androgenetic, gynogenetic, and normal fertilized embryos. Expression data reveal that the Bex1/Rex3 gene is initially transcribed at the 2-cell stage, transiently induced at the 8-cell stage, and then increases in expression again at the blastocyst stage. Very little expression is observed in isolated inner cell masses, indicating selective expression in the trophectoderm. Comparisons of Bex1/Rex3 mRNA expression between male and female androgenetic and control embryos and gynogenetic embros failed to reveal any significant difference in expression between the different classes of embryos at the 8-cell stage, or the expanding blastocyst stage (121 hr post-hCG). At the late blastocyst stage (141 hr post-hCG), expression was significantly lower in XY control embryos as compared with XX controls. Bex1/Rex3 mRNA expression did not differ between XX and XY androgenones at the blastocyst stage or between gynogenones and XX control embryos. Thus, the Bex1/Rex3 gene does not appear to be regulated directly by genomic imprinting during the preimplantation period, just as it is not regulated by imprinting at later stages. Apparent differences in gene expression may arise through the effects of trophectoderm-specific expression coupled with differences in timing of trophectoderm differentiation between the different classes of embryos and effects of preferential paternal X chromosome inactivation (XCI).


Assuntos
Blastocisto/metabolismo , Proteínas de Ligação a DNA/genética , Ectoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Ligação Genética/genética , Proteínas do Tecido Nervoso/genética , Cromossomo X/genética , Animais , Técnicas de Cultura , Feminino , Ordem dos Genes , Genótipo , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Trofoblastos/metabolismo
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