RESUMO
Duplication of the genome in mammalian cells occurs in a defined temporal order referred to as its replication-timing (RT) program. RT changes dynamically during development, regulated in units of 400-800 kb referred to as replication domains (RDs). Changes in RT are generally coordinated with transcriptional competence and changes in subnuclear position. We generated genome-wide RT profiles for 26 distinct human cell types, including embryonic stem cell (hESC)-derived, primary cells and established cell lines representing intermediate stages of endoderm, mesoderm, ectoderm, and neural crest (NC) development. We identified clusters of RDs that replicate at unique times in each stage (RT signatures) and confirmed global consolidation of the genome into larger synchronously replicating segments during differentiation. Surprisingly, transcriptome data revealed that the well-accepted correlation between early replication and transcriptional activity was restricted to RT-constitutive genes, whereas two-thirds of the genes that switched RT during differentiation were strongly expressed when late replicating in one or more cell types. Closer inspection revealed that transcription of this class of genes was frequently restricted to the lineage in which the RT switch occurred, but was induced prior to a late-to-early RT switch and/or down-regulated after an early-to-late RT switch. Analysis of transcriptional regulatory networks showed that this class of genes contains strong regulators of genes that were only expressed when early replicating. These results provide intriguing new insight into the complex relationship between transcription and RT regulation during human development.
Assuntos
Linhagem da Célula , Período de Replicação do DNA , Perfilação da Expressão Gênica/métodos , Células-Tronco Pluripotentes/fisiologia , Diferenciação Celular , Células Cultivadas , Análise por Conglomerados , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Genoma Humano , Humanos , Células-Tronco Pluripotentes/citologiaRESUMO
Human embryonic stem cells (hESCs) have received considerable attention due to their therapeutic potential and usefulness in understanding early development and cell fate commitment. In order to appreciate the unique properties of these pluripotent, self-renewing cells, we have performed an in-depth multidimensional fractionation followed by LC-MS/MS analysis of the hESCs harvested from defined media to elucidate expressed, phosphorylated, O-linked ß-N-acetylglucosamine (O-GlcNAc) modified, and secreted proteins. From the triplicate analysis, we were able to assign more than 3000 proteins with less than 1% false-discovery rate. This analysis also allowed us to identify nearly 500 phosphorylation sites and 68 sites of O-GlcNAc modification with the same high confidence. Investigation of the phosphorylation sites allowed us to deduce the set of kinases that are likely active in these cells. We also identified more than 100 secreted proteins of hESCs that likely play a role in extracellular matrix formation and remodeling, as well as autocrine signaling for self-renewal and maintenance of the undifferentiated state. Finally, by performing in-depth analysis in triplicate, spectral counts were obtained for these proteins and posttranslationally modified peptides, which will allow us to perform relative quantitative analysis between these cells and any derived cell type in the future.
Assuntos
Células-Tronco Embrionárias/metabolismo , Proteoma/análise , Acetilglucosamina/análise , Acetilglucosamina/metabolismo , Fracionamento Celular , Linhagem Celular , Células-Tronco Embrionárias/química , Humanos , Fosforilação , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , Proteômica , Espectrometria de Massas em TandemRESUMO
To identify evolutionarily conserved features of replication timing and their relationship to epigenetic properties, we profiled replication timing genome-wide in four human embryonic stem cell (hESC) lines, hESC-derived neural precursor cells (NPCs), lymphoblastoid cells, and two human induced pluripotent stem cell lines (hiPSCs), and compared them with related mouse cell types. Results confirm the conservation of coordinately replicated megabase-sized "replication domains" punctuated by origin-suppressed regions. Differentiation-induced replication timing changes in both species occur in 400- to 800-kb units and are similarly coordinated with transcription changes. A surprising degree of cell-type-specific conservation in replication timing was observed across regions of conserved synteny, despite considerable species variation in the alignment of replication timing to isochore GC/LINE-1 content. Notably, hESC replication timing profiles were significantly more aligned to mouse epiblast-derived stem cells (mEpiSCs) than to mouse ESCs. Comparison with epigenetic marks revealed a signature of chromatin modifications at the boundaries of early replicating domains and a remarkably strong link between replication timing and spatial proximity of chromatin as measured by Hi-C analysis. Thus, early and late initiation of replication occurs in spatially separate nuclear compartments, but rarely within the intervening chromatin. Moreover, cell-type-specific conservation of the replication program implies conserved developmental changes in spatial organization of chromatin. Together, our results reveal evolutionarily conserved aspects of developmentally regulated replication programs in mammals, demonstrate the power of replication profiling to distinguish closely related cell types, and strongly support the hypothesis that replication timing domains are spatially compartmentalized structural and functional units of three-dimensional chromosomal architecture.
Assuntos
Evolução Biológica , Cromatina/genética , Replicação do DNA , Animais , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Humanos , CamundongosRESUMO
Three new bicyclic C(21) terpenoids, clathric acid (1) and two N-acyl taurine derivatives, clathrimides A (2) and B (3), were isolated from the marine sponge Clathria compressa. The structures of these compounds were elucidated by interpretation of spectroscopic data. Clathric acid showed mild antibacterial activity against several Gram-positive bacteria.
Assuntos
Antibacterianos/isolamento & purificação , Poríferos/química , Terpenos/isolamento & purificação , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Bactérias Gram-Positivas/efeitos dos fármacos , Biologia Marinha , Testes de Sensibilidade Microbiana , Estrutura Molecular , Oceanos e Mares , Terpenos/química , Terpenos/farmacologiaRESUMO
Two new briarane diterpenoids briareolate esters J (1) and K (2) were isolated from the methanolic extract of the octocoral Briareum asbestinum collected off the coast of Boca Raton, Florida. The structures of briaranes 1 and 2 were elucidated by interpretation of spectroscopic data. Briareolate ester K (2) showed weak growth inhibition activity against human embryonic stem cells (BG02).
Assuntos
Antozoários/química , Diterpenos/farmacologia , Células-Tronco Embrionárias/efeitos dos fármacos , Ésteres/farmacologia , Animais , Diterpenos/isolamento & purificação , Células-Tronco Embrionárias/metabolismo , Ésteres/isolamento & purificação , Florida , Humanos , Análise EspectralRESUMO
The temporal order of DNA replication is regulated during development and is highly correlated with gene expression, histone modifications and 3D genome architecture. We tracked changes in replication timing, gene expression, and chromatin conformation capture (Hi-C) A/B compartments over the first two cell cycles during differentiation of human embryonic stem cells to definitive endoderm. Remarkably, transcriptional programs were irreversibly reprogrammed within the first cell cycle and were largely but not universally coordinated with replication timing changes. Moreover, changes in A/B compartment and several histone modifications that normally correlate strongly with replication timing showed weak correlation during the early cell cycles of differentiation but showed increased alignment in later differentiation stages and in terminally differentiated cell lines. Thus, epigenetic cell fate transitions during early differentiation can occur despite dynamic and discordant changes in otherwise highly correlated genomic properties.
Assuntos
Reprogramação Celular/genética , Cromatina/genética , Período de Replicação do DNA , Células-Tronco/metabolismo , Transcrição Gênica , Ciclo Celular/genética , Diferenciação Celular/genética , Linhagem da Célula/genética , Cromatina/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Perfilação da Expressão Gênica , Humanos , Modelos Biológicos , Células-Tronco/citologiaRESUMO
Apelin, the endogenous ligand of the G protein-coupled APJ receptor has been shown to promote tumor angiogenesis. However, the effect of apelin on inducing angiogenesis in adipose tissue has not been investigated. In this review, we propose a putative role for apelin in promoting angiogenesis in adipose tissue. We further propose that targeting adipose tissue vasculature by blocking apelin signaling with anti-apelin antibodies will lead not only to inhibition of angiogenesis in adipose tissue but also to decreased adiposity.
Assuntos
Tecido Adiposo/irrigação sanguínea , Tecido Adiposo/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Modelos Biológicos , Neovascularização Patológica/metabolismo , Obesidade/etiologia , Obesidade/metabolismo , Animais , Apelina , HumanosRESUMO
BACKGROUND: Much of our current knowledge of the molecular expression profile of human embryonic stem cells (hESCs) is based on transcriptional approaches. These analyses are only partly predictive of protein expression however, and do not shed light on post-translational regulation, leaving a large gap in our knowledge of the biology of pluripotent stem cells. RESULTS: Here we describe the use of two large-scale western blot assays to identify over 600 proteins expressed in undifferentiated hESCs, and highlight over 40 examples of multiple gel mobility variants, which are suspected protein isoforms and/or post-translational modifications. Twenty-two phosphorylation events in cell signaling molecules, as well as potential new markers of undifferentiated hESCs were also identified. We confirmed the expression of a subset of the identified proteins by immunofluorescence and correlated the expression of transcript and protein for key molecules in active signaling pathways in hESCs. These analyses also indicated that hESCs exhibit several features of polarized epithelia, including expression of tight junction proteins. CONCLUSION: Our approach complements proteomic and transcriptional analysis to provide unique information on human pluripotent stem cells, and is a framework for the continued analyses of self-renewal.
Assuntos
Células-Tronco Embrionárias/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Western Blotting , Imunofluorescência , Humanos , Imuno-Histoquímica , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Ocludina , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Processamento de Proteína Pós-Traducional , Proteoma/classificação , Proteoma/genética , Transcrição Gênica , Proteína da Zônula de Oclusão-1RESUMO
The number of human embryonic stem cell (hESC) lines available to federally funded U.S. researchers is currently limited. Thus, determining their basic characteristics and disseminating these lines is important. In this report, we recovered and expanded the earliest available cryopreserved stocks of the BG01, BG02, and BG03 hESC lines. These cultures exhibited multiple definitive characteristics of undifferentiated cells, including long-term self-renewal, expression of markers of pluripotency, maintenance of a normal karyotype, and differentiation to mesoderm, endoderm, and ectoderm. Each cell line exhibited a unique genotype and human leukocyte antigen (HLA) isotype, confirming that they were isolated independently. BG01, BG02, and BG03 maintained in feederfree conditions demonstrated self-renewal, maintenance of normal karyotype, and gene expression indicative of undifferentiated pluripotent stem cells. A survey of gene expression in BG02 cells using massively parallel signature sequencing generated a digital read-out of transcript abundance and showed that this line was similar to other hESC lines. BG01, BG02, and BG03 hESCs are therefore independent, undifferentiated, and pluripotent lines that can be maintained without accumulation of karyotypic abnormalities.
Assuntos
Técnicas de Cultura de Células , Linhagem Celular , Embrião de Mamíferos/citologia , Genótipo , Cariotipagem , Células-Tronco Pluripotentes/citologia , Células-Tronco/citologia , Fosfatase Alcalina/metabolismo , Diferenciação Celular , Linhagem da Célula , Criopreservação , Citogenética/métodos , DNA Complementar/metabolismo , Regulação da Expressão Gênica , Teste de Histocompatibilidade , Humanos , Microscopia de Fluorescência , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Transdução de SinaisRESUMO
We have reported relatively efficient methods for somatic cell nuclear transfer and for knocking out the alpha(1,3)-galactosyltransferase (alpha1,3-GT) gene in porcine fetal fibroblasts using a nonisogenic promoterless construct approach. Here we report the production of alpha1,3-GT gene knockout pigs using these procedures. Seven alpha1,3-GT gene knockout cell clones were identified by long-range PCR from 108 neomycin resistant (neo(R)) colonies, giving a 6.5% targeting efficiency. Three cell clones were used for nuclear transfer. Nuclear transfer was performed using a fusion before activation protocol using in vitro-matured adult oocytes. Between 51 and 110 fused couplets were transferred to 10 recipients synchronized 1 day behind the embryos. Parturition was induced on day 115, and piglets were delivered by caesarean section. Four recipients gave birth to a total of 18 live piglets. All pigs were female, and all three clones resulted in the birth of live pigs. alpha1,3-GT gene knockout pigs were identified by long-range PCR and confirmed by Southern blot analysis. The efficiency (embryos transferred/piglets born) of our cloning protocol was 1.9% for all transfers and 4.6% for animals that gave birth.
Assuntos
Fibroblastos , Galactosiltransferases/genética , Deleção de Genes , Técnicas de Transferência Nuclear , Oócitos , Suínos/genética , Animais , Animais Geneticamente Modificados/genética , Feminino , GravidezRESUMO
PURPOSE: To explore a karyotypically abnormal variant human embryonic stem cell (hESC) line, BG01V, as a potential model for studies of dopaminergic neuronal differentiation. METHODS: The properties of BG01V cells were compared to those of normal BG01 cells using immunocytochemistry, RT-PCR, focused microarrays and in vitro differentiation, including dopaminergic differentiation, by culturing with the stromal cell line PA6. RESULTS: Despite the karyotypic abnormality (49, +12, +17 and XXY), undifferentiated BG01V cells expressed pluripotent ESC markers similar to BG01 cells, and retained the ability to differentiate into cell types characteristic of all three germ layers. When co-cultured with the stromal cell line PA6, BG01V cells differentiated into dopaminergic cells which exhibited properties similar to those of mature dopaminergic neurons. CONCLUSIONS: BG01V cells were easier to maintain in culture than karyotypically normal BG01 cells and can be used as an alternative pluripotent hESC type for in vitro developmental studies.
Assuntos
Diferenciação Celular/fisiologia , Proliferação de Células , Dopamina/metabolismo , Células-Tronco/fisiologia , Northern Blotting/métodos , Linhagem Celular , Aberrações Cromossômicas , Dopamina/genética , Embrião de Mamíferos , Regulação da Expressão Gênica/fisiologia , Humanos , Imuno-Histoquímica/métodos , Neurônios/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Embryonic development is characterized by dynamic changes in gene expression, yet the role of chromatin remodeling in these cellular transitions remains elusive. To address this question, we profiled the transcriptome and select chromatin modifications at defined stages during pancreatic endocrine differentiation of human embryonic stem cells. We identify removal of Polycomb group (PcG)-mediated repression on stage-specific genes as a key mechanism for the induction of developmental regulators. Furthermore, we discover that silencing of transitory genes during lineage progression associates with reinstatement of PcG-dependent repression. Significantly, in vivo- but not in vitro-differentiated endocrine cells exhibit close similarity to primary human islets in regard to transcriptome and chromatin structure. We further demonstrate that endocrine cells produced in vitro do not fully eliminate PcG-mediated repression on endocrine-specific genes, probably contributing to their malfunction. These studies reveal dynamic chromatin remodeling during developmental lineage progression and identify possible strategies for improving cell differentiation in culture.
Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , Células-Tronco Embrionárias/citologia , Pâncreas/citologia , Proteínas do Grupo Polycomb/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Montagem e Desmontagem da Cromatina/genética , Células-Tronco Embrionárias/metabolismo , Células Endócrinas/citologia , Células Endócrinas/metabolismo , Endoderma/citologia , Endoderma/metabolismo , Humanos , Camundongos , Proteínas do Grupo Polycomb/genéticaRESUMO
Development of a human embryonic stem cell (hESC)-based therapy for type 1 diabetes will require the translation of proof-of-principle concepts into a scalable, controlled, and regulated cell manufacturing process. We have previously demonstrated that hESC can be directed to differentiate into pancreatic progenitors that mature into functional glucose-responsive, insulin-secreting cells in vivo. In this study we describe hESC expansion and banking methods and a suspension-based differentiation system, which together underpin an integrated scalable manufacturing process for producing pancreatic progenitors. This system has been optimized for the CyT49 cell line. Accordingly, qualified large-scale single-cell master and working cGMP cell banks of CyT49 have been generated to provide a virtually unlimited starting resource for manufacturing. Upon thaw from these banks, we expanded CyT49 for two weeks in an adherent culture format that achieves 50-100 fold expansion per week. Undifferentiated CyT49 were then aggregated into clusters in dynamic rotational suspension culture, followed by differentiation en masse for two weeks with a four-stage protocol. Numerous scaled differentiation runs generated reproducible and defined population compositions highly enriched for pancreatic cell lineages, as shown by examining mRNA expression at each stage of differentiation and flow cytometry of the final population. Islet-like tissue containing glucose-responsive, insulin-secreting cells was generated upon implantation into mice. By four- to five-months post-engraftment, mature neo-pancreatic tissue was sufficient to protect against streptozotocin (STZ)-induced hyperglycemia. In summary, we have developed a tractable manufacturing process for the generation of functional pancreatic progenitors from hESC on a scale amenable to clinical entry.
Assuntos
Técnicas de Cultura Celular por Lotes/métodos , Diferenciação Celular/fisiologia , Diabetes Mellitus Tipo 1/terapia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/transplante , Células Secretoras de Insulina/citologia , Análise de Variância , Animais , Criopreservação/métodos , Células-Tronco Embrionárias/fisiologia , Citometria de Fluxo , Imunofluorescência , Perfilação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos SCID , EstreptozocinaRESUMO
Three new briarane diterpenoids, briareolate esters L-N (1-3), have been isolated from a gorgonian Briareum asbestinum. Briareolate esters L (1) and M (2) are the first natural products possessing a 10-membered macrocyclic ring with a (E,Z)-dieneone and exhibit growth inhibition activity against both human embryonic stem cells (BG02) and a pancreatic cancer cell line (BxPC-3). Briareolate ester L (1) was found to contain a "spring-loaded" (E,Z)-dieneone Michael acceptor group that can form a reversible covalent bond to model sulfur-based nucleophiles.
Assuntos
Antozoários/química , Diterpenos/química , Animais , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Linhagem Celular , Diterpenos/isolamento & purificação , Humanos , Modelos Moleculares , Estrutura MolecularRESUMO
Novocell, Inc. is a stem cell engineering company creating, delivering and commercializing cell and drug therapies for diabetes and other chronic diseases. The use of human embryonic stem cells provides a scalable source of any differentiated lineage that has potential for cell replacement therapy, as well as tools for drug discovery to create regenerative medicines.
Assuntos
Biotecnologia/métodos , Biotecnologia/organização & administração , Pesquisas com Embriões , Células-Tronco Embrionárias/citologia , Técnicas de Transferência Nuclear , Medicina Regenerativa , Engenharia Tecidual/métodos , Animais , Diferenciação Celular/fisiologia , Endoderma/metabolismo , HumanosRESUMO
Pluripotent cells can be isolated from the human blastocyst and maintained in culture as self-renewing, undifferentiated, human ESCs (hESCs). These cells are a valuable model of human development in vitro and are the focus of substantial research aimed at generating differentiated populations for cellular therapies. The extracellular markers that have been used to characterize hESCs are primarily carbohydrate epitopes on proteoglycans or sphingolipids, such as stage-specific embryonic antigen (SSEA)-3 and -4. The expression of SSEA-3 and -4 is tightly regulated during preimplantation development and on hESCs. Although this might imply a molecular function in undifferentiated cells, it has not yet been tested experimentally. We used inhibitors of sphingolipid and glycosphingolipid (GSL) biosynthesis to block the generation of SSEA-3 and -4 in hESCs. Depletion of these antigens and their precursors was confirmed using immunostaining, flow cytometry, and tandem mass spectroscopy. Transcriptional analysis, immunostaining, and differentiation in vitro and in teratomas indicated that other properties of pluripotency were not noticeably affected by GSL depletion. These experiments demonstrated that the GSLs recognized as SSEA-3 and -4 do not play critical functional roles in maintaining the pluripotency of hESCs, but instead suggested roles for this class of molecules during cellular differentiation.
Assuntos
Antígenos Glicosídicos Associados a Tumores/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Glicoesfingolipídeos/fisiologia , Células-Tronco Pluripotentes/fisiologia , Células Cultivadas , Citometria de Fluxo , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Glicoesfingolipídeos/deficiência , Glicoesfingolipídeos/genética , Humanos , Células-Tronco Pluripotentes/citologia , Antígenos Embrionários Estágio-EspecíficosRESUMO
Despite progress in developing defined conditions for human embryonic stem cell (hESC) cultures, little is known about the cell-surface receptors that are activated under conditions supportive of hESC self-renewal. A simultaneous interrogation of 42 receptor tyrosine kinases (RTKs) in hESCs following stimulation with mouse embryonic fibroblast (MEF) conditioned medium (CM) revealed rapid and prominent tyrosine phosphorylation of insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R); less prominent tyrosine phosphorylation of epidermal growth factor receptor (EGFR) family members, including ERBB2 and ERBB3; and trace phosphorylation of fibroblast growth factor receptors. Intense IGF1R and IR phosphorylation occurred in the absence of MEF conditioning (NCM) and was attributable to high concentrations of insulin in the proprietary KnockOut Serum Replacer (KSR). Inhibition of IGF1R using a blocking antibody or lentivirus-delivered shRNA reduced hESC self-renewal and promoted differentiation, while disruption of ERBB2 signaling with the selective inhibitor AG825 severely inhibited hESC proliferation and promoted apoptosis. A simple defined medium containing an IGF1 analog, heregulin-1beta (a ligand for ERBB2/ERBB3), fibroblast growth factor-2 (FGF2), and activin A supported long-term growth of multiple hESC lines. These studies identify previously unappreciated RTKs that support hESC proliferation and self-renewal, and provide a rationally designed medium for the growth and maintenance of pluripotent hESCs.
Assuntos
Proliferação de Células , Células-Tronco Embrionárias/metabolismo , Células-Tronco Pluripotentes/metabolismo , Receptor ErbB-2/metabolismo , Receptor IGF Tipo 2/metabolismo , Transdução de Sinais/fisiologia , Animais , Anticorpos Monoclonais/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Benzotiazóis/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Meios de Cultivo Condicionados , Células-Tronco Embrionárias/citologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Camundongos , Neuregulina-1/farmacologia , Fosforilação/efeitos dos fármacos , Células-Tronco Pluripotentes/citologia , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-3/antagonistas & inibidores , Receptor ErbB-3/metabolismo , Receptor IGF Tipo 2/antagonistas & inibidores , Receptor de Insulina/antagonistas & inibidores , Receptor de Insulina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tirfostinas/farmacologiaRESUMO
Human embryonic stem (hES) cells originate during an embryonic period of active epigenetic remodeling. DNA methylation patterns are likely to be critical for their self-renewal and pluripotence. We compared the DNA methylation status of 1536 CpG sites (from 371 genes) in 14 independently isolated hES cell lines with five other cell types: 24 cancer cell lines, four adult stem cell populations, four lymphoblastoid cell lines, five normal human tissues, and an embryonal carcinoma cell line. We found that the DNA methylation profile clearly distinguished the hES cells from all of the other cell types. A subset of 49 CpG sites from 40 genes contributed most to the differences among cell types. Another set of 25 sites from 23 genes distinguished hES cells from normal differentiated cells and can be used as biomarkers to monitor differentiation. Our results indicate that hES cells have a unique epigenetic signature that may contribute to their developmental potential.
Assuntos
Metilação de DNA , Embrião de Mamíferos/citologia , Epigênese Genética , Células-Tronco/metabolismo , Diferenciação Celular , Linhagem Celular , Linhagem Celular Tumoral , Linhagem da Célula , Análise por Conglomerados , Feminino , Humanos , Masculino , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Células-Tronco/citologiaRESUMO
Human pluripotent embryonic stem (ES) cells have important potential in regenerative medicine and as models for human preimplantation development; however, debate continues over whether embryos should be destroyed to produce human ES cells. We have derived four ES cell lines on mouse embryonic fibroblast cells in medium supplemented with basic fibroblast growth factor, human recombinant leukemia inhibitory factor, and fetal bovine serum. The source of these cell lines was poor-quality embryos that in the course of routine clinical practice would have been discarded. After continuous proliferation in vitro for more than 12 months, these ES cell lines maintained their developmental potential to form trophoblast and somatic cells, including cardiac muscle and neuronal tissue.