RESUMO
The signaling mechanisms controlling somatic cell reprogramming are not fully understood. In this study, we report a novel role for mitochondrial Akt1 signaling that enhanced somatic cell reprogramming efficiency. The role of mitochondrial Akt1 in somatic cell reprogramming was investigated by transducing fibroblasts with the four reprogramming factors (Oct4, Sox2, Klf4, c-Myc) in conjunction with Mito-Akt1, Mito-dnAkt1, or control virus. Mito-Akt1 enhanced reprogramming efficiency whereas Mito-dnAkt1 inhibited reprogramming. The resulting iPSCs formed embryoid bodies in vitro and teratomas in vivo. Moreover, Oct4 and Nanog promoter methylation was reduced in the iPSCs generated in the presence of Mito-Akt1. Akt1 was activated and translocated into mitochondria after growth factor stimulation in embryonic stem cells (ESCs). To study the effect of mitochondrial Akt in ESCs, a mitochondria-targeting constitutively active Akt1 (Mito-Akt1) was expressed in ESCs. Gene expression profiling showed upregulation of genes that promote stem cell proliferation and survival and down-regulation of genes that promote differentiation. Analysis of cellular respiration indicated similar metabolic profile in the resulting iPSCs and ESCs, suggesting comparable bioenergetics. These findings showed that activation of mitochondrial Akt1 signaling was required during somatic cell reprogramming.
Assuntos
Diferenciação Celular , Reprogramação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 4 Semelhante a Kruppel , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Ativação TranscricionalRESUMO
The ability to realize the full potential of human pluripotent stem cells (hPSCs) as tools for -understanding human development and advancing the field of regenerative medicine is dependent on efficient methods to genetically manipulate these cells. There are several methods for introducing foreign DNA into cells such as electroporation, lipid-based transfection technology, and viral transduction. We describe here a method to transfect human embryonic stem cells (hESCs) using nucleofection technology. This unique method uses the Nucleofector II Device that combines the use of a cell type-specific Nucleofector Solution and preprogrammed electrical parameters to efficiently deliver DNA into the cell nucleus. The use of this technology allows high-efficiency transfer of nucleic acids into hESCs enabling both transient and stable manipulation of gene expression in these cells.
Assuntos
Núcleo Celular/metabolismo , Eletroporação/métodos , Células-Tronco Embrionárias/metabolismo , Transfecção/métodos , Animais , Células Cultivadas , Células-Tronco Embrionárias/citologia , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Indicadores e Reagentes , Camundongos , Sus scrofaRESUMO
BACKGROUND: Human embryonic stem cells (hESC) are stem cells capable of differentiating into cells representative of the three primary embryonic germ layers. There has been considerable interest in understanding the mechanisms regulating stem cell pluripotency, which will ultimately lead to development of more efficient methods to derive and culture hESC. In particular, Oct4, Sox2 and Nanog are transcription factors known to be important in maintenance of hESC. However, many of the downstream targets of these transcription factors are not well characterized. Furthermore, it remains unknown whether additional novel stem cell factors are involved in the establishment and maintenance of the stem cell state. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that a novel gene, L1TD1 (also known as FLJ10884 or ECAT11), is abundantly expressed in undifferentiated hESC. Differentiation of hESC via embryoid body (EB) formation or BMP4 treatment results in the rapid down-regulation of L1TD1 expression. Furthermore, populations of undifferentiated and differentiated hESC were sorted using the stem cell markers SSEA4 and TRA160. Our results show that L1TD1 is enriched in the SSEA4-positive or TRA160-positive population of hESC. Using chromatin immunoprecipitation we found enriched association of Nanog to the predicted promoter region of L1TD1. Furthermore, siRNA-mediated knockdown of Nanog in hESC also resulted in downregulation of L1TD1 expression. Finally, using luciferase reporter assay we demonstrated that Nanog can activate the L1TD1 upstream promoter region. Altogether, these results provide evidence that L1TD1 is a downstream target of Nanog. CONCLUSION/SIGNIFICANCE: Taken together, our results suggest that L1TD1 is a downstream target of Nanog and represents a useful marker for identifying undifferentiated hESC.
Assuntos
Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Proteínas/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem Celular , Separação Celular , Imunoprecipitação da Cromatina , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Modelos Biológicos , Proteína Homeobox Nanog , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismoRESUMO
Autosomal dominant facioscapulohumeral muscular dystrophy (FSHD) is likely caused by epigenetic alterations in chromatin involving contraction of the D4Z4 repeat array near the telomere of chromosome 4q. The precise mechanism by which deletions of D4Z4 influence gene expression in FSHD is not yet resolved. Regulatory models include a cis effect on proximal gene transcription (position effect), DNA looping, non-coding RNA, nuclear localization and trans-effects. To directly test whether deletions of D4Z4 affect gene expression in cis, nascent RNA was examined in single myonuclei so that transcription from each allele could be measured independently. FSHD and control myotubes (differentiated myoblasts) were subjected to sequential RNA-DNA FISH. A total of 16 genes in the FSHD region (FRG2, TUBB4Q, FRG1, FAT1, F11, KLKB1, CYP4V2, TLR3, SORBS2, PDLIM3 (ALP), LRP2BP, ING2, SNX25, SLC25A4 (ANT1), HELT and IRF2) were examined for interallelic variation in RNA expression within individual myonuclei. Sequential DNA hybridization with a unique 4q35 chromosome probe was then applied to confirm the localization of nascent RNA to 4q. A D4Z4 probe, labeled with a third fluorochrome, distinguished between the deleted and normal allele in FSHD nuclei. Our data do not support an FSHD model in which contracted D4Z4 arrays induce altered transcription in cis from 4q35 genes, even for those genes (FRG1, FRG2 and SLC25A4 (ANT1)) for which such an effect has been proposed.
Assuntos
Cromatina/genética , DNA/genética , Hibridização in Situ Fluorescente , Distrofia Muscular Facioescapuloumeral/genética , RNA/genética , Transcrição Gênica , Alelos , Núcleo Celular/genética , Células Cultivadas , Mapeamento Cromossômico , Cromossomos Humanos Par 4/genética , Humanos , Microscopia de Fluorescência , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Sequências de Repetição em Tandem , Telômero/genéticaRESUMO
Facioscapulohumeral dystrophy (FSHD) is an autosomal dominant muscular dystrophy in which no mutation of pathogenic gene(s) has been identified. Instead, the disease is, in most cases, genetically linked to a contraction in the number of 3.3 kb D4Z4 repeats on chromosome 4q. How contraction of the 4qter D4Z4 repeats causes muscular dystrophy is not understood. In addition, a smaller group of FSHD cases are not associated with D4Z4 repeat contraction (termed "phenotypic" FSHD), and their etiology remains undefined. We carried out chromatin immunoprecipitation analysis using D4Z4-specific PCR primers to examine the D4Z4 chromatin structure in normal and patient cells as well as in small interfering RNA (siRNA)-treated cells. We found that SUV39H1-mediated H3K9 trimethylation at D4Z4 seen in normal cells is lost in FSHD. Furthermore, the loss of this histone modification occurs not only at the contracted 4q D4Z4 allele, but also at the genetically intact D4Z4 alleles on both chromosomes 4q and 10q, providing the first evidence that the genetic change (contraction) of one 4qD4Z4 allele spreads its effect to other genomic regions. Importantly, this epigenetic change was also observed in the phenotypic FSHD cases with no D4Z4 contraction, but not in other types of muscular dystrophies tested. We found that HP1gamma and cohesin are co-recruited to D4Z4 in an H3K9me3-dependent and cell type-specific manner, which is disrupted in FSHD. The results indicate that cohesin plays an active role in HP1 recruitment and is involved in cell type-specific D4Z4 chromatin regulation. Taken together, we identified the loss of both histone H3K9 trimethylation and HP1gamma/cohesin binding at D4Z4 to be a faithful marker for the FSHD phenotype. Based on these results, we propose a new model in which the epigenetic change initiated at 4q D4Z4 spreads its effect to other genomic regions, which compromises muscle-specific gene regulation leading to FSHD pathogenesis.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Histonas/metabolismo , Distrofia Muscular Facioescapuloumeral/metabolismo , Animais , Cricetinae , Eucromatina/metabolismo , Células HeLa , Heterocromatina/metabolismo , Humanos , Metilação , Metiltransferases/metabolismo , Camundongos , Modelos Moleculares , Distrofia Muscular Facioescapuloumeral/genética , Reação em Cadeia da Polimerase , Proteínas Repressoras/metabolismo , Sequências de Repetição em Tandem , Células Tumorais Cultivadas , CoesinasRESUMO
High-efficiency genetic modification of human embryonic stem (hES) cells would enable manipulation of gene activity, routine gene targeting, and development of new human disease models and treatments. Chemical transfection, nucleofection, and electroporation of hES cells result in low transfection efficiencies. Viral transduction is efficient but has significant drawbacks. Here we describe techniques to transiently and stably express transgenes in hES cells with high efficiency using a widely available vector system. The technique combines nucleofection of single hES cells with improved methods to select hES cells at clonal density. As validation, we reduced Oct4 and Nanog expression using siRNAs and shRNA vectors in hES cells. Furthermore, we derived many hES cell clones with either stably reduced alkaline phosphatase activity or stably overexpressed green fluorescent protein. These clones retained stem cell characteristics (normal karyotype, stem cell marker expression, self-renewal, and pluripotency). These studies will accelerate efforts to interrogate gene function and define the parameters that control growth and differentiation of hES cells. Disclosure of potential conflicts of interest is found at the end of this article.
Assuntos
Células-Tronco Embrionárias/fisiologia , Regulação da Expressão Gênica , Transfecção/métodos , Transgenes , Fosfatase Alcalina/genética , Técnicas de Cultura de Células , Sobrevivência Celular , Citomegalovirus/genética , DNA/genética , Células-Tronco Embrionárias/citologia , Citometria de Fluxo , Vetores Genéticos , Humanos , Cariotipagem , Reação em Cadeia da Polimerase/métodos , RNA Interferente Pequeno/genética , RNA Viral/genéticaRESUMO
Growth of human embryonic stem (hES) cells as a pluripotent population requires a balance between survival, proliferation and self-renewal signals. Here we demonstrate that hES cells express receptors of the tropomyosin-related kinase (TRK) family, which mediate antiapoptotic signals. We show that three TRK ligands, brain-derived neurotrophic factor, neurotrophin 3 and neurotrophin 4, are survival factors for hES cells. Addition of neurotrophins to hES cell cultures effects a 36-fold improvement in their clonal survival. hES cell cultures maintained in medium containing neurotrophins remain diploid and retain full developmental potency. In the presence of neurotrophins, TRK receptors in hES cells are phosphorylated; TRK receptor inhibition leads to hES cell apoptosis. The survival activity of neurotrophins in hES cells is mediated by the phosphatidylinositol-3-kinase pathway but not the mitogen-activated protein kinase pathway. Neurotrophins improve hES cell survival and may facilitate their manipulation and the development of high-throughput screens to identify factors responsible for hES cell differentiation.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Fatores de Crescimento Neural/metabolismo , Neurotrofina 3/metabolismo , Células-Tronco/citologia , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células Clonais/efeitos dos fármacos , Células Clonais/metabolismo , Relação Dose-Resposta a Droga , Humanos , Fatores de Crescimento Neural/farmacologia , Neurotrofina 3/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Receptor trkB/metabolismo , Receptor trkC/metabolismoRESUMO
Global gene-expression analyses of human embryonic stem cells confirm the involvement of some known genes in stem-cell function and identify some new candidate regulators of stem-cell growth. Support remains elusive, however, for the concept of 'stemness'--a pattern of expression of genes that is common to all stem cells.