RESUMO
Pluripotent cells can be derived from fibroblasts by ectopic expression of defined transcription factors. A fundamental unresolved question is whether terminally differentiated cells can be reprogrammed to pluripotency. We utilized transgenic and inducible expression of four transcription factors (Oct4, Sox2, Klf4, and c-Myc) to reprogram mouse B lymphocytes. These factors were sufficient to convert nonterminally differentiated B cells to a pluripotent state. However, reprogramming of mature B cells required additional interruption with the transcriptional state maintaining B cell identity by either ectopic expression of the myeloid transcription factor CCAAT/enhancer-binding-protein-alpha (C/EBPalpha) or specific knockdown of the B cell transcription factor Pax5. Multiple iPS lines were clonally derived from both nonfully and fully differentiated B lymphocytes, which gave rise to adult chimeras with germline contribution, and to late-term embryos when injected into tetraploid blastocysts. Our study provides definite proof for the direct nuclear reprogramming of terminally differentiated adult cells to pluripotency.
Assuntos
Linfócitos B/citologia , Diferenciação Celular , Células-Tronco Pluripotentes/citologia , Animais , Núcleo Celular/genética , Células-Tronco Embrionárias/citologia , Humanos , Fator 4 Semelhante a Kruppel , Camundongos , Fatores de Transcrição/metabolismoRESUMO
There is growing recognition that mammalian cells produce many thousands of large intergenic transcripts. However, the functional significance of these transcripts has been particularly controversial. Although there are some well-characterized examples, most (>95%) show little evidence of evolutionary conservation and have been suggested to represent transcriptional noise. Here we report a new approach to identifying large non-coding RNAs using chromatin-state maps to discover discrete transcriptional units intervening known protein-coding loci. Our approach identified approximately 1,600 large multi-exonic RNAs across four mouse cell types. In sharp contrast to previous collections, these large intervening non-coding RNAs (lincRNAs) show strong purifying selection in their genomic loci, exonic sequences and promoter regions, with greater than 95% showing clear evolutionary conservation. We also developed a functional genomics approach that assigns putative functions to each lincRNA, demonstrating a diverse range of roles for lincRNAs in processes from embryonic stem cell pluripotency to cell proliferation. We obtained independent functional validation for the predictions for over 100 lincRNAs, using cell-based assays. In particular, we demonstrate that specific lincRNAs are transcriptionally regulated by key transcription factors in these processes such as p53, NFkappaB, Sox2, Oct4 (also known as Pou5f1) and Nanog. Together, these results define a unique collection of functional lincRNAs that are highly conserved and implicated in diverse biological processes.
Assuntos
Cromatina/genética , Sequência Conservada , Mamíferos/genética , RNA/genética , Animais , Sequência de Bases , Células Cultivadas , Sequência Conservada/genética , DNA Intergênico , Éxons/genética , Camundongos , Regiões Promotoras Genéticas/genética , Reprodutibilidade dos Testes , Fatores de Transcrição/metabolismoRESUMO
Human and mouse embryonic stem cells (ESCs) are derived from blastocyst-stage embryos but have very different biological properties, and molecular analyses suggest that the pluripotent state of human ESCs isolated so far corresponds to that of mouse-derived epiblast stem cells (EpiSCs). Here we rewire the identity of conventional human ESCs into a more immature state that extensively shares defining features with pluripotent mouse ESCs. This was achieved by ectopic induction of Oct4, Klf4, and Klf2 factors combined with LIF and inhibitors of glycogen synthase kinase 3beta (GSK3beta) and mitogen-activated protein kinase (ERK1/2) pathway. Forskolin, a protein kinase A pathway agonist which can induce Klf4 and Klf2 expression, transiently substitutes for the requirement for ectopic transgene expression. In contrast to conventional human ESCs, these epigenetically converted cells have growth properties, an X-chromosome activation state (XaXa), a gene expression profile, and a signaling pathway dependence that are highly similar to those of mouse ESCs. Finally, the same growth conditions allow the derivation of human induced pluripotent stem (iPS) cells with similar properties as mouse iPS cells. The generation of validated "naïve" human ESCs will allow the molecular dissection of a previously undefined pluripotent state in humans and may open up new opportunities for patient-specific, disease-relevant research.
Assuntos
Desdiferenciação Celular/fisiologia , Células-Tronco Embrionárias/fisiologia , Células-Tronco Pluripotentes/fisiologia , Ativação Transcricional/fisiologia , Animais , Colforsina/farmacologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Perfilação da Expressão Gênica , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/metabolismo , Especificidade da Espécie , Ativação Transcricional/efeitos dos fármacosRESUMO
Pluripotent cells can be derived from different types of somatic cells by nuclear reprogramming through the ectopic expression of four transcription factors, Oct3/4, Sox2, Klf4, and c-Myc. However, it is unclear whether postmitotic neurons are susceptible to direct reprogramming. Here, we show that postnatal cortical neurons, the vast majority of which are postmitotic, are amenable to epigenetic reprogramming. However, ectopic expression of the four canonical reprogramming factors is not sufficient to reprogram postnatal neurons. Efficient reprogramming was only achieved after forced cell proliferation by p53 suppression. Additionally, overexpression of repressor element-1 silencing transcription, a suppressor of neuronal gene activity, increased reprogramming efficiencies in combination with the reprogramming factors. Our findings indicate that terminally differentiated postnatal neurons are able to acquire the pluripotent state by direct epigenetic reprogramming, and this process is made more efficient through the suppression of lineage specific gene expression.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Neurônios/citologia , Proteínas Repressoras/metabolismo , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Blastocisto/citologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Técnicas de Cocultura , Transferência Embrionária , Fibroblastos/citologia , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Homeodomínio/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/transplante , Fator 4 Semelhante a Kruppel , Antígenos CD15/metabolismo , Camundongos , Proteína Homeobox Nanog , Neurônios/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Regiões Promotoras Genéticas , Teratoma/patologia , Quimeras de TransplanteRESUMO
Overexpression of transcription factors has been used to directly reprogram somatic cells into a range of other differentiated cell types, including multipotent neural stem cells (NSCs), that can be used to generate neurons and glia. However, the ability to maintain the NSC state independent of the inducing factors and the identity of the somatic donor cells remain two important unresolved issues in transdifferentiation. Here we used transduction of doxycycline-inducible transcription factors to generate stable tripotent NSCs. The induced NSCs (iNSCs) maintained their characteristics in the absence of exogenous factor expression and were transcriptionally, epigenetically, and functionally similar to primary brain-derived NSCs. Importantly, we also generated tripotent iNSCs from multiple adult cell types, including mature liver and B cells. Our results show that self-maintaining proliferative neural cells can be induced from nonectodermal cells by expressing specific combinations of transcription factors.
Assuntos
Linfócitos B/citologia , Linhagem da Célula , Transdiferenciação Celular , Hepatócitos/citologia , Células-Tronco Neurais/citologia , Animais , Linfócitos B/metabolismo , Linhagem da Célula/genética , Transdiferenciação Celular/genética , Reprogramação Celular , Análise por Conglomerados , Epigênese Genética , Expressão Gênica , Perfilação da Expressão Gênica , Hepatócitos/metabolismo , Camundongos , Células-Tronco Neurais/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Autophagy dysfunction has been implicated in misfolded protein accumulation and cellular toxicity in several diseases. Whether alterations in autophagy also contribute to the pathology of lipid-storage disorders is not clear. Here, we show defective autophagy in Niemann-Pick type C1 (NPC1) disease associated with cholesterol accumulation, where the maturation of autophagosomes is impaired because of defective amphisome formation caused by failure in SNARE machinery, whereas the lysosomal proteolytic function remains unaffected. Expression of functional NPC1 protein rescues this defect. Inhibition of autophagy also causes cholesterol accumulation. Compromised autophagy was seen in disease-affected organs of Npc1 mutant mice. Of potential therapeutic relevance is that HP-ß-cyclodextrin, which is used for cholesterol-depletion treatment, impedes autophagy, whereas stimulating autophagy restores its function independent of amphisome formation. Our data suggest that a low dose of HP-ß-cyclodextrin that does not perturb autophagy, coupled with an autophagy inducer, may provide a rational treatment strategy for NPC1 disease.
Assuntos
Autofagia , Glicoproteínas de Membrana/metabolismo , Doença de Niemann-Pick Tipo C/metabolismo , Animais , Células Cultivadas , Colesterol/deficiência , Colesterol/metabolismo , Células HEK293 , Humanos , Lisossomos/metabolismo , Glicoproteínas de Membrana/genética , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Proteína C1 de Niemann-Pick , Doença de Niemann-Pick Tipo C/genética , Ratos , Proteínas SNARE/metabolismo , beta-Ciclodextrinas/farmacologiaRESUMO
Recent advances in somatic cell reprogramming have highlighted the plasticity of the somatic epigenome, particularly through demonstrations of direct lineage reprogramming of one somatic cell type to another by defined factors. However, it is not clear to what extent this type of reprogramming is able to generate fully functional differentiated cells. In addition, the activity of the reprogrammed cells in cell transplantation assays, such as those envisaged for cell-based therapy of Parkinson's disease (PD), remains to be determined. Here we show that ectopic expression of defined transcription factors in mouse tail tip fibroblasts is sufficient to induce Pitx3+ neurons that closely resemble midbrain dopaminergic (DA) neurons. In addition, transplantation of these induced DA (iDA) neurons alleviates symptoms in a mouse model of PD. Thus, iDA neurons generated from abundant somatic fibroblasts by direct lineage reprogramming hold promise for modeling neurodegenerative disease and for cell-based therapies of PD.
Assuntos
Diferenciação Celular , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Fibroblastos/citologia , Animais , Neurônios Dopaminérgicos/transplante , Perfilação da Expressão Gênica , Camundongos , Doença de Parkinson/terapia , Fatores de Transcrição/metabolismoRESUMO
We compared two genetically highly defined transgenic systems to identify parameters affecting reprogramming of somatic cells to a pluripotent state. Our results demonstrate that the level and stoichiometry of reprogramming factors during the reprogramming process strongly influence the resulting pluripotency of iPS cells. High expression of Oct4 and Klf4 combined with lower expression of c-Myc and Sox2 produced iPS cells that efficiently generated "all-iPSC mice" by tetraploid (4n) complementation, maintained normal imprinting at the Dlk1-Dio3 locus, and did not create mice with tumors. Loss of imprinting (LOI) at the Dlk1-Dio3 locus did not strictly correlate with reduced pluripotency though the efficiency of generating "all-iPSC mice" was diminished. Our data indicate that stoichiometry of reprogramming factors can influence epigenetic and biological properties of iPS cells. This concept complicates efforts to define a "generic" epigenetic state of iPSCs and ESCs and should be considered when comparing different iPS and ES cell lines.
Assuntos
Reprogramação Celular/fisiologia , Epigênese Genética , Células-Tronco Pluripotentes Induzidas/fisiologia , Animais , Biomarcadores/metabolismo , Linhagem Celular , Células-Tronco Pluripotentes Induzidas/citologia , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , TransgenesRESUMO
Targeted genetic engineering of human pluripotent cells is a prerequisite for exploiting their full potential. Such genetic manipulations can be achieved using site-specific nucleases. Here we engineered transcription activator-like effector nucleases (TALENs) for five distinct genomic loci. At all loci tested we obtained human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) clones carrying transgenic cassettes solely at the TALEN-specified location. Our data suggest that TALENs employing the specific architectures described here mediate site-specific genome modification in human pluripotent cells with similar efficiency and precision as do zinc-finger nucleases (ZFNs).
Assuntos
Células-Tronco Embrionárias/fisiologia , Endonucleases/metabolismo , Marcação de Genes/métodos , Engenharia Genética/métodos , Células-Tronco Pluripotentes Induzidas/fisiologia , Fatores de Transcrição/metabolismo , Sequência de Bases , Endonucleases/genética , Proteínas de Homeodomínio/genética , Humanos , Dados de Sequência Molecular , Fosfatase de Miosina-de-Cadeia-Leve/genética , Fator 3 de Transcrição de Octâmero/genética , Fatores de Transcrição/genética , Dedos de ZincoRESUMO
Proviruses carrying drug-inducible Oct4, Sox2, Klf4 and c-Myc used to derive 'primary' induced pluripotent stem (iPS) cells were segregated through germline transmission, generating mice and cells carrying subsets of the reprogramming factors. Drug treatment produced 'secondary' iPS cells only when the missing factor was introduced. This approach creates a defined system for studying reprogramming mechanisms and allows screening of genetically homogeneous cells for compounds that can replace any transcription factor required for iPS cell derivation.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Doxiciclina/farmacologia , Técnicas Genéticas , Fatores de Transcrição/genética , Animais , Células Cultivadas , Quimera/genética , Quimera/metabolismo , Feminino , Fibroblastos/metabolismo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Provírus/genética , Provírus/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição/efeitos dos fármacosRESUMO
Embryonic stem cells (ESCs) are isolated from the inner cell mass (ICM) of blastocysts, whereas epiblast stem cells (EpiSCs) are derived from the postimplantation epiblast and display a restricted developmental potential. Here we characterize pluripotent states in the nonobese diabetic (NOD) mouse strain, which prior to this study was considered "nonpermissive" for ESC derivation. We find that NOD stem cells can be stabilized by providing constitutive expression of Klf4 or c-Myc or small molecules that can replace these factors during in vitro reprogramming. The NOD ESCs and iPSCs appear to be "metastable," as they acquire an alternative EpiSC-like identity after removal of the exogenous factors, while their reintroduction converts the cells back to ICM-like pluripotency. Our findings suggest that stem cells from different genetic backgrounds can assume distinct states of pluripotency in vitro, the stability of which is regulated by endogenous genetic determinants and can be modified by exogenous factors.
Assuntos
Hemostasia , Células-Tronco Pluripotentes/citologia , Animais , Desdiferenciação Celular , Células-Tronco Embrionárias/citologia , Camadas Germinativas/citologia , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-myc/genéticaRESUMO
It has recently been demonstrated that mouse and human fibroblasts can be reprogrammed into an embryonic stem cell-like state by introducing combinations of four transcription factors. However, the therapeutic potential of such induced pluripotent stem (iPS) cells remained undefined. By using a humanized sickle cell anemia mouse model, we show that mice can be rescued after transplantation with hematopoietic progenitors obtained in vitro from autologous iPS cells. This was achieved after correction of the human sickle hemoglobin allele by gene-specific targeting. Our results provide proof of principle for using transcription factor-induced reprogramming combined with gene and cell therapy for disease treatment in mice. The problems associated with using retroviruses and oncogenes for reprogramming need to be resolved before iPS cells can be considered for human therapy.
Assuntos
Anemia Falciforme/terapia , Reprogramação Celular , Fibroblastos/citologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Pluripotentes/citologia , Anemia Falciforme/sangue , Anemia Falciforme/fisiopatologia , Animais , Diferenciação Celular , Células Cultivadas , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Células-Tronco Embrionárias/citologia , Contagem de Eritrócitos , Genes myc , Globinas/genética , Hematopoese , Hemoglobina A/análise , Hemoglobina Falciforme/análise , Humanos , Capacidade de Concentração Renal , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Masculino , Camundongos , Fator 3 de Transcrição de Octâmero/genética , Fatores de Transcrição SOXB1 , Transativadores/genética , Transdução GenéticaRESUMO
Neurodegeneration in Huntington disease is described by neuronal loss in which the probability of cell death remains constant with time. However, the quantitative connection between the kinetics of cell death and the molecular mechanism initiating neurodegeneration remains unclear. One hypothesis is that nucleation of protein aggregates containing exon I fragments of the mutant huntingtin protein (mhttex1), which contains an expanded polyglutamine region in patients with the disease, is the explanation for the infrequent but steady occurrence of neuronal death, resulting in adult onset of the disease. Recent in vitro evidence suggests that sufficiently long polyglutamine peptides undergo a unimolecular conformational change to form a nucleus that seeds aggregation. Here we use this nucleation mechanism as the basis to derive a stochastic mathematical model describing the probability of aggregate formation in cells as a function of time and mhttex1 protein concentration, and validate the model experimentally. These findings suggest that therapeutic strategies for Huntington disease predicated on reducing the rate of mhttex1 aggregation need only make modest reductions in huntingtin expression level to substantially increase the delay time until aggregate formation.
Assuntos
Núcleo Celular/metabolismo , Modelos Biológicos , Complexos Multiproteicos/biossíntese , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Morte Celular/genética , Éxons , Proteínas de Fluorescência Verde/genética , Humanos , Proteína Huntingtina , Cinética , Complexos Multiproteicos/genética , Mutação , Degeneração Neural/genética , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Peptídeos/genética , Dobramento de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Processos Estocásticos , Fatores de Tempo , Leveduras/genética , Leveduras/metabolismoRESUMO
Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an expansion in the number of polyglutamine-encoding CAG repeats in the gene that encodes the huntingtin (htt) protein. A property of the mutant protein that is intimately involved in the development of the disease is the propensity of the glutamine-expanded protein to misfold and generate an N-terminal proteolytic htt fragment that is toxic and prone to aggregation. Intracellular antibodies (intrabodies) against htt have been shown to reduce htt aggregation by binding to the toxic fragment and inactivating it or preventing its misfolding. Intrabodies may therefore be a useful gene-therapy approach to treatment of the disease. However, high levels of intrabody expression have been required to obtain even limited reductions in aggregation. We have engineered a single-domain intracellular antibody against htt for robust aggregation inhibition at low expression levels by increasing its affinity in the absence of a disulfide bond. Furthermore, the engineered intrabody variable light-chain (V(L))12.3, rescued toxicity in a neuronal model of HD. We also found that V(L)12.3 inhibited aggregation and toxicity in a Saccharomyces cerevisiae model of HD. V(L)12.3 is significantly more potent than earlier anti-htt intrabodies and is a potential candidate for gene therapy treatment for HD. To our knowledge, this is the first attempt to improve affinity in the absence of a disulfide bond to improve intrabody function. The demonstrated importance of disulfide bond-independent binding for intrabody potency suggests a generally applicable approach to the development of effective intrabodies against other intracellular targets.