RESUMEN
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.
Asunto(s)
Linfocitos B/citología , Linaje de la Célula , Transdiferenciación Celular , Hepatocitos/citología , Células-Madre Neurales/citología , Animales , Linfocitos B/metabolismo , Linaje de la Célula/genética , Transdiferenciación Celular/genética , Reprogramación Celular , Análisis por Conglomerados , Epigénesis Genética , Expresión Génica , Perfilación de la Expresión Génica , Hepatocitos/metabolismo , Ratones , Células-Madre Neurales/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Induced pluripotent stem cells (iPSCs) are commonly generated by transduction of Oct4, Sox2, Klf4, and Myc (OSKM) into cells. Although iPSCs are pluripotent, they frequently exhibit high variation in terms of quality, as measured in mice by chimera contribution and tetraploid complementation. Reliably high-quality iPSCs will be needed for future therapeutic applications. Here, we show that one major determinant of iPSC quality is the combination of reprogramming factors used. Based on tetraploid complementation, we found that ectopic expression of Sall4, Nanog, Esrrb, and Lin28 (SNEL) in mouse embryonic fibroblasts (MEFs) generated high-quality iPSCs more efficiently than other combinations of factors including OSKM. Although differentially methylated regions, transcript number of master regulators, establishment of specific superenhancers, and global aneuploidy were comparable between high- and low-quality lines, aberrant gene expression, trisomy of chromosome 8, and abnormal H2A.X deposition were distinguishing features that could potentially also be applicable to human.
Asunto(s)
Reprogramación Celular , Células Madre Pluripotentes Inducidas/metabolismo , Factores de Transcripción/metabolismo , Animales , Línea Celular , Quimera , Cromosomas Humanos Par 8/genética , Metilación de ADN/genética , Células Madre Embrionarias/metabolismo , Elementos de Facilitación Genéticos/genética , Perfilación de la Expresión Génica , Genoma/genética , Histonas/metabolismo , Humanos , Factor 4 Similar a Kruppel , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , ARN Mensajero/genética , ARN Mensajero/metabolismo , Trisomía/genéticaRESUMEN
Embryonic stem cells (ESCs) of mice and humans have distinct molecular and biological characteristics, raising the question of whether an earlier, "naive" state of pluripotency may exist in humans. Here we took a systematic approach to identify small molecules that support self-renewal of naive human ESCs based on maintenance of endogenous OCT4 distal enhancer activity, a molecular signature of ground state pluripotency. Iterative chemical screening identified a combination of five kinase inhibitors that induces and maintains OCT4 distal enhancer activity when applied directly to conventional human ESCs. These inhibitors generate human pluripotent cells in which transcription factors associated with the ground state of pluripotency are highly upregulated and bivalent chromatin domains are depleted. Comparison with previously reported naive human ESCs indicates that our conditions capture a distinct pluripotent state in humans that closely resembles that of mouse ESCs. This study presents a framework for defining the culture requirements of naive human pluripotent cells.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Células Madre Pluripotentes/citología , Supervivencia Celular , Cromatina/metabolismo , Elementos de Facilitación Genéticos/genética , Perfilación de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Datos de Secuencia Molecular , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Células Madre Pluripotentes/metabolismo , TransgenesRESUMEN
Sertoli cells are considered the "supporting cells" of the testis that play an essential role in sex determination during embryogenesis and in spermatogenesis during adulthood. Their essential roles in male fertility along with their immunosuppressive and neurotrophic properties make them an attractive cell type for therapeutic applications. Here we demonstrate the generation of induced embryonic Sertoli-like cells (ieSCs) by ectopic expression of five transcription factors. We characterize the role of specific transcription factor combinations in the transition from fibroblasts to ieSCs and identify key steps in the process. Initially, transduced fibroblasts underwent a mesenchymal to epithelial transition and then acquired the ability to aggregate, formed tubular-like structures, and expressed embryonic Sertoli-specific markers. These Sertoli-like cells facilitated neuronal differentiation and self-renewal of neural progenitor cells (NPCs), supported the survival of germ cells in culture, and cooperated with endogenous embryonic Sertoli and primordial germ cells in the generation of testicular cords in the fetal gonad.
Asunto(s)
Reprogramación Celular/genética , Embrión de Mamíferos/citología , Fibroblastos/citología , Células de Sertoli/citología , Animales , Agregación Celular/genética , Diferenciación Celular/genética , Movimiento Celular/genética , Proliferación Celular , Supervivencia Celular/genética , Embrión de Mamíferos/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Transición Epitelial-Mesenquimal/genética , Fibroblastos/metabolismo , Factor de Transcripción GATA4/metabolismo , Perfilación de la Expresión Génica , Cariotipificación , Masculino , Ratones , Neovascularización Fisiológica/genética , Neuronas/citología , Neuronas/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Factor de Transcripción SOX9/metabolismo , Células de Sertoli/metabolismo , Factor Esteroidogénico 1/metabolismo , Testículo/citología , Testículo/embriología , Factores de Transcripción/metabolismoRESUMEN
During cellular reprogramming, only a small fraction of cells become induced pluripotent stem cells (iPSCs). Previous analyses of gene expression during reprogramming were based on populations of cells, impeding single-cell level identification of reprogramming events. We utilized two gene expression technologies to profile 48 genes in single cells at various stages during the reprogramming process. Analysis of early stages revealed considerable variation in gene expression between cells in contrast to late stages. Expression of Esrrb, Utf1, Lin28, and Dppa2 is a better predictor for cells to progress into iPSCs than expression of the previously suggested reprogramming markers Fbxo15, Fgf4, and Oct4. Stochastic gene expression early in reprogramming is followed by a late hierarchical phase with Sox2 being the upstream factor in a gene expression hierarchy. Finally, downstream factors derived from the late phase, which do not include Oct4, Sox2, Klf4, c-Myc, and Nanog, can activate the pluripotency circuitry.
Asunto(s)
Reprogramación Celular , Células Madre Pluripotentes Inducidas/metabolismo , Análisis de la Célula Individual , Transcriptoma , Animales , Línea Celular , Embrión de Mamíferos/citología , Células Madre Embrionarias , Fibroblastos/citología , Fibroblastos/metabolismo , Marcadores Genéticos , Células Madre Pluripotentes Inducidas/citología , Factor 4 Similar a Kruppel , Ratones , Técnicas Analíticas Microfluídicas , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción/metabolismoRESUMEN
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.
Asunto(s)
Reprogramación Celular/fisiología , Epigénesis Genética , Células Madre Pluripotentes Inducidas/fisiología , Animales , Biomarcadores/metabolismo , Línea Celular , Células Madre Pluripotentes Inducidas/citología , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Masculino , Ratones , Ratones Transgénicos , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , TransgenesRESUMEN
The Tet family of enzymes (Tet1/2/3) converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Mouse embryonic stem cells (mESCs) highly express Tet1 and have an elevated level of 5hmC. Tet1 has been implicated in ESC maintenance and lineage specification in vitro but its precise function in development is not well defined. To establish the role of Tet1 in pluripotency and development, we have generated Tet1 mutant mESCs and mice. Tet1(-/-) ESCs have reduced levels of 5hmC and subtle changes in global gene expression, and are pluripotent and support development of live-born mice in tetraploid complementation assay, but display skewed differentiation toward trophectoderm in vitro. Tet1 mutant mice are viable, fertile, and grossly normal, though some mutant mice have a slightly smaller body size at birth. Our data suggest that Tet1 loss leading to a partial reduction in 5hmC levels does not affect pluripotency in ESCs and is compatible with embryonic and postnatal development.