RESUMEN
The role of miRNAs in neuroectoderm specification is largely unknown. We screened miRNA profiles that are differentially changed when human embryonic stem cells (hESCs) were differentiated to neuroectodermal precursors (NEP), but not to epidermal (EPI) cells and found that two miRNA families, miR-200 and miR-96, were uniquely downregulated in the NEP cells. We confirmed zinc-finger E-box-binding homeobox (ZEB) transcription factors as a target of the miR-200 family members and identified paired box 6 (PAX6) transcription factor as the new target of miR-96 family members via gain- and loss-of-function analyses. Given the essential roles of ZEBs and PAX6 in neural induction, we propose a model by which miR-200 and miR-96 families coordinate to regulate neural induction.
Asunto(s)
Células Madre Embrionarias/metabolismo , MicroARNs/metabolismo , Placa Neural/citología , Animales , Diferenciación Celular , Línea Celular , Linaje de la Célula , Regulación hacia Abajo , Células Madre Embrionarias/citología , Células Epidérmicas , Epidermis/metabolismo , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , MicroARNs/genética , Placa Neural/metabolismo , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Tiempo , Transcripción Genética , Caja Homeótica 2 de Unión a E-Box con Dedos de ZincRESUMEN
The mechanisms by which transcription factors control stepwise lineage restriction during the specification of cortical neurons remain largely unknown. Here, we investigated the role of forebrain embryonic zinc finger like (Fezf2) in this process by generating Fezf2 knockdown and tetracycline-inducible Fezf2 overexpression mouse embryonic stem cell (mESC) lines. The overexpression of Fezf2 at early time points significantly increased the generation of rostral forebrain progenitors (Foxg1(+), Six3(+)) and inhibited the expression of transcription factors which are expressed by the midbrain and caudal diencephalon (En1(+), Irx(+)). This effect was partially achieved by the regulation of Wnt signaling during this critical early time window. The role of Fezf2 in regulating the rostrocaudal patterning was further confirmed by the significant decrease in the expression of Foxg1 and Six3 and the increase in the expression of En1 when Fezf2 was knocked down. In addition, Fezf2 overexpression at later time points had little effect on the expression of Foxg1 and Six3. Instead, Fezf2 promotes the generation of dorsal telencephalic progenitors and deep-layer cortical neurons at later stages. Collectively, our data suggest that Fezf2 controls the specification of telencephalic progenitors from mESCs through differentially regulating the expression of rostrocaudal and dorsoventral patterning genes.
Asunto(s)
Diferenciación Celular/genética , Diferenciación Celular/fisiología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Células-Madre Neurales/fisiología , Telencéfalo/embriología , Animales , Western Blotting , Línea Celular , Proteínas de Unión al ADN/biosíntesis , Proteínas del Ojo/genética , Factores de Transcripción Forkhead/genética , Técnicas de Silenciamiento del Gen , Vectores Genéticos , Proteínas de Homeodominio/genética , Inmunohistoquímica , Lentivirus/genética , Ratones , Proteínas del Tejido Nervioso/biosíntesis , Prosencéfalo/citología , Prosencéfalo/embriología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Telencéfalo/citología , Factores de Transcripción/genética , Transfección , Vía de Señalización Wnt/fisiología , Proteína Homeobox SIX3RESUMEN
Botulinum neurotoxins (BoNTs), the most poisonous protein toxins known, represent a serious bioterrorism threat but are also used as a unique and important bio-pharmaceutical to treat an increasing myriad of neurological disorders. The only currently accepted detection method by the United States Food and Drug Administration for biological activity of BoNTs and for potency determination of pharmaceutical preparations is the mouse bioassay (MBA). Recent advances have indicated that cell-based assays using primary neuronal cells can provide an equally sensitive and robust detection platform as the MBA to reliably and quantitatively detect biologically active BoNTs. This study reports for the first time a BoNT detection assay using mouse embryonic stem cells to produce a neuronal cell culture. The data presented indicate that this assay can reliably detect BoNT/A with a similar sensitivity as the MBA.
Asunto(s)
Bioterrorismo , Toxinas Botulínicas Tipo A/análisis , Células Madre Embrionarias/citología , Neurogénesis , Neuronas/química , Animales , Bioensayo , Toxinas Botulínicas Tipo A/toxicidad , Recuento de Células , Técnicas de Cultivo de Célula , Células Cultivadas , Ratones , Neuronas/citología , Neuronas/efectos de los fármacos , Sensibilidad y EspecificidadRESUMEN
To circumvent the silencing effect of transgene expression in human embryonic stem cells (hESCs), we employed the Cre recombination-mediated cassette exchange strategy to target the silencing-resistant site in the genome. We have identified new loci that sustain transgene expression during stem cell expansion and differentiation to cells representing the three germ layers in vitro and in vivo. The built-in double loxP cassette in the established master hESC lines was specifically replaced by a targeting vector containing the same loxP sites, using the cell-permeable Cre protein transduction method, resulting in successful generation of new hESC lines with constitutive functional gene expression, inducible transgene expression, and lineage-specific reporter gene expression. This strategy and the master cell lines allow for rapid production of transgenic hESC lines in ordinary laboratories.
Asunto(s)
Células Madre Embrionarias/metabolismo , Integrasas/metabolismo , Mutagénesis Insercional , Recombinación Genética/genética , Transgenes/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular , Línea Celular , Permeabilidad de la Membrana Celular , Células Madre Embrionarias/citología , Regulación de la Expresión Génica , Silenciador del Gen , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Neuronas/metabolismo , Factor de Transcripción 2 de los Oligodendrocitos , Especificidad de Órganos , TransfecciónRESUMEN
Specification of distinct cell types from human embryonic stem cells (hESCs) is key to the potential application of these naïve pluripotent cells in regenerative medicine. Determination of the nontarget differentiated populations, which is lacking in the field, is also crucial. Here, we show an efficient differentiation of motor neurons ( approximately 50%) by a simple sequential application of retinoid acid and sonic hedgehog (SHH) in a chemically defined suspension culture. We also discovered that purmorphamine, a small molecule that activates the SHH pathway, could replace SHH for the generation of motor neurons. Immunocytochemical characterization indicated that cells differentiated from hESCs were nearly completely restricted to the ventral spinal progenitor fate (NKX2.2+, Irx3+, and Pax7-), with the exception of motor neurons (HB9+) and their progenitors (Olig2+). Thus, the directed neural differentiation system with small molecules, even without further purification, will facilitate basic and translational studies using human motoneurons at a minimal cost.
Asunto(s)
Diferenciación Celular , Evolución Molecular Dirigida/métodos , Células Madre Embrionarias/citología , Neuronas Motoras/citología , Neuronas Motoras/fisiología , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Línea Celular , Células Cultivadas , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/fisiología , Proteínas Hedgehog/biosíntesis , Proteínas Hedgehog/genética , Proteínas Hedgehog/fisiología , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio , Humanos , Ratones , Morfolinas/farmacología , Neuronas Motoras/efectos de los fármacos , Proteínas Nucleares , Purinas/farmacología , Médula Espinal/citología , Médula Espinal/efectos de los fármacos , Médula Espinal/fisiología , Células Madre/citología , Células Madre/efectos de los fármacos , Células Madre/fisiología , Factores de Transcripción , Tretinoina/farmacología , Tretinoina/fisiologíaRESUMEN
An understanding of how mammalian stem cells produce specific neuronal subtypes remains elusive. Here we show that human embryonic stem cells generated early neuroectodermal cells, which organized into rosettes and expressed Pax6 but not Sox1, and then late neuroectodermal cells, which formed neural tube-like structures and expressed both Pax6 and Sox1. Only the early, but not the late, neuroectodermal cells were efficiently posteriorized by retinoic acid and, in the presence of sonic hedgehog, differentiated into spinal motoneurons. The in vitro-generated motoneurons expressed HB9, HoxC8, choline acetyltransferase and vesicular acetylcholine transporter, induced clustering of acetylcholine receptors in myotubes, and were electrophysiologically active. These findings indicate that retinoic acid action is required during neuroectoderm induction for motoneuron specification and suggest that stem cells have restricted capacity to generate region-specific projection neurons even at an early developmental stage.
Asunto(s)
Diferenciación Celular/fisiología , Proteínas del Ojo/metabolismo , Proteínas de Homeodominio/metabolismo , Neuronas Motoras/fisiología , Proteínas Represoras/metabolismo , Células Madre/citología , Células Madre/fisiología , Ingeniería de Tejidos/métodos , Tretinoina/metabolismo , Biomarcadores/metabolismo , Línea Celular , Proteínas de Unión al ADN/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas del Grupo de Alta Movilidad/metabolismo , Humanos , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box , Factores de Transcripción SOXB1RESUMEN
Electrophysiology of excitable cells, including muscle cells and neurons, has been measured by making direct contact with a single cell using a micropipette electrode. To increase the assay throughput, optical devices such as microscopes and microplate readers have been used to analyze electrophysiology of multiple cells. We have established a high-throughput (HTP) analysis of action potentials (APs) in highly enriched motor neurons and cardiomyocytes (CMs) that are differentiated from human induced pluripotent stem cells (iPSCs). A multichannel electric field stimulation (EFS) device enabled the ability to electrically stimulate cells and measure dynamic changes in APs of excitable cells ultra-rapidly (>100 data points per second) by imaging entire 96-well plates. We found that the activities of both neurons and CMs and their response to EFS and chemicals are readily discerned by our fluorescence imaging-based HTP phenotyping assay. The latest generation of calcium (Ca2+) indicator dyes, FLIPR Calcium 6 and Cal-520, with the HTP device enables physiological analysis of human iPSC-derived samples highlighting its potential application for understanding disease mechanisms and discovering new therapeutic treatments.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento , Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/citología , Neuronas/citología , Imagen Óptica , Calcio/metabolismo , Células Cultivadas , Estimulación Eléctrica/instrumentación , Electrodos , Ensayos Analíticos de Alto Rendimiento/instrumentación , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/metabolismo , Neuronas/metabolismo , Imagen Óptica/instrumentación , FenotipoRESUMEN
OBJECTIVE: Tissue-specific stem cells from differentiating embryonic stem (ES) cells are both pluripotent and genetically flexible. Recent observations indicate that ES cells can differentiate into hepatocytes. Therefore, cell-based therapy can potentially be a therapeutic alternative to liver transplantation. In this study the treatment of acute liver failure in rats by transplantation of hepatocyte nuclear factor 4 (HNF4)-overexpressing ES cells was investigated. METHODS: The HNF4 was transfected into ES cells and ES cell clones overexpressing HNF4 were selected. The levels of markers of hepatocyte differentiation, including albumin, transthyretin, glucose-6-phosphates (G-6-P) and SAPK/ERK kinase-1 (SEK1) mRNA, were tested in spontaneously differentiated HNF4-overexpressing ES cells by reverse transcription-polymerase chain reaction (RT-PCR). The ultrastructure of the spontaneously differentiated HNF4-overexpressing ES cells was examined by electron microscopy. To induce acute liver failure, Sprague-Dawley rats were subjected to 90% hepatectomy and given 5% oral dextrose. The rats were divided into three groups. The rats in the treatment group (n = 12) received intraliver injection of 2 x 10(7) undifferentiated HNF4-overexpressing ES cells from the same clone, the rats in control group 1 (n = 12) received 2 x 10(7) undifferentiated ES cells, and the rats in control group 2 (n = 12) received the same volume of media without any cells. RESULTS: All rats in control group 1 and control group 2 died within 72 h, while 33% of rats that received undifferentiated HNF4-overexpressing ES cells transplantation survived more than 1 month. Spontaneously differentiated HNF4-overexpressing ES cells only expressed transthyretin mRNA. The cells were rich in mitochondrion and catalase-containing peroxisomes in ultrastructure. CONCLUSIONS: Transplantation of ES cells could be a potential treatment in supporting life during acute liver insufficiency and could be a bridge to orthotopic liver transplantation.
Asunto(s)
Hepatectomía/efectos adversos , Factor Nuclear 4 del Hepatocito/biosíntesis , Factor Nuclear 4 del Hepatocito/uso terapéutico , Fallo Hepático Agudo/etiología , Fallo Hepático Agudo/terapia , Trasplante de Células Madre , Animales , Biomarcadores/sangre , Diferenciación Celular , Modelos Animales de Enfermedad , Factor Nuclear 4 del Hepatocito/ultraestructura , Hepatocitos/metabolismo , Hepatocitos/ultraestructura , Fallo Hepático Agudo/mortalidad , Fallo Hepático Agudo/patología , Masculino , Mitocondrias Hepáticas/metabolismo , Mitocondrias Hepáticas/ultraestructura , Peroxisomas/metabolismo , Peroxisomas/ultraestructura , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Células Madre/metabolismo , Células Madre/ultraestructura , Regulación hacia ArribaRESUMEN
Human pluripotent stem cells (hPSCs) have opened new opportunities for understanding human development, modelling disease processes and developing new therapeutics. However, these applications are hindered by the low efficiency and heterogeneity of cell types, such as motorneurons (MNs), differentiated from hPSCs as well as our inability to maintain the potency of lineage-committed progenitors. Here by using a combination of small molecules that regulate multiple signalling pathways, we develop a method to guide human embryonic stem cells to a near-pure population (>95%) of motor neuron progenitors (MNPs) in 12 days, and an enriched population (>90%) of functionally mature MNs in an additional 16 days. More importantly, the MNPs can be expanded for at least five passages so that a single MNP can be amplified to 1 × 10(4). This method is reproducible in human-induced pluripotent stem cells and is applied to model MN-degenerative diseases and in proof-of-principle drug-screening assays.
Asunto(s)
Neuronas Motoras/citología , Células-Madre Neurales/citología , Células Madre Pluripotentes/citología , Técnicas de Cultivo de Célula , Humanos , Unión Neuromuscular/citologíaRESUMEN
As a part of a basic research project on Xeno-transplantion, we have been engaged in the derivation of embryonic stem cell lines from Chinese mini swine. Here, we reported for the first time the establishment of two porcine EG cell lines (BPEG1 and BPEG2) from primordial germ cells of genital ridges of a 28 and a 27 d embryos respectively. Their pluripotent nature has been identified by colony morphology, marker characterization as well as by in vitro and in vivo differentiation. These porcine EG cells are potentially useful for further basic studies.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Embrión de Mamíferos/citología , Células Germinativas/citología , Factores de Transcripción , Fosfatasa Alcalina/análisis , Animales , Antígenos de Superficie/inmunología , Biomarcadores/análisis , Diferenciación Celular/fisiología , Proteínas de Unión al ADN/inmunología , Embrión de Mamíferos/química , Embrión de Mamíferos/ultraestructura , Femenino , Células Germinativas/química , Células Germinativas/ultraestructura , Inmunohistoquímica , Cariotipificación , Masculino , Microscopía Electrónica , Factor 3 de Transcripción de Unión a Octámeros , Células Madre/química , Células Madre/citología , Células Madre/ultraestructura , Porcinos , Porcinos EnanosRESUMEN
Embryonic stem (ES) cells can in theory produce all cell types of a living organism while renewing themselves with a stable genetic background. These unique features make ES cells a favorable tool for biomedical researches as well as a potential source for therapeutic application. A first step for approaching to ES cells is the directed differentiation to cells of interest, such as the neural cell lineage. Here, we summarize the up and down sides of each category of neural differentiation protocols that have so far been used in mouse and human ES cells, and introduce an efficient and plausible method used in our laboratory for derivation of neuroectodermal cells from human ES cells. This synthesis has led to our suggestions on issues for future design of neural differentiation protocols.
Asunto(s)
Diferenciación Celular , Neuronas/citología , Células Madre/citología , Animales , División Celular , Embrión de Mamíferos , Factor 2 de Crecimiento de Fibroblastos/fisiología , Humanos , Sistema Nervioso/embriología , Transducción de SeñalRESUMEN
Human Embryonic stem cells (hESCs) offer an invaluable tool for revealing human biology and a potential source of functional cells/tissues for regenerative medicine. The utility of hESCs will likely be significantly enhanced and broadened by our ability to build versatile genetically modified hESC lines. Here, we describe an efficient lentiviral vector mediated method to establish stable transgenic hESCs.
Asunto(s)
Células Madre Embrionarias/metabolismo , Vectores Genéticos , Lentivirus/genética , Transgenes , Línea Celular , Humanos , Regiones Promotoras Genéticas , Medicina Regenerativa , TransfecciónRESUMEN
The transcriptional regulation of neuroectoderm (NE) specification is unknown. Here we show that Pax6 is uniformly expressed in early NE cells of human fetuses and those differentiated from human embryonic stem cells (hESCs). This is in contrast to the later expression of Pax6 in restricted mouse brain regions. Knockdown of Pax6 blocks NE specification from hESCs. Overexpression of either Pax6a or Pax6b, but not Pax6triangle upPD, triggers hESC differentiation. However, only Pax6a converts hESCs to NE. In contrast, neither loss nor gain of function of Pax6 affects mouse NE specification. Both Pax6a and Pax6b bind to pluripotent gene promoters but only Pax6a binds to NE genes during human NE specification. These findings indicate that Pax6 is a transcriptional determinant of the human NE and suggest that Pax6a and Pax6b coordinate with each other in determining the transition from pluripotency to the NE fate in human by differentially targeting pluripotent and NE genes.
Asunto(s)
Diferenciación Celular/fisiología , Proteínas del Ojo/metabolismo , Proteínas de Homeodominio/metabolismo , Placa Neural/citología , Placa Neural/metabolismo , Factores de Transcripción Paired Box/metabolismo , Proteínas Represoras/metabolismo , Animales , Diferenciación Celular/genética , Línea Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Proteínas del Ojo/genética , Proteínas de Homeodominio/genética , Humanos , Técnicas In Vitro , Ratones , Ratones SCID , Modelos Biológicos , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/genética , Proteínas Represoras/genética , Teratoma/patologíaRESUMEN
We have developed a four-part protocol to differentiate human embryonic stem cells (hESCs) to oligodendrocyte progenitor cells (OPCs) according to developmental principles. In the first 2 weeks, hESCs are induced to differentiate into neuroepithelial cells, which form neural tube-like rosettes. In the following 10 d, these neuroepithelial cells are specified to OLIG2-expressing progenitors in the presence of retinoic acid (RA) and sonic hedgehog (SHH). Upon treatment with fibroblast growth factor 2 (FGF2) for another 10 d, these progenitors convert to OLIG2 and NKX2.2-expressing pre-OPCs. Finally, the pre-OPCs take 8-9 weeks to differentiate into OPCs, which express additional markers of oligodendrocytes, such as SOX10, platelet-derived growth factor receptor alpha (PDGFRalpha) and NG2. The unique aspects of the protocol are the use of FGF2 to promote the differentiation of gliogenic pre-OPCs in the third part and the removal of FGF2 during the transition of pre-OPCs to OPCs. This 3-month differentiation protocol consistently yields OPCs of high purity capable of producing myelin sheaths in vivo.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Oligodendroglía/citología , Células Madre Pluripotentes/citología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Proteínas Hedgehog/farmacología , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio/metabolismo , Humanos , Proteínas del Tejido Nervioso/metabolismo , Células Neuroepiteliales/citología , Células Neuroepiteliales/efectos de los fármacos , Células Neuroepiteliales/metabolismo , Proteínas Nucleares , Factor de Transcripción 2 de los Oligodendrocitos , Oligodendroglía/efectos de los fármacos , Oligodendroglía/metabolismo , Células Madre Pluripotentes/efectos de los fármacos , Células Madre Pluripotentes/metabolismo , Factores de Transcripción/metabolismo , Tretinoina/farmacología , Proteínas de Pez CebraRESUMEN
Embryonic mesenchymal stem cells (eMSCs) were first derived from human embryonic stem cells (hESCs) overexpressing green fluorescence protein (GFP). They expressed CD29, CD44, CD73, CD105, CD166 and nestin, but not CD34, CD45, CD106 SSEA-4 or Oct3/4. Twenty million eMSCs in 1 mL of phosphate-buffered saline (PBS) were injected into the femoral veins of spontaneously hypertensive rats after transient middle cerebral artery occlusion. The migration and differentiation of the eMSCs in the ischemic brain were analyzed. The results revealed that eMSCs migrated to the infarction region and differentiated into neurons, which were positive for beta-tubulin III, microtubule-associated protein 2 (MAP2), HuC, neurofilament and human nuclear antibody, and to vascular endothelial cells, which were positive for von Willebrand factor (vWF). The transplanted cells survived in the infarction region for at least 4 weeks. Adhesive removal function significantly improved in the first week after cell transplantation, and rotarod motor function significantly improved starting from the second week. The infarction volume in the eMSC group was significantly smaller than that in the PBS control group at 4 weeks after infusion. The results of this study show that when administered intravenously, eMSCs differentiated into neuronal and endothelial cells, reduced the infarction volume, and improved behavioral functional outcome significantly in transient focal cerebral ischemia.
Asunto(s)
Células Madre Embrionarias/citología , Ataque Isquémico Transitorio/terapia , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/fisiología , Animales , Diferenciación Celular , Movimiento Celular , Células Endoteliales/citología , Vena Femoral , Proteínas Fluorescentes Verdes , Humanos , Inmunofenotipificación , Infarto de la Arteria Cerebral Media , Células Madre Mesenquimatosas/citología , Neuronas/citología , Ratas , Ratas Endogámicas SHR , Trasplante HeterólogoRESUMEN
Human embryonic stem cells (hESCs) offer a platform to bridge what we have learned from animal studies to human biology. Using oligodendrocyte differentiation as a model system, we show that sonic hedgehog (SHH)-dependent sequential activation of the transcription factors OLIG2, NKX2.2 and SOX10 is required for sequential specification of ventral spinal OLIG2-expressing progenitors, pre-oligodendrocyte precursor cells (pre-OPCs) and OPCs from hESC-derived neuroepithelia, indicating that a conserved transcriptional network underlies OPC specification in human as in other vertebrates. However, the transition from pre-OPCs to OPCs is protracted. FGF2, which promotes mouse OPC generation, inhibits the transition of pre-OPCs to OPCs by repressing SHH-dependent co-expression of OLIG2 and NKX2.2. Thus, despite the conservation of a similar transcriptional network across vertebrates, human stem/progenitor cells may respond differently to those of other vertebrates to certain extrinsic factors.
Asunto(s)
Diferenciación Celular , Células Madre Embrionarias/citología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Proteínas Hedgehog/metabolismo , Oligodendroglía/efectos de los fármacos , Oligodendroglía/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Células Cultivadas , Regulación de la Expresión Génica , Proteínas Hedgehog/genética , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Microscopía Electrónica , Vaina de Mielina/metabolismo , Vaina de Mielina/ultraestructura , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Proteínas Nucleares , Factor de Transcripción 2 de los Oligodendrocitos , Oligodendroglía/citología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Pez CebraRESUMEN
To dissect out interactions between the transcription factor Olig2 and other intrinsic and extrinsic factors in neural cell fate determination, we established a mouse embryonic stem (ES) cell line with induced expression of Olig2 along neural differentiation. During neuronal differentiation, both the control and Olig2-induced groups produced a similar proportion of HB9-expressing motoneurons in the presence of retinoic acid (RA) and sonic hedgehog (SHH), but both generated few motoneurons in the absence of SHH. Induced Olig2 expression did not alter the pattern of gene transcription without SHH, suggesting that Olig2 requires cooperation with RA and SHH for motoneuron specification. During glial differentiation, the Olig2-induced group generated significantly more oligodendrocytes and fewer neurons and astrocytes than the control group. This effect was not blocked by inhibition of SHH signaling, suggesting that Olig2 bypasses the need of SHH in oligodendrocyte specification. However, treatment with ciliary neurotropic factor (CNTF) markedly increased astrocyte and decreased oligodendrocyte differentiation even when Olig2 is sustained in the nuclei, suggesting that Olig2 cannot bypass the CNTF-STAT signaling to repress astrocyte differentiation.
Asunto(s)
Astrocitos/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Neuronas Motoras/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Oligodendroglía/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diferenciación Celular/efectos de los fármacos , Línea Celular , Embrión de Pollo , Factor Neurotrófico Ciliar/farmacología , Embrión de Mamíferos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Proteínas Hedgehog/farmacología , Proteínas de Homeodominio/metabolismo , Inmunohistoquímica/métodos , Ratones , Proteínas del Tejido Nervioso/genética , Factor de Transcripción 2 de los Oligodendrocitos , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Células Madre/efectos de los fármacos , Células Madre/fisiología , Factores de Transcripción/metabolismo , Transfección/métodos , Tretinoina/farmacologíaRESUMEN
Several putative Oct-4 downstream genes from mouse embryonic stem (ES) cells have been identified using the suppression-subtractive hybridization method. In this study, one of the novel genes encoding an ES cell and germ cell specific protein (ESGP) was cloned by rapid amplification of cDNA ends. ESGP contains 801 bp encoding an 84 amino acid small protein and has no significant homology to any known genes. There is a signal peptide at the N-terminal of ESGP protein as predicted by SeqWeb (GCG) (SeqWeb version 2.0.2, http://gcg.biosino.org:8080/). The result of immunofluorescence assay suggested that ESGP might encode a secretory protein. The expression pattern of ESGP is consistent with the expression of Oct-4 during embryonic development. ESGP protein was detected in fertilized oocyte, from 3.5 day postcoital (dpc) blastocyst to 17.5 dpc embryo, and was only detected in testis and ovary tissues in adult. In vitro, ESGP was only expressed in pluripotent cell lines, such as embryonic stem cells, embryonic caoma cells and embryonic germ cells, but not in their differentiated progenies. Despite its specific expression, forced expression of ESGP is not indispensable for the effect of Oct-4 on ES cell self-renewal, and does not affect the differentiation to three germ layers.
Asunto(s)
Células Germinativas/metabolismo , Proteínas de Membrana de los Lisosomas/química , Proteínas de Membrana de los Lisosomas/fisiología , Células Madre/metabolismo , Secuencia de Aminoácidos , Animales , Diferenciación Celular/genética , Células Cultivadas , Clonación Molecular , Regulación del Desarrollo de la Expresión Génica , Proteínas de Membrana de los Lisosomas/genética , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , Células Madre/citología , Células Tumorales CultivadasRESUMEN
This study is designed to immortalize endothelial cells differentiated from embryonic stem cells. The embryoid bodies (EB) formed in vitro from embryonic stem cells, were induced to differentiate into many "round cells" (the precursor of endothelial cells) by retinoic acid (RA) and transforming growth factor-beta1 (TGF-beta1). These "round cells" later formed the vascular tube-like structures. Studies by scanning electronic microscopy and light microscopy and immunocytochemistry, demonstrated that these tube-like structures were constituted by a large number of round and flat cells, which were positive for "vWF" and "CD34"staining. These results indicate they are vascular endothelial cells. To immortalize these cells, human telomerase reverse transcriptase (hTERT) cDNA was transfected into "round cells" by lipofectine. hTERT mRNA expression in transfected cells was confirmed by Dot blot, RT-PCR. Furthermore, 95% these transfected cells maintain the characteristic of endothelial cell, can proliferate in large quantity in vitro, and are able to form tubular structures. These results suggest that hTERT cDNA transfection can immortalize the induced endothelial cells and therefore may provide a new source of seed cells for vascular engineering.