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
Regulatable transgene expression in human pluripotent stem cells (hPSCs) and their progenies is often necessary to dissect gene function in a temporal and spatial manner. However, hPSC lines with inducible transgene expression, especially in differentiated progenies, have not been established due to silencing of randomly inserted genes during stem cell expansion and/or differentiation. Here, we report the use of transcription activator-like effector nucleases-mediated targeting to AAVS1 site to generate versatile conditional hPSC lines. Transgene (both green fluorescent protein and a functional gene) expression in hPSCs and their derivatives was not only sustained but also tightly regulated in response to doxycycline both in vitro and in vivo. We modified the donor construct so that any gene of interest can be readily inserted to produce hPSC lines with conditional transgene expression. This technology will substantially improve the way we study human stem cells.
Asunto(s)
Expresión Génica/genética , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes/metabolismo , Transgenes/genética , Animales , Astrocitos/citología , Astrocitos/metabolismo , Western Blotting , Doxiciclina/farmacología , Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Inmunohistoquímica , Células Madre Pluripotentes Inducidas/citología , Ratones SCID , Microscopía Confocal , Neuronas/citología , Neuronas/metabolismo , Células Madre Pluripotentes/citologíaRESUMEN
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
Differentiation of astrocytes from human pluripotent stem cells (hPSCs) is a tedious and variable process. This hampers the study of hPSC-generated astrocytes in disease processes and drug development. By using CRISPR/Cas9-mediated inducible expression of NFIA or NFIA plus SOX9 in hPSCs, we developed a method to efficiently generate astrocytes in 4-7 weeks. The astrocytic identity of the induced cells was verified by their characteristic molecular and functional properties as well as after transplantation. Furthermore, we developed a strategy to generate region-specific astrocyte subtypes by combining differentiation of regional progenitors and transgenic induction of astrocytes. This simple and efficient method offers a new opportunity to study the fundamental biology of human astrocytes and their roles in disease processes.
Asunto(s)
Astrocitos/citología , Células Madre Pluripotentes/citología , Astrocitos/metabolismo , Diferenciación Celular , Humanos , Factores de Transcripción NFI/metabolismo , Proyección Neuronal , Células Madre Pluripotentes/metabolismo , Factor de Transcripción SOX9/metabolismoRESUMEN
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
Nanog is a recently discovered homeodomain transcription factor that sustains the pluripotency of embryonic stem (ES) cells and blocks their differentiation into endoderm. The murine F9 embryonal carcinoma cell line is a well-documented model system for endoderm cell lineage differentiation. Here, we examined the function of Nanog in F9 cell endoderm differentiation. Over-expression of Nanog returns the F9 cells to the early status of ES cells and represses the differentiation of primitive endoderm and parietal endoderm in F9 cells, whereas it has no effect on the differentiation of visceral endoderm. In contrast, the expression of C-terminal domain-truncated Nanog spontaneously promotes endoderm differentiation in F9 cells. These data suggest that Nanog is required to sustain the proper undifferentiated status of F9 cells, and the C-terminal domain of Nanog transduces the most effects in repressing primitive endoderm and parietal endoderm differentiation in F9 cells.
Asunto(s)
Diferenciación Celular/fisiología , Proteínas de Unión al ADN/metabolismo , Endodermo/citología , Proteínas de Homeodominio/metabolismo , Células Madre Neoplásicas/fisiología , Secuencia de Aminoácidos , Animales , Antineoplásicos/metabolismo , Biomarcadores/metabolismo , Forma de la Célula , Células Cultivadas , Células Madre de Carcinoma Embrionario , Endodermo/fisiología , Ratones , Datos de Secuencia Molecular , Proteína Homeótica Nanog , Células Madre Neoplásicas/citología , Tretinoina/metabolismoRESUMEN
Spinal muscular atrophy (SMA) presents severe muscle weakness with limited motor neuron (MN) loss at an early stage, suggesting potential functional alterations in MNs that contribute to SMA symptom presentation. Using SMA induced pluripotent stem cells (iPSCs), we found that SMA MNs displayed hyperexcitability with increased membrane input resistance, hyperpolarized threshold, and larger action potential amplitude, which was mimicked by knocking down full length survival motor neuron (SMN) in non-SMA MNs. We further discovered that SMA MNs exhibit enhanced sodium channel activities with increased current amplitude and facilitated recovery, which was corrected by restoration of SMN1 in SMA MNs. Together we propose that SMN reduction results in MN hyperexcitability and impaired neurotransmission, the latter of which exacerbate each other via a feedback loop, thus contributing to severe symptoms at an early stage of SMA.
Asunto(s)
Neuronas Motoras/metabolismo , Atrofia Muscular Espinal/fisiopatología , Diferenciación Celular/genética , Línea Celular , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Potenciales de la Membrana , Neuronas Motoras/citología , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , ARN Mensajero/genética , Canales de Sodio/metabolismo , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismo , Potenciales SinápticosRESUMEN
Astrocytes are integral components of the homeostatic neural network as well as active participants in pathogenesis of and recovery from nearly all neurological conditions. Evolutionarily, compared with lower vertebrates and nonhuman primates, humans have an increased astrocyte-to-neuron ratio; however, a lack of effective models has hindered the study of the complex roles of human astrocytes in intact adult animals. Here, we demonstrated that after transplantation into the cervical spinal cords of adult mice with severe combined immunodeficiency (SCID), human pluripotent stem cell-derived (PSC-derived) neural progenitors migrate a long distance and differentiate to astrocytes that nearly replace their mouse counterparts over a 9-month period. The human PSC-derived astrocytes formed networks through their processes, encircled endogenous neurons, and extended end feet that wrapped around blood vessels without altering locomotion behaviors, suggesting structural, and potentially functional, integration into the adult mouse spinal cord. Furthermore, in SCID mice transplanted with neural progenitors derived from induced PSCs from patients with ALS, astrocytes were generated and distributed to a similar degree as that seen in mice transplanted with healthy progenitors; however, these mice exhibited motor deficit, highlighting functional integration of the human-derived astrocytes. Together, these results indicate that this chimeric animal model has potential for further investigating the roles of human astrocytes in disease pathogenesis and repair.
Asunto(s)
Astrocitos/fisiología , Células-Madre Neurales/trasplante , Esclerosis Amiotrófica Lateral/patología , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Apoptosis , Diferenciación Celular , Movimiento Celular , Proliferación Celular , Células Cultivadas , Humanos , Células Madre Pluripotentes Inducidas/trasplante , Ratones SCID , Neuronas Motoras/fisiología , Fuerza Muscular , Médula Espinal/patologíaRESUMEN
Dorsal root avulsion results in permanent impairment of sensory functions due to disconnection between the peripheral and central nervous system. Improved strategies are therefore needed to reconnect injured sensory neurons with their spinal cord targets in order to achieve functional repair after brachial and lumbosacral plexus avulsion injuries. Here, we show that sensory functions can be restored in the adult mouse if avulsed sensory fibers are bridged with the spinal cord by human neural progenitor (hNP) transplants. Responses to peripheral mechanical sensory stimulation were significantly improved in transplanted animals. Transganglionic tracing showed host sensory axons only in the spinal cord dorsal horn of treated animals. Immunohistochemical analysis confirmed that sensory fibers had grown through the bridge and showed robust survival and differentiation of the transplants. Section of the repaired dorsal roots distal to the transplant completely abolished the behavioral improvement. This demonstrates that hNP transplants promote recovery of sensorimotor functions after dorsal root avulsion, and that these effects are mediated by spinal ingrowth of host sensory axons. These results provide a rationale for the development of novel stem cell-based strategies for functionally useful bridging of the peripheral and central nervous system.
Asunto(s)
Axones/fisiología , Células Madre Embrionarias Humanas/fisiología , Regeneración Nerviosa/fisiología , Células Receptoras Sensoriales/fisiología , Traumatismos de la Médula Espinal/fisiopatología , Raíces Nerviosas Espinales/fisiología , Células Madre/fisiología , Animales , Ganglios Espinales/fisiología , Humanos , Masculino , Ratones , Médula Espinal/fisiologíaRESUMEN
Precise temporal control of gene expression or deletion is critical for elucidating gene function in biological systems. However, the establishment of human pluripotent stem cell (hPSC) lines with inducible gene knockout (iKO) remains challenging. We explored building iKO hPSC lines by combining CRISPR/Cas9-mediated genome editing with the Flp/FRT and Cre/LoxP system. We found that "dual-sgRNA targeting" is essential for biallelic knockin of FRT sequences to flank the exon. We further developed a strategy to simultaneously insert an activity-controllable recombinase-expressing cassette and remove the drug-resistance gene, thus speeding up the generation of iKO hPSC lines. This two-step strategy was used to establish human embryonic stem cell (hESC) and induced pluripotent stem cell (iPSC) lines with iKO of SOX2, PAX6, OTX2, and AGO2, genes that exhibit diverse structural layout and temporal expression patterns. The availability of iKO hPSC lines will substantially transform the way we examine gene function in human cells.
Asunto(s)
Sistemas CRISPR-Cas/genética , Técnicas de Inactivación de Genes , Ingeniería Genética/métodos , Células Madre/metabolismo , Secuencia de Bases , Diferenciación Celular , Exones/genética , Regulación de la Expresión Génica , Técnicas de Sustitución del Gen , Marcación de Gen , Homocigoto , Humanos , Datos de Secuencia Molecular , Mutagénesis Insercional/genética , Factores de Transcripción Otx/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Células Madre Pluripotentes/metabolismo , ARN Guía de Kinetoplastida/metabolismoRESUMEN
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
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
Amyotrophic lateral sclerosis (ALS) presents motoneuron (MN)-selective protein inclusions and axonal degeneration but the underlying mechanisms of such are unknown. Using induced pluripotent cells (iPSCs) from patients with mutation in the Cu/Zn superoxide dismutase (SOD1) gene, we show that spinal MNs, but rarely non-MNs, exhibited neurofilament (NF) aggregation followed by neurite degeneration when glia were not present. These changes were associated with decreased stability of NF-L mRNA and binding of its 3' UTR by mutant SOD1 and thus altered protein proportion of NF subunits. Such MN-selective changes were mimicked by expression of a single copy of the mutant SOD1 in human embryonic stem cells and were prevented by genetic correction of the SOD1 mutation in patient's iPSCs. Importantly, conditional expression of NF-L in the SOD1 iPSC-derived MNs corrected the NF subunit proportion, mitigating NF aggregation and neurite degeneration. Thus, NF misregulation underlies mutant SOD1-mediated NF aggregation and axonal degeneration in ALS MNs.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Modelos Biológicos , Neuronas Motoras/metabolismo , Proteínas Mutantes/metabolismo , Superóxido Dismutasa/metabolismo , Esclerosis Amiotrófica Lateral/patología , Humanos , Células Madre Pluripotentes Inducidas/patología , Proteínas Mutantes/genética , Mutación , Superóxido Dismutasa/genética , Superóxido Dismutasa-1RESUMEN
Postnatal and adult human and monkey fibroblasts were infected with Sendai virus containing the Yamanaka factors for 24 hr, then they were cultured in a chemically defined medium containing leukemia inhibitory factor (LIF), transforming growth factor (TGF)-ß inhibitor SB431542, and glycogen synthase kinase (GSK)-3ß inhibitor CHIR99021 at 39°C for inactivation of the virus. Induced neural progenitor (iNP) colonies appeared as early as day 13 and can be expanded for >20 passages. Under the same defined condition, no induced pluripotent stem cell (iPSC) colonies formed at either 37°C or 39°C. The iNPs predominantly express hindbrain genes and differentiate into hindbrain neurons, and when caudalized, they produced an enriched population of spinal motor neurons. Following transplantation into the forebrain, the iNP-derived cells retained the hindbrain identity. The ability to generate defined, integration-free iNPs from adult primate fibroblasts under a defined condition with predictable fate choices will facilitate disease modeling and therapeutic development.
Asunto(s)
Fibroblastos/citología , Células-Madre Neurales/citología , Animales , Benzamidas/farmacología , Diferenciación Celular , Dioxoles/farmacología , Fibroblastos/efectos de los fármacos , Haplorrinos , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/trasplante , Factor Inhibidor de Leucemia/farmacología , Neuroglía/citología , Neuroglía/metabolismo , Neuronas/citología , Neuronas/metabolismo , Prosencéfalo/metabolismo , Piridinas/farmacología , Pirimidinas/farmacología , Rombencéfalo/metabolismo , TemperaturaRESUMEN
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.