Induction of cells expressing markers of GABAergic neurons by transcription factors
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Minoru Ko
PROVIDER: E-GEOD-78951 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Publications
Induction of specific neuron types by overexpression of single transcription factors.
Teratani-Ota Yusuke Y Yamamizu Kohei K Piao Yulan Y Sharova Lioudmila L Amano Misa M Yu Hong H Schlessinger David D Ko Minoru S H MS Sharov Alexei A AA
In vitro cellular & developmental biology. Animal 20160601 9
Specific neuronal types derived from embryonic stem cells (ESCs) can facilitate mechanistic studies and potentially aid in regenerative medicine. Existing induction methods, however, mostly rely on the effects of the combined action of multiple added growth factors, which generally tend to result in mixed populations of neurons. Here, we report that overexpression of specific transcription factors (TFs) in ESCs can rather guide the differentiation of ESCs towards specific neuron lineages. Analys ...[more]
Similar Datasets
Project description:Specific neuronal types derived from embryonic stem cells (ESCs) can facilitate mechanistic studies and potentially aid in regenerative medicine. Existing induction methods, however, mostly rely on the effects of growth factors, which generally tend to result in mixed populations of neurons. Here we report that over-expression of specific transcription factors (TFs) in ESCs can rather guide the differentiation of ESCs towards specific neuron types. Analysis of published data on gene expression changes early (two days) after induction of each of 185 induced TFs implicated candidate TFs for further ESC differentiation studies. After induction for 6 days four of them (Ascl1, Smad7, Nr2f1, and Ascl2) generated a high proportion (>35%) of cells with neural progenitor marker PSA-NCAM and clear neural morphology on day 14. The capacity of these TFs to induce neural differentiation is inferred to be most likely linked to early activation of the Notch signaling pathway. Among the neuron-like cells, GABA-positive cells were most abundant (32-97% for 4 top TFs), whereas Isl1-positive cells and TH-positive cells were less abundant (<12% and <5%, respectively). Enrichment of cells obtained with the induction of Ascl1, Smad7, and Nr2f1 using beads with anti-PSA-NCAM antibody resulted in essentially pure population of neuron-like cells with expression profiles similar to neural tissues and highly expressed markers of GABAergic neurons. A time-course experiment with induction of Ascl1 showed early upregulation of most neural-specific and GABAergic-specific mRNA and miRNAs. We identified mRNA and miRNAs, whose expression depended on the induction of Ascl1, and showed that they were enriched in Ascl1 target genes. In summary, this study indicates that induction of transcription factors is a promising approach to generate candidate specific neural cell types from ESCs. Individual transcription factors (TFs) (Ascl1, Smad7, and Nr2f1) were induced in mouse ESCs to facilitate neural differentiation. Expression of transgenic TFs was repressed by doxycycline (Dox); thus, TFs were induced in Dox- conditions, whereas Dox+ conditions represent control cells with no expression of a transgene. For neural differentiation, cells were cultured 3 days in alpha-MEM medium and then in NeuroCult neural differentiation medium. Cells marked as PSANCAM+ were enriched by magnetic microbeads with anty-PSA-NCAM antibody (Miltenyi Biotec) on day 6 of culturing, whereas remaining cells are marked as PSANCAM-. Both PSANCAM+ and PSANCAM- cells were then cultured for another 8 days (total 14 days in differentiation). A time course experiment with Ascl1 induction did not include cell enrichment procedure. RNA was extracted with either Trizol (invitrogen) or mirVana kit (Thermo Fisher Scientific).
2016-07-21 | E-GEOD-78949 | biostudies-arrayexpress
Project description:Specific neuronal types derived from embryonic stem cells (ESCs) can facilitate mechanistic studies and potentially aid in regenerative medicine. Existing induction methods, however, mostly rely on the effects of growth factors, which generally tend to result in mixed populations of neurons. Here we report that over-expression of specific transcription factors (TFs) in ESCs can rather guide the differentiation of ESCs towards specific neuron types. Analysis of published data on gene expression changes early (two days) after induction of each of 185 induced TFs implicated candidate TFs for further ESC differentiation studies. After induction for 6 days four of them (Ascl1, Smad7, Nr2f1, and Ascl2) generated a high proportion (>35%) of cells with neural progenitor marker PSA-NCAM and clear neural morphology on day 14. The capacity of these TFs to induce neural differentiation is inferred to be most likely linked to early activation of the Notch signaling pathway. Among the neuron-like cells, GABA-positive cells were most abundant (32-97% for 4 top TFs), whereas Isl1-positive cells and TH-positive cells were less abundant (<12% and <5%, respectively). Enrichment of cells obtained with the induction of Ascl1, Smad7, and Nr2f1 using beads with anti-PSA-NCAM antibody resulted in essentially pure population of neuron-like cells with expression profiles similar to neural tissues and highly expressed markers of GABAergic neurons. A time-course experiment with induction of Ascl1 showed early upregulation of most neural-specific and GABAergic-specific mRNA and miRNAs. We identified mRNA and miRNAs, whose expression depended on the induction of Ascl1, and showed that they were enriched in Ascl1 target genes. In summary, this study indicates that induction of transcription factors is a promising approach to generate candidate specific neural cell types from ESCs. Transcription factor Ascl1 was induced in mouse ESCs to facilitate neural differentiation. Expression of transgenic Ascl1 was repressed by doxycycline (Dox); thus, it were induced in Dox- conditions, whereas Dox+ conditions represent control cells with no expression of Ascl1 transgene. For neural differentiation, cells were cultured 3 days in alpha-MEM medium and then - in NeuroCult neural differentiation medium for 2-11 days (total up to 14 days). RNA was extracted with mirVana kit (Thermo Fisher Scientific).
2016-07-21 | E-GEOD-78950 | biostudies-arrayexpress
Project description:Comparison among ES, EC, TS, NS, differentiated neural cells derived from NS and placenta in addition to ES-N2B27 neural induction. Comparison among ES, EC, TS, NS, differentiated neural cells derived from NS and placenta in addition to ES-N2B27 neural induction.
2010-06-11 | E-GEOD-4077 | biostudies-arrayexpress
Project description:Defining a developmental path to neural fate by global expression profiling of mouse embryonic stem cells and adult neural stem/progenitor cells. This SuperSeries is composed of the following subset Series: GSE4075: Neural induction (Embrioid bodies-Retinoic acid) GSE4076: Neural induction (N2B27 monolayer cluture) GSE4077: ES, EC, NS, TS, Placenta and Neural induction (N2B27) comaprison Keywords: SuperSeries Refer to individual Series
2010-06-11 | E-GEOD-4082 | biostudies-arrayexpress
Project description:Biologists rely on morphology, function, and specific markers to define the differentiation status of cells. Transcript profiling has expanded the repertoire of these markers by providing the snapshot of cellular status that reflects the activity of all genes. However, such data have been used only to assess relative similarities and differences of these cells. Here we show that principal component analysis (PCA) of global gene expression profiles map cells in multidimensional transcript profile space and the positions of differentiating cells progress in a stepwise manner along trajectories starting from undifferentiated embryonic stem (ES) cells located in the apex. We present three cell lineage trajectories, which represent the differentiation of ES cells into the first three lineages in mammalian development: primitive endoderm, trophoblast, and primitive ectoderm/neural ectoderm. The positions of the cells along these trajectories seem to reflect the developmental potency of cells and can be used as a scale for the potential of cells. Indeed, we show that embryonic germ (EG) cells and induced pluripotent (iPS) cells are mapped near the origin of the trajectories, whereas mouse embryo fibroblast (MEF) and fibroblast cell lines are mapped near the far end of the trajectories. We propose that this method can be used as the non-operational semi-quantitative definition of cell differentiation status and developmental potency. Furthermore, the global expression profiles of cell lineages provide a framework for the future study of in vitro and in vivo cell differentiation. Keywords: cell type comparison design,reference design,replicate design,time series design Most of the cells and RNA samples used in this study were described in detail previously (See paper's citation associated with this dataset). To maximize the uniformity of the microarray data, all the samples, including ones analyzed by DNA microarray previously, were hybridized to the same platform (the NIA Mouse 44K Microarray manufactured by Agilent Technologies: AMADID #015087). The intensity of each gene feature per array was extracted from scanned microarray images using Feature Extraction Software V9.5.
2008-12-27 | E-GEOD-11523 | biostudies-arrayexpress
Project description:The generation of induced pluripotent stem cells (iPSCs) by the forced expression of defined transcription factors in somatic cells holds great promise for the future of regenerative medicine. However, due to a poor understanding of the initial reprogramming mechanism, it is difficult to control the efficiency and quality of the iPSCs. Here we show that Zscan4, expressed transiently in 2-cell embryos and embryonic stem cells (ESCs), efficiently produces iPSCs from mouse embryo fibroblasts when coexpressed with Klf4, Oct4, and Sox2. Unlike other factors, Zscan4 is required only for the first few days of iPSC formation. Microarray analysis revealed transient and early induction of preimplantation-specific genes in a Zscan4-dependent manner. Our work indicates that Zscan4 is a previously unidentified potent natural factor that facilitates the reprogramming process through the recapitulation of the early embryonic program. Secondary MEFs were isolated from E13.5 embryos, which were harvested by tetraploid complementation. Secondary MEFs were plated on gelatin-coated 6-well plates at a density of 1x105 cells/well in the complete ES medium. After 24 h incubation, secondary MEFs were fed with complete ES medium with or without Dox (1.5 ?g/ml) and with or without 200 nM Tmx. Culture medium was changed every day. Withdrawal of drugs (Dox or Tmx) was always followed by 1x washing by PBS before changing culture medium. We cotransfected MEFs with PB-Tet-MKOS and either a PB-Tet-Zscan4 or PB-Tet-DsRed (control). These cells were subsequently cultured in the standard iPSC generation condition (Dox+). Unexpectedly, we found that MEFs transfected with a PB-Tet-Zscan4 show consistently higher efficiency of iPSC generation than MEFs transfected with a PB-Tet-DsRed (control). All selected iPSCs expressed pluripotent marker genes according to RT-PCR analysis and were positive for ALP, Nanog, and SSEA-1. These iPSCs also formed embryoid bodies in hanging drops without LIF and could be differentiated in vitro to cells of all three germ layers. We are currently studying whether the presence of Zscan4 has the beneficial effects on iPSCs in terms of genome stability.
2012-01-06 | E-GEOD-28436 | biostudies-arrayexpress
Project description:B-type lamins, the major structural component of the nuclear lamina, is thought to play a repressive role in the expression of many of its bound genes whose silencing are known to be critical for cell differentiation during early development. Using global mapping of lamin-B-chromatin interaction, we find that the binding to lamin-B indeed correlated with gene silencing in mouse embryonic stem cells (ESC) and ESC-derived trophectoderm (TE) lineage cells. To address whether lamin-B directly suppresses the expression of the bound genes, we have derived ESCs from lamin-B-null mouse blastocysts. Unexpectedly, microarray analyses of ESCs and TE cells show that B-type lamins do not repress the expression of their bound genes. Furthermore, the knockout mice that do not express any B-type lamins can develop to term but exhibit small body size and perinatal lethality. Our studies demonstrate that B-type lamins are not required for cell differentiation during early embryonic development. Instead, they are required for proper late embryonic and perinatal development. The availability of the null ESCs and mice opens the door to further define the functions of B-type lamins. The Dam and Dam-lamin B1 (Lmnb1) lentivirus generated from 293T cells were concentrated by spinning at 25,000 rpm with the SW28Ti rotor (Beckman) for 3 hours. The viral pellets were resuspended in 1x PBS. For ESCs, freshly trypsinized ESCs were plated into a 6-well plate with 2 ml complete GMEM medium containing either Dam or Dam-Lmnb1 lentivirus, spin-infected at 700g for 45 min, and incubated for a total of 48 hours. For TE cells, TSCs were differentiated toward TE cells for 6 days. On day 6, the TE cells were transduced by spin-infection as above with a fresh TS medium containing either Dam or Dam-Lmnb1 lentivirus. The TE cells were incubated for 48 hours after viral transduction. The adenine-methylated DNAs were amplified from the genomic DNA by adaptor-mediated PCR. The amplified DNAs were labeled by the Dual color DNA labeling kit (Roche). The labeled DNAs were hybridized with 2.1M mouse whole-genome tiling arrays (NimbleGen, 05327911001), which covers non-repetitive regions in approximately 250 bp intervals, according to the manufacturer's recommendation. This submission represents the gene expression component of the study.
2011-10-31 | E-GEOD-24532 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2013-06-12 | E-GEOD-26278 | biostudies-arrayexpress
Project description:The gold standard for examining pluripotency of stem cells is to see whether cells can contribute to entire body of animals. Here we show that the increased frequency of Zscan4 activation in mouse ES cells not only enhances, but also maintains their developmental potency in long-term cell culture. As the potency increases, even a whole animal can be produced from a single ES cell injected into 4N blastocyst at unusually high success rate. Although Zscan4-activated cells express genes that are also expressed in 2-cell stage mouse embryos, transiently Zscan4-activated state of ES cells is not associated with the high potency of ES cells. It is thus concluded that ES cells acquire higher potency by going through transient Zscan4 activation state more frequently than the regular state. Taken together, our results indicate that frequent activation of Zscan4 can rejuvenate pluripotent stem cells. Previously, we have shown that constitutive and strong expression of Zscan4 arrest the proliferation of ES cells (refs). We sought for the system to mimic the transient expression of Zscan4 and decided to use ERT2 - Tamoxifen-inducible system (refs). The system allows one to keep a transgene off in the absence of Tmx and turn on in the presence of Tmx at will (refs). We first made a plasmid construct pCAG-Zscan4-ERT2, in which ZSCAN4 ORF fused with ERT2 domain can be driven by a strong ubiquitous promoter CAG. To further investigate this, we transfected pCAG-Zscan4-ERT2 plasmid into an F1 hybrid (C57BL/6J vs. 129) ES cell line V6.5 and isolated multiple cell clones. Based on the qRT-PCR analysis of Zscan4 ORF, we found that cell clones showed a variety of Zscan4 expression levels. We selected the clone that showed the highest Zscan4 expression levels (clone #18) and a clone #2 with the background Zscan4 level.
2013-06-12 | E-GEOD-26162 | biostudies-arrayexpress
Project description:Human tissue based proteomics projects are challenging due to low abundance of proteins and tissue specificity of protein expression. In this study, we aimed to develop a cell-based approach to profile the male specific region of the Y chromosome (MSY) proteins. First, we profiled the expression of 23 Y chromosome genes and 15 of their X-linked homologues during neural cell differentiation from NT2 cells at three different developmental stages using qRT-PCR, western blotting and immunofluorescent (IF) techniques. The expression level of 12 Y-linked genes significantly increased over neural differentiation. Including RBMY1, EIF1AY, DDX3Y1, HSFY1, BPY2, PCDH11Y, UTY, RPS4Y1, USP9Y, SRY, PRY, and ZFY. Subsequently, DDX3Y was selected as a candidate for knockdown as it was significantly expressed in neural progenitor cells and it is known to be expressed in a gender specific manner and play a role in spermatogenesis. A siRNA-mediated DDX3Y knockdown in neural progenitor cells impaired cell cycle progression and increased apoptosis, consequently interrupting differentiation. Label-free quantitative shotgun proteomics based on a spectral counting approach was then used to characterize the proteomic profile of the cells after DDX3Y knockdown. Among 920 reproducibly identified proteins detected, 74 proteins were differentially expressed following DDX3Y siRNA treatment compared to mock treated cells. Functional grouping indicated these proteins were associated with cell cycle, cell-to-cell signaling, apoptosis and other important networks such as RNA processing and transcription regulation. Disease-based analysis confirmed DDX3Y involvement primarily in neurological and RNA metabolism disorders. Our results confirm that MSY genes are expressed in male neuronal cells, and demonstrate that Y linked DDX3 (DDX3Y) could play a multifunctional role in neural cell development in a sexually dimorphic manner.
2015-09-17 | PXD002428 | Pride