Neuronal differentiation of human embryonic stem cells
Ontology highlight
ABSTRACT: Human embryonic stem cells were differentiated to early Pax6 positive neural epithelial progenitor cells and were differentiated further in neurospheres. On indicated days (hESC: n=3, day 0: n=3, day 5 n=3, day 15: n=2, day 22: n=1 and day 25: n=1) cells were extracted, mRNA prepared and hybridized in triplicates to Illumina Sentrix HumanHT-12 BeadChip gene arrays. More experimental detail is available under Hoelting et al., Archives of Toxicology.
Project description:We used microarrays to detail the global programme of gene expression underlying neuronal differentiation of SVZ-neurospheres when manipulating Pax6 expression in the presence or absence of Pbx1
Project description:Axon guidance is required for the establishment of brain circuits. Whether much of the molecular basis of axon guidance is known from animal models, the molecular machinery coordinating axon growth and pathfinding in humans remains to be elucidated. The use of induced pluripotent stem cells (iPSC) from human donors has revolutionized in vitro studies of the human brain. iPSC can be differentiated into neuronal stem cells which can be used to generate neural tissue-like cultures, known as neurospheres, that reproduce, in many aspects, the cell types and molecules present in the brain. Here, we analyzed quantitative changes in the proteome of neurospheres during differentiation. Relative quantification was performed at early time points during differentiation using iTRAQ-based labeling and LC-MS/MS analysis. We identified 6438 proteins, from which 433 were downregulated and 479 were upregulated during differentiation. We show that human neurospheres have a molecular profile that correlates to the fetal brain. During differentiation, upregulated pathways are related to neuronal development and differentiation, cell adhesion and axonal guidance whereas cell proliferation pathways were downregulated. We developed a functional assay to check for neurite outgrowth in neurospheres and confirmed that neurite outgrowth potential is increased after 10 days of differentiation and is enhanced by increasing cyclic AMP levels. The proteins identified here represent a resource to monitor neurosphere differentiation and coupled to the neurite outgrowth assay can be used to functionally explore neurological disorders using human neurospheres as a model.
Project description:Stable Pax6 and Luciferase knockdown human embryonic stem cell lines (H9) were made through lentiviral infection. Two different Pax6 RNAi lines and two Luciferase RNAi lines were then differentiated to neuroectoderm cells for 6 days. mRNA pooled from these two individual lines were then subjected to gene expression profiling analysis using affymetrix U133 plus 2.0 array.
Project description:Purpose: We find that Wnt7a-PAX6 axis determine corneal epithelial cell fate. To obtain global evidence for successful cell fate conversion, we performed gene expression profiling by RNA-seq on CECs, SECs, and LSCs after knocking down PAX6 and on SESCs transduced with PAX6 upon 3-D differentiation. Methods: Under 3-D culture condition, limbal stem cell (LSCs) can be differentiated to Cornea epithelial cells (CECs), and skin epithelial stem cells (SESCs) can be differentiated to skin epithelial cells (SECs). Total RNA was isolated from CECs, SECs, and LSCs after knocking down PAX6 (3-D shPAX6 LSCs) and on SESCs transduced with PAX6 (3-D PAX6+ SESCs) upon 3-D differentiation. Libraries were prepared following published standard protocol (Fox-Walsh K et al., 2011, genomics, 266-71). mRNA profiles were generated by deep sequencing, in duplicate, using Illumina HiSeq 2000. Results: Following optimized decoding and mapping workfollow, we mapped about 5 million sequence reads to the human genome and identified more than 23659 transcripts per sample. Conclusions: Hierarchical clustering analysis of differentially expressed gene signatures revealed that the gene expression pattern of SESCs with PAX6 transduction was strikingly similar to that of CECs, whereas the profile of LSCs with PAX6 knockdown was highly related to that in SECs upon differentiation. These data therefore provided global evidence for a decisive role of the WNT7A/PAX6 axis in cell fate conversion from SESCs to CECs. RNA-seq on CECs, SECs, and LSCs after knocking down PAX6 and on SESCs transduced with PAX6 upon 3-D differentiation, using Illumina HiSeq 2000
Project description:Cerebral cortical-derived neurosphere cultures were exposed to ethanol to model heavy alcohol exposure during the period of cortical neurogenesis Keywords: Compared differentiated neurospheres pre-exposed to ethanol to untreated differentiated neurospheres
Project description:Pax6 is one of the important transcription factors involved in regional specification and neurogenesis in the developing cortex. To identify candidate target genes of Pax6, we performed transcriptome analyses of wild-type (WT) and Pax6 homozygous mutant rats (rSey2/rSey2) telencephalons at E11.5 within a day of onset of Pax6 expression. In our transcriptome analyses, down-regulated genes in the rSey2/rSey2 rat exhibited larger fold changes, whereas up-regulated genes had relatively small fold changes. Total RNA was prepared using from 13 telencephalon dissected from E11.5 WT or rSey2/rSey2 rats embryos. Experiments using 13 telencephaon were repeated twice in each genotype.
Project description:Protocol to differentiate iPSC control lines into astrocytes. We performed the RNA sequencing of cells at different time point: embryoid bodies ; neurospheres at passage 1 ; neurospheres at passage 2 and differentiated astrocytes (iPast) for the 201B7 iPSC and iPast stage for WD39.
Project description:Monocytes differentiated in the presence of farnesol from 4 different donors where harvested after 3 and 6 days of differentiation and after exposure to LPS for 6h (at day 6) and analyzed on an Illumina HumanHT-12 v4 Expression BeadChip.
Project description:This study identifies differentially expression genes in the sputum of people with eosinophilic, neutrophilic and paucigranulocytic asthma. A selection of markers identified using this microarray were further validated using qPCR on a wider sample set. Gene expression profiles were generated from induced sputum samples from 47 asthma patients and were grouped by the inflammatory phenotype assigned using sputum cell counts into neutrophilic asthma (n=12), eosinophilic asthma (n=17) and paucigranulocytic asthma (n=18). RNA was extracted, amplified and hybridised to Illumina Sentrix HumanRef-8 Version 2 Expression BeadChips, and genes that were differentially expressed between asthma inflammatory phenotypes were compared.