Microarray analysis to detect whether the genome-wide gene expression profile of fibroblast-induced neurons resembles primary neurons
ABSTRACT: To explore more details in the similarities and differences between ABN+Rarg+Nr5a2 iN cells and primary neurons, we compared the global gene expression pattern of matured iN cells 12 days post infection with mouse primary neurons and MEFs by microarray analysis. In the study presented here, a total of 26423 unique genes were detected in primary mouse embryonic fibroblasts, induced neurons from MEFs by Ascl1+Brn2+Ngn2+Rarg+Nr5a2 at 12 days after infection and primary neurons from postnatal day 0 hippocampus, leading to more details about the similarities and differences among the three groups.
Project description:Compared the global gene expression profiles of HD- and CON-iPSC-derived neurons We used microarrays to detail the global programme of gene expression for comparing the global gene expression profiles of HD- and CON-iPSC-derived neurons and facilitating studies of medium spiny neurons (MSN)-degenerative processes of Huntington's Disease (HD). By using a step-wise in vitro differentiation protocol combining EB formation, neural induction by small molecules, treatment with inhibitors of the TGFß pathway (SB431542) and the BMP pathway (LDN193189), and mechanical isolation/purification of neural progenitors and neurons, we induced 60-70% of control iPSCs or HD-iPSCs to differentiate into GABA- and DARPP-32- double positive neurons.
Project description:Induced pluripotent stem cell (iPSC)-derived cortical neurons present a powerful new model of neurological disease. Previous work has established that differentiation protocols produce cortical neurons but little has been done to characterise these at cellular resolution. In particular, it is unclear to what extent in vitro two-dimensional, relatively disordered culture conditions recapitulate the development of in vivo cortical layer identity. Single cell multiplex RT-qPCR was used to interrogate the expression of genes previously implicated in cortical layer or phenotypic identity in individual cells. Unexpectedly, 22.7% of neurons analysed frequently co-expressed canonical fetal deep and upper cortical layer markers, and this co-expression was also present at the level of translated protein. By comparing our results to available single cell RNA-seq data from human fetal and adult brain, we observed that this co-expression of layer markers was also seen in primary tissue. These results suggest that establishing neuronal layer identity in iPSC-derived or primary cortical neurons using canonical marker genes transcripts is unlikely to be informative. Single cell RNA-seq of 16 iPSC-derived cortical neurons. This dataset was used for normalization purposes for GSE67835.
Project description:Expression response after induction of putative phrenic neuronal determinants in ES cell-derived motor neurons was compared to a pre-determined list of genes over-expressed in FACS-sorted primary. Transcription factor Pou3f1 was identified as a major determinant of phrenic identity. Expression in induced cell lines were compared to YFP controls, and over-representation of phrenic genes was computed for the list of differentially expressed genes in each indiced cell lines.
Project description:Somatic cells can be reprogrammed to Induced Pluripotent Stem Cells (iPSCs) by expressing four transcription factors, Oct4, Sox2, Klf4 and c-Myc. Co-expressing Rarg (retinoic acid receptor gamma) and Lrh-1 (liver receptor homolog 1, Nr5a2) with the four factors greatly accelerated reprogramming so that reprogramming of mouse embryonic fibroblast cells (MEFs) to ground state iPSCs requires only four days’ induction of these six factors. The six-factor combination readily reprogrammed primary human neonatal and adult fibroblast cells to exogenous-factor-independent iPSCs, which resembled ground state mouse ES cells in growth properties, gene expression and signalling dependency. Our findings demonstrate that signalling through RARs has critical roles in molecular reprogramming and the synergistic interaction between Rarg and Lrh1 directs reprogramming towards ground state pluripotency. SH-iPS20-1, -3 and -9 lines were derived from HDFa (439656), SH-iPS24-1, -2 and -11 lines were derived from HDFa (709590), and SH-iPS28-23, -25 and -27 lines were derived from HDFn (617769). Parental HDF lines were used as control. SH-iPS15-5 and -10 lines were derived from HDFn (617769, Invitrogen). H1 hESC were obtained from Wicell (Madison, USA). SH-iPSCs were cultured in both 2i+LIF and FGFconditions. Expression pattern of SH-iPSCs was compared to H1 hESC cultured in FGF conditions. S1, S2 and S3 are three indpendent subcultures of a cell line.
Project description:LSD1n regulates transcriptional elongation by removing H4K20 methylation. Identification of genome-wide binding sites of LSD1, and examination of the histone modifications upon LSD1n deletion in primary cortical neurons
Project description:To investigate the genetic relationship between two major grain length loci GS3 and qGL3, we developed the near-isogenic lines (NILs), NIL-GS3 (GS3/qGL3), NIL-qgl3 (gs3/qgl3), NIL-GS3/qgl3 (GS3/qgl3) in the background of 93-11 (gs3/qGL3) by crossing and MAS approach. Four samples was analyzed: three near-isogenic lines (NILs), NIL-GS3 (GS3/qGL3), NIL-qgl3 (gs3/qgl3), NIL-GS3/qgl3 (GS3/qgl3) and their background of 93-11. Every sample had three independed duplications. And the primary panicle with 3-6 cm length from the three NILs and 93-11 were used for RNA preparation and hybrid with Rice Genome OneArray Microarray (Phalanx Biotech Group).
Project description:CHD5 is frequently deleted in neuroblastoma, and appears to be a tumor suppressor gene; however, little is known about the role of CHD5. We found CHD5 mRNA was restricted to brain; by contrast most other remodeling ATPases were broadly expressed. CHD5 protein isolated from mouse brain was associated with HDAC2, p66, MTA3 and RbAp46 in a megadalton complex. CHD5 protein was detected in several rat brain regions and appeared to be enriched in neurons. CHD5 protein was predominantly nuclear in primary rat neurons and brain sections. Microarray analysis revealed genes that were upregulated and downregulated when CHD5 was depleted from primary neurons. CHD5 depletion altered expression of neuronal genes, transcription factors, and brain-specific subunits of the SWI/SNF remodeling enzyme. Aging and Alzheimers gene sets were strongly affected by CHD5 depletion from primary neurons. Chromatin immunoprecipitation revealed CHD5 bound to these genes, suggesting the regulation was direct. Together, these results indicate that CHD5 is found in a NuRD-like multi-protein complex. CHD5 is restricted to the brain, unlike the closely related family members CHD3 and CHD4. CHD5 regulates expression of neuronal genes, cell cycle genes and remodeling genes. CHD5 is linked to regulation of aging and Alzheimer’s genes. CHD5 KD shRNA sequences were designed according to the instructions for the pLKO.1 system (Addgene). Control was as described (Sci307-1098,2005) Scramble, clone 1864 from Addgene). Virus was packaged using HEK-293T cells, pLKO.1 vector with shRNA inserts for CHD5, and the control. 48 hours after transfection of 293 cells, medium containing virus was filtered (0.45 micron), then applied for 6 hours to primary cortical neurons one day after the neurons were plated (Day 1). Medium was removed, and replaced with Neural Basal Medium, and the cells were cultured until Day 5, 9 or 12. RNA was harvested from 3 replicates of the treated primary cortical neurons at each time point. RNA was isolated using RNAeasy Kit (Qiagen), Quality and quantity of the total RNA was checked with the Agilent 2100 bioanalyzer using RNA 6000 Nano chips. RNA was labeled using the standard Illumina protocol and Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX, cat # IL1791) Biotin labeled cRNA was hybridized to Illumina's Sentrix Rat Ref-12 v1 Expression BeadChips.
Project description:This experiment comprises RNA-seq data used to study evolutionary differences between humans and mice in neuronal activity-dependent transcriptional responses. Activity-dependent transcriptional responses in developing human stem cell-derived cortical neurons were compared with those induced in developing primary- or stem cell-derived mouse cortical neurons 4 hours after KCl-induced membrane depolarisation. Activity-dependent transcriptional responses were also measured in aneuploid mouse neurons carrying human chromosome 21, allowing study of the regulation of Hsa21 genes, plus their mouse orthologs, side-by-side in the same cellular environment of a mouse primary neuron.
Project description:Primary cortical neurons were isolated from E15 mice and after 5 days in vitro were untreated or treated for 24 h with mesenchymal stem cell conditioned medium and then untreated or treated for a further 24 h with NMDA. Neuron gene expression was profiled and compared between the four different conditions (neurons, neurons+MSC cm, neurons+NMDA, neurons+MSC cm+NMDA) to investigate the molecular mechanisms of MSC neuroprotection. Mesenchymal stem cells (MSC) promote functional recovery in experimental models of central nervous system (CNS) pathology and are currently being tested in clinical trials for stroke, multiple sclerosis and CNS injury. Their beneficial effects are attributed to activation of endogenous CNS repair processes and immune regulation but their mechanisms of action are poorly understood. Here we investigated the neuroprotective effects of MSC in simplified MSC-neuron co-culture systems and in mice using models of glutamate excitotoxicity. MSC protected primary cortical neurons against glutamate (NMDA) receptor-induced death and conditioned medium from MSC (MSC cm), but not control NIH3T3 cells, was sufficient for this effect. MSC cm neuroprotection in mouse cortical neurons was reduced by neutralizing antibodies to bFGF and associated with altered gene expression in neurons towards an immature phenotype as well as reduced neuronal Grin1, Grin2a and Grin2b mRNA levels in response to NMDA stimulation. Further, MSC cm neuroprotection in rat retinal ganglion cells was associated with absence of glutamate-induced calcium influx. Adoptive transfer of EGFP+MSC in a mouse kainic acid seizure model reduced CA3 neuron damage and hippocampal astrocytosis and resulted in the increased expression of neuronal genes that are upregulated by MSC cm, Bmi1, Ddx4, Ezh1, in the hippocampus. These results show that MSC mediate direct neuroprotection against glutamate excitotoxicity by secreting bFGF, reducing glutamate receptor expression and function and altering neuron gene expression towards an immature pattern, and provide evidence for a link between the therapeutic effects of MSC and the activation of endogenous repair processes following CNS injury. In vitro cultures primary cortical neurons from mice were protected from glutamate excitotoxicity when pre-treated with MSC cm. Global gene expression changes induced in neurons before and after treatment with MSC cm and/or NMDA were investigated using a cDNA spotted macroarray filter. Four samples were analysed in duplicate: neurons alone (untreated), neurons+MSC cm, neurons+NMDA, neurons+MSC cm+NMDA.
Project description:Identification of new small molecules that regulate the step-wise differentiation of hPSC into dopaminergic neurons population. Furthermore, the naturally occurring steroid, guggulsterone, was found to be the most effective inducer of neural stem cells into dopaminergic neurons. Total RNA extracted at different stages of neural differentiation from human pluripotent stem cells