Comparison of gene expression signature between nascent Lhx6-GFP+ cells from mouse embryonic brain and differentiated Lhx6-GFP+ cells from ES cells (as well as comparing those of Lhx6-GFP+ and Lhx6-GFP- cells from ES cells)
ABSTRACT: There was a remarkable similarity in the molecular properties of the MGE-GFP+ and ES-GFP+ cells. In particular, genes that are important for medial ganglionic eminence (MGE) and cortical interneurons development are both high in expression in both MGE-Lhx6-GFP+ and ES-Lhx6-GFP+ cells (compared to ES-Lhx6-GFP- cells). To investigate how closely ES cells-derived Lhx6-GFP+ cells resembled authentic Lhx6+ MGE cells, and to define the molecular properties of the Lhx6-GFP+ and Lhx6-GFP- cells from differentiated ES cells, we compared their gene expression profiles. We used FACS to purify GFP+ cells from the E12.5 MGE of Lhx6-GFP transgenic mice. ES-Lhx6-GFP+ cells and ES-Lhx6-GFP- cells (both from D12 EB aggregates) were also isolated by fluorescent activated cell sorting (FACS) and all of the RNA samples were subjected to RNA expression microarray analyses.
Project description:J14 ES cells differentiated into MGE-like cells. Three groups of single-cell preparations were analyzed: ES cells (undifferentiated), differentiated cells (unsorted, of which less than 10% are GFP+) and GFP+ differentiated cells. These are specified in the "group" sample characteristic, with values "ES", "Unsorted" and "GFP+" respectively. The "SAMPLE_ID" sample characteristic is a sample identifier internal to Genentech. The ID of this project in Genentech's ExpressionPlot database is PRJ0007904 Overall design: J14 ES cells differentiated into MGE-like cells
Project description:To identify the activity-induced gene expression programs in inhibitory and excitatory neurons, we analyzed RNA extracted from cultured E14 mouse MGE- and CTX-derived neurons (DIV 10) after these cultures were membrane-depolarized for 0, 1 and 6 hrs with 55mM extracellular KCl. To identify the gene programs regulated in these cells by the activity-induced early-response transcription factor Npas4, we repeated the same experiment in the MGE- and CTX-cultures lacking Npas4 (Npas4-KO). Littermate mouse E14 MGE- or CTX-derived neurons (WT or KO for Npas4) were cultured for 9 days, quieted overnight with TTX and AP-5 and then membrane-depolarized for 0, 1 or 6 hours by raising the extracellular KCl-concentration to 55mM. RNA was then extracted and analyzed using Affymetrix GeneChip Mouse Expression Set 430 2.0 microarray platform.
Project description:Transcriptional programming of cell identity promises to open up new frontiers in regenerative medicine by enabling the efficient production of clinically relevant cell types. We examine if such cellular programming is accomplished by transcription factors that each have an independent and additive effect on cellular identity, or if programming factors synergize to produce an effect that is not independently obtainable. The combinations of Ngn2-Isl1-Lhx3 and Ngn2-Isl1-Phox2a transcription factors program embryonic stem cells to express a spinal or cranial motor neuron identity respectively. The two alternate expression programs are determined by recruitment of Isl1/Lhx3 and Isl1/Phox2a pairs to distinct genomic locations characterized by two alternative dimeric homeobox motifs. These results suggest that the function of programming modules relies on synergistic interactions among transcription factors and thus cannot be extrapolated from the study of individual transcription factors in a different cellular context. In this study, we functionally characterize induced motor neurons that have been directly generated from ES cells via the forced expression of two different combinations of three transcription factors. Spinal motor neurons are induced via the expression of Ngn2, Isl1, and Lhx3 (iNIL), while cortical motor neurons are induced via the expression of Ngn2, Isl1, and Phox2a (iNIP). Here we profile the gene expression patterns of both types of induced motor neurons, directed differentiation motor neurons, and control cells. In all, 20 microarray experiments are provided in this submission, including 3 replicates of a control condition, 3 replicates of cells that have 24hrs induction of iNIL, 2 replicates of induced spinal motor neurons (induction of iNIL for 48hrs) that have been Hb9-GFP sorted, 3 replicates of induced spinal motor neurons exposed to retinoic acid that have been Hb9-GFP sorted, 3 replicates of motor neurons that have been differentiated in vitro using RA and Hh signalling, 3 replicates of induced cortical motor neurons (induction of iNIP for 48hrs), and 3 replicates of cells in which Isl1 in induced alone (induction of iI for 48hrs). For ChIP-Seq Samples: In this study, we functionally characterize induced motor neurons that have been directly generated from ES cells via the forced expression of two different combinations of three transcription factors. Spinal motor neurons are induced via the expression of Ngn2, Isl1, and Lhx3 (iNIL), while cortical motor neurons are induced via the expression of Ngn2, Isl1, and Phox2a (iNIP). The genome-wide binding of some of the programming factors is characterized here using ChIP-seq. We characterize the binding of Lhx3 and Isl1/2 in iNIL cells, Phox2a and Isl1/2 in iNIP cells, and Isl1/2 in cells in which Isl1 is induced alone (iI). There are 7 Illumina sequence datasets in this submission; one replicate for each of iLhx3-V5 and Isl1/2 in iNIL cells, two replicates for each of iPhox2a-V5 and Isl1/2 in iNIP cells, and one replicate for Isl1/2 in iI cells. An appropriate pseudo-IP control experiment is included.
Project description:Human embryonic stem cells with a GFP reporter knock-in into the NKX2.1 locus were differentiated and purified by FACS sorting for global gene expression analysis. Directed differentiation from human pluripotent stem cells (hPSCs) has seen significant progress in recent years. Most differentiated populations, however, exhibit immature properties of an early embryonic stage, raising concerns about their ability to model and treat disease. Here, we report the directed differentiation of hPSCs into medial ganglionic eminence (MGE)-like progenitors and their maturation into forebrain type interneurons. We find that early stage progenitors progress via a radial glial-like stem cell enriched in the human fetal brain. Both in vitro and post-transplantation into the rodent cortex, the MGE-like cells develop into GABAergic interneuron subtypes with mature physiological properties along a prolonged intrinsic timeline of up to seven months, mimicking endogenous human neural development. MGE-derived cortical interneuron deficiencies are implicated in a broad range of neurodevelopmental and degenerative disorders, highlighting the importance of these results for modeling human neural development and disease. Human embryonic stem cells with a GFP reporter knock-in into the NKX2.1 locus were differentiated for 20, 35, and 55 days in vitro and GFP+ cells were purified by FACS sorting. Total RNA was prepared from each timepoint and compared to undifferentiated human embryonic stem cells. hESC = one sample and three technical replicates. D20 = three independent samples. D35 = one sample and two technical replicates. D55 = one sample and one technical replicate.
Project description:To identify the activity-induced gene expression programs in inhibitory neurons, we analyzed RNA extracted from cultured E14 mouse MGE-derived neurons (DIV 10) after these cultures were membrane-depolarized for 0, 1 and 6 hrs with 55mM extracellular KCl. Mouse E14 MGE-derived neurons were cultured for 9 days, quieted overnight with TTX and AP-5 and then membrane-depolarized for 0, 1 or 6 hours by raising the extracellular KCl-concentration to 55mM. RNA was then extracted and WT RNA-Seq was performed on ABi SOLiD
Project description:To isolate photoreceptor precursors fit for transplantation, we adapted a 3D differentiation protocol that generates neuroretina from mouse ES cells. We used an adeno-associated viral vector (pseudotype2/9)carrying a GFP reporter under the control of a Rhodopsin promoter (AAV2/9.Rhop.GFP)to select rod precursors. AAV2/9.Rhop.GFP+ rods were sorted at days 26 and 34 and post natal day P12.
Project description:We analyzed the role of the histone lysine methyltransferase Set7/9 in the differentiation of human embryonic stem (ES) cells. Human ES cell lines expressing a control short hairpin and a short hairpin against Set7/9 were established and the genome wide expression profile was compared between both cell lines at different days during differentiation. Analysis of both profiles indicates that in the absence of Set7/9 there is a delay in the silencing of self-renewal factors as well as in the induction of differentiation markers. These results indicate that Set7/9 plays an active role in the differentiation of human ES cells. control and set7/9 shRNA hESCs were differentiated, and profiling was realized at days 0, 8 and 15.
Project description:We aim to understand the role that Cdx2 plays in specifying the rostro-caudal identity of differentiating motor neurons. We find that expressing Cdx2 in combination with FGF signaling is sufficient to produce motor neurons with a more caudal identity. ChIP-seq analysis of Cdx2 finds that it binds extensively throughout the Hox regions in progenitor motor neurons. Analysis of polycomb-associated chromatin over Hox regions in the subsequently generated motor neurons finds that Cdx2 binding corresponds to chromatin domains encompassing de-repressed caudal Hox genes. These results suggest a direct role for Cdx2 in specifying caudal motor neuron identity. Expression studies: Affymetrix arrays are used to profile gene expression in ES cells, RA/Hh-derived Day 5 motor neurons, and RA/Hh-derived motor neurons that have also been exposed to Dox (to activate iCdx2) and FGF.
Project description:The homeodomain protein Meis1 is essential for definitive hematopoiesis and vascular patterning in the mouse embryo. Meis2, another member of the same family, shares 82% protein identities with Meis1. Our present study suggested Meis2 exerts two distinguishable effects in differentiating ES cells. First, it increases the numbers of hematopoietic progenitors and extends their persistence in culture. Second, Meis2 skews hematopoietic differentiation by suppressing erythroid while enhancing megakaryocytic progenitor differentiation. To identify the underlying transcriptional bases of these actions, we carried out microarray analysis to compare the various populations of cells developing in ES differentiation cultures in the presence and absence of Meis2 induction. ES cells with dox-inducible Meis2 (A2lox.Meis2) were differentiated as embryoid bodies (EBs) for 6 days before plating on OP9-GFP cell monolayers and cytokines, and treated with (+) or without (-) doxycycline (dox). Cells were purified by cell sorting on day 7 or 8 into various populations based on levels of CD41 expression: GFP-CD41-, GFP-CD41+ (day 7) and GFP-CD41-,GFP-CD41int, and GFP-CD41hi (day 8). Gene expression of these purified populations was determined by microarray analysis.