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Project description:Single cell transcriptome of human embryonic stem cell-derived neurons spanning the rostrocaudal and dorsoventral axes
| PRJNA775086 | ENA
Project description:Transsynaptic circuit tracing using genetically modified rabies virus (RV) is an emerging technology for identifying synaptic connections between neurons. Complementing this methodology, it is now possible to assay the basic molecular and cellular properties of neuronal lineages derived from embryonic stem cells (ESCs) in vitro, and these properties are under intense investigation toward devising cell replacement therapies. Here, we report the generation of a novel mouse ESC (mESC) line that harbors the genetic elements to allow RV-mediated transsynaptic circuit tracing in ESC-derived neurons and their synaptic networks. To facilitate transsynaptic tracing, we have engineered a new reporter allele by introducing cDNA encoding tdTomato, the Rabies-G glycoprotein, and the avian TVA receptor into the ROSA26 locus by gene targeting. We demonstrate high-efficiency differentiation of these novel mESCs into functional neurons, show their capacity to synaptically connect with primary neuronal cultures as evidenced by immunohistochemistry and electrophysiological recordings, and show their ability to act as source cells for presynaptic tracing of neuronal networks in vitro and in vivo. Together, our data highlight the potential for using genetically engineered stem cells to investigate fundamental mechanisms of synapse and circuit formation with unambiguous identification of presynaptic inputs onto neuronal populations of interest.
| S-EPMC3458586 | biostudies-literature
Project description:ObjectiveGenerating myocyte grafts that bridge across infarcts could maximize their functional impact and best utilize small numbers of stem cells. To date, however, graft survival within acute infarcts has not been feasible. To enhance intrainfarct graft viability, human embryonic stem cell-derived cardiomyocytes (hESC-CMs) were pretreated before implantation with cobalt protoporphyrin (CoPP), a pharmacologic inducer of cytoprotective heme oxygenase-1.MethodsAfter preculturing with CoPP (vs phosphate-buffered saline), hESC-CMs were injected intramyocardially into acutely infarcted rat hearts, using directed injections to span the infarct. A further group received CoPP-pretreated hESC-CMs plus 4 weekly doses of systemic CoPP to prolong exposure to cytoprotectants. Two control groups with infarcts received vehicle-only intramyocardial injections or weekly systemic CoPP without cell therapy. Postinfarct ventricular function was gauged by echocardiography and graft size quantified at 8 weeks by histomorphometry.ResultsCoPP-preconditioned hESC-CMs formed stable grafts deep within infarcted myocardium, while grafts without CoPP exposure survived mainly at the infarct periphery. Fractional shortening was improved at 4 and 8 weeks in all hearts receiving cell therapies (P < .01 vs vehicle-only injections). CoPP treatment of both graft hESC-CMs and recipient animals resulted in the largest grafts, highest fractional shortening, preserved wall thickness, and reduced infarct dimensions.ConclusionsCellular therapy delivered acutely after infarction significantly improved postinfarct ventricular function at 1 and 2 months. CoPP pretreatment of cells resulted in stable hESC-CM grafts within infarcted myocardium. This design enables construction of directionally oriented, infarct-spanning bands of new cardiomyocytes that might further improve functional restoration as engrafted myocytes proliferate and mature.
| S-EPMC4250416 | biostudies-literature
Project description:BackgroundCharacterization of gene expression signatures for cardiomyocytes derived from embryonic stem cells will help to define their early biologic processes.ResultsA transgenic alpha-myosin heavy chain (MHC) embryonic stem cell lineage was generated, exhibiting puromycin resistance and expressing enhanced green fluorescent protein (EGFP) under the control of the alpha-MHC promoter. A puromycin-resistant, EGFP-positive, alpha-MHC-positive cardiomyocyte population was isolated with over 92% purity. RNA was isolated after electrophysiological characterization of the cardiomyocytes. Comprehensive transcriptome analysis of alpha-MHC-positive cardiomyocytes in comparison with undifferentiated alpha-MHC embryonic stem cells and the control population from 15-day-old embryoid bodies led to identification of 884 upregulated probe sets and 951 downregulated probe sets in alpha-MHC-positive cardiomyocytes. A subset of upregulated genes encodes cytoskeletal and voltage-dependent channel proteins, and proteins that participate in aerobic energy metabolism. Interestingly, mitosis, apoptosis, and Wnt signaling-associated genes were downregulated in the cardiomyocytes. In contrast, annotations for genes upregulated in the alpha-MHC-positive cardiomyocytes are enriched for the following Gene Ontology (GO) categories: enzyme-linked receptor protein signaling pathway (GO:0007167), protein kinase activity (GO:0004672), negative regulation of Wnt receptor signaling pathway (GO:0030178), and regulation of cell size (O:0008361). They were also enriched for the Biocarta p38 mitogen-activated protein kinase signaling pathway and Kyoto Encyclopedia of Genes and Genomes (KEGG) calcium signaling pathway.ConclusionThe specific pattern of gene expression in the cardiomyocytes derived from embryonic stem cells reflects the biologic, physiologic, and functional processes that take place in mature cardiomyocytes. Identification of cardiomyocyte-specific gene expression patterns and signaling pathways will contribute toward elucidating their roles in intact cardiac function.
| S-EPMC1896009 | biostudies-literature
Project description:Proximal spinal muscular atrophy (SMA) is an early onset, autosomal recessive motor neuron disease caused by loss of or mutation in SMN1 (survival motor neuron 1). Despite understanding the genetic basis underlying this disease, it is still not known why motor neurons (MNs) are selectively affected by the loss of the ubiquitously expressed SMN protein. Using a mouse embryonic stem cell (mESC) model for severe SMA, the RNA transcript profiles (transcriptomes) between control and severe SMA (SMN2+/+;mSmn-/-) mESC-derived MNs were compared in this study using massively parallel RNA sequencing (RNA-Seq). The MN differentiation efficiencies between control and severe SMA mESCs were similar. RNA-Seq analysis identified 3,094 upregulated and 6,964 downregulated transcripts in SMA mESC-derived MNs when compared against control cells. Pathway and network analysis of the differentially expressed RNA transcripts showed that pluripotency and cell proliferation transcripts were significantly increased in SMA MNs while transcripts related to neuronal development and activity were reduced. The differential expression of selected transcripts such as Crabp1, Crabp2 and Nkx2.2 was validated in a second mESC model for SMA as well as in the spinal cords of low copy SMN2 severe SMA mice. Furthermore, the levels of these selected transcripts were restored in high copy SMN2 rescue mouse spinal cords when compared against low copy SMN2 severe SMA mice. These findings suggest that SMN deficiency affects processes critical for normal development and maintenance of MNs.
| S-EPMC4156416 | biostudies-literature
Project description:Embryonic stem cells (ESCs) hold great potential for replacing neurons following injury or disease. The therapeutic and diagnostic potential of ESCs may be hindered by heterogeneity in ESC-derived populations. Drug selection has been used to purify ESC-derived cardiomyocytes and endothelial cells but has not been applied to specific neural lineages. In this study we investigated positive selection of progenitor motor neurons (pMNs) through transgenic expression of the puromycin resistance enzyme, puromycin N-acetyl-transferase (PAC), under the Olig2 promoter. The protein-coding region in one allele of Olig2 was replaced with PAC to generate the P-Olig2 cell line. This cell line provided specific puromycin resistance in cells that express Olig2, while Olig2(-) cells were killed by puromycin. Positive selection significantly enriched populations of Olig2(+) pMNs. Committed motoneurons (MNs) expressing Hb9, a common progeny of pMNs, were also enriched by the end of the selection period. Selected cells remained viable and differentiated into mature cholinergic MNs and oligodendrocyte precursor cells. Drug resistance may provide a scalable and inexpensive method for enriching desired neural cell types for use in research applications.
| S-EPMC3319638 | biostudies-literature
Project description:Ferroptosis is a regulated form of cell death driven by the lethal accumulation of lipid peroxides in cell membranes. Several regulators of ferroptosis have been identified using cancer cell lines. However, the cellular pathways of ferroptosis in neurons remain poorly characterized. In this study, we used a mouse embryonic stem cell-derived motor neuron model to investigate how motor neurons respond to ferroptosis inducers. Pharmacological and genetic inhibition of glutathione peroxidase 4 (GPx4) induced ferroptosis in motor neurons, while system xc - inhibition by erastin had no effect. RNA-seq analysis showed that the expression levels of several genes were altered during RSL3-induced ferroptosis. Subsequent bioinformatic analysis revealed alterations in several biological pathways during ferroptosis, including synaptogenesis and calcium signaling. Finally, we found that edaravone, an FDA-approved drug for treating amyotrophic lateral sclerosis (ALS) disease, rescued motor neurons from RSL3-induced ferroptosis. Our data highlight the crucial role of GPx4 in ferroptosis regulation and demonstrate that stem cell-derived motor neuron culture is a valuable model to study ferroptosis at the single-cell level in a neuronal context.
| S-EPMC9989922 | biostudies-literature
Project description:Embryonic stem cells (ESCs) represent a promising source of midbrain dopaminergic (DA) neurons for applications in Parkinson disease. However, ESC-based transplantation paradigms carry a risk of introducing inappropriate or tumorigenic cells. Cell purification before transplantation may alleviate these concerns and enable identification of the specific DA neuron stage most suitable for cell therapy. Here, we used 3 transgenic mouse ESC reporter lines to mark DA neurons at 3 stages of differentiation (early, middle, and late) following induction of differentiation using Hes5::GFP, Nurr1::GFP, and Pitx3::YFP transgenes, respectively. Transplantation of FACS-purified cells from each line resulted in DA neuron engraftment, with the mid-stage and late-stage neuron grafts being composed almost exclusively of midbrain DA neurons. Mid-stage neuron cell grafts had the greatest amount of DA neuron survival and robustly induced recovery of motor deficits in hemiparkinsonian mice. Our data suggest that the Nurr1+ stage (middle stage) of neuronal differentiation is particularly suitable for grafting ESC-derived DA neurons. Moreover, global transcriptome analysis of progeny from each of the ESC reporter lines revealed expression of known midbrain DA neuron genes and also uncovered previously uncharacterized midbrain genes. These data demonstrate remarkable fate specificity of ESC-derived DA neurons and outline a sequential stage-specific ESC reporter line paradigm for in vivo gene discovery.
| S-EPMC3408729 | biostudies-literature
Project description:Cholinergic neurotransmission is essential for many important functions in the brain, including cognitive mechanisms. Here we demonstrate that human embryonic stem (hES) cells differentiate into a population of neuronal cells that express the cholinergic enzyme choline acetyltransferase and homeobox proteins specifying neuronal progenitors of ventral telencephalic lineage. These differentiated cells express transcripts for cholinergic alpha(3), alpha(4) and alpha(7) nicotinic acetylcholine (ACh) receptor subunits and for M1, M2 and M3 muscarinic acetylcholine receptor (mAChR) subtypes. Stimulation with brain-derived neurotrophic factor, neurotrophin-3, ciliary neurotrophic factor and nerve growth factor increases the proportion of cholinergic neurons. These cholinergic receptors also mediate ACh-evoked increase in cytosolic calcium levels, and this response was unaffected by extracellular calcium removal and was abolished by the mAChR antagonist scopolamine. Our findings demonstrate expression of functional cholinergic receptors on hES cell-derived neurons, which may provide a source of expandable cells to facilitate screening of novel cholinergic drugs and useful for evaluating cell transplantation in animal models of cholinergic dysfunction.
| S-EPMC3829014 | biostudies-literature
Project description:Given the complex nature of Alzheimer's disease (AD), a cell-based model that recapitulates the physiological properties of the target neuronal population would be extremely valuable for discovering improved drug candidates and chemical probes to uncover disease mechanisms. We established phenotypic neuronal assays for the biogenesis and synaptic action of amyloid β peptide (Aβ) based on embryonic stem cell-derived neurons (ESNs). ESNs enriched with pyramidal neurons were robust, scalable, and amenable to a small-molecule screening assay, overcoming the apparent limitations of neuronal models derived from human pluripotent cells. Small-molecule screening of clinical compounds identified four compounds capable of reducing Aβ levels in ESNs derived from the Tg2576 mouse model of AD. Our approach is therefore highly suitable for phenotypic screening in AD drug discovery and has the potential to identify therapeutic candidates with improved efficacy and safety potential.
| S-EPMC3780781 | biostudies-literature