MicroRNA profiling during neural differentiation of induced pluripotent stem cells
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ABSTRACT:
ORGANISM(S): synthetic construct Homo sapiens
PROVIDER: GSE134061 | GEO | 2019/07/11
REPOSITORIES: GEO
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Project description:The differentiation capability of induced pluripotent stem cells (iPSCs) towards certain cell types for disease modelling and drug screening assays might be influenced by their somatic cell of origin. Here, we have compared the neural induction of human iPSCs generated from either fetal neural stem cells (fNSCs), dermal fibroblasts or CD34+ hematopoietic stem cells. Neural progenitor cells (NSCs) and neurons could be generated at similar efficiencies from all iPSCs. Whole genome transcriptome analysis and an expression analysis of neural genes in iPSC-derived NSCs revealed a separation of neuroectoderm- from mesoderm-derived cells. Furthermore, we found genes, which were similarly expressed between fNSCs and neuroectoderm- but not mesoderm-derived NSCs. Notably, these neural signatures were retained after transplantation into the cortex of mice and paralleled with increased survival and integration of neuroectoderm-derived cells in vivo. These results indicated distinct origin- dependent neural cell identities in differentiated human iPSCs both in vitro and in vivo. RNA samples for microarray analysis were prepared using RNeasy columns (Qiagen, Germany) with on-column DNA digestion. 300 ng of total RNA per sample were used as the input in the linear amplification protocol (Ambion), which involved the synthesis of T7-linked double-stranded cDNAs and 12hrs of in vitro transcription incorporating biotin-labeled nucleotides. Purified and labeled cRNA was then hybridized for 18 hrs onto HumanHT-12 v4 expression BeadChips (Illumina, USA) following the manufacturer's instructions. After the recommended washing, the chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and the accompanying software. The samples were exclusively hybridized as biological replicates. The RNA samples from in vivo transplanted and FACS sorted cells were amplified using the TargetAmp 2-Round Biotin-aRNA Amplification Kit 3.0 (Epicentre, USA) from 100 pg to 50 microg. 43 samples were analyzed Fib, Human fibroblast F134, 2 replicates CD34+, Human Cord Blood CD34+ Hematopoyetic Stem Cell (HSC), 1 replicate fNSC, Human fetal Neural Stem Cells (NSC), 1 replicate fNSC-1FiPSCa-NSC, Human one factor (POU5F1) fetal Neural Stem Cell (NSC) induced reprogrammed cell (iPSC) clone a, in vitro differentiated to NSC, 2 replicates fNSC-1FiPSCb-NSC, Human one factor (POU5F1) fetal Neural Stem Cell (NSC) induced reprogrammed cell (iPSC) clone b, in vitro differentiated to NSC, 2 replicates fNSC-2FiPSCa-NSC, Human two factors (POU5F1, KLF4) fetal Neural Stems Cell (NSC) induced reprogrammed cell (iPSC) clone a, in vitro differentiated to NSC, 2 replicates CD34-2FiPSCa-NSC, Human two factors (POU5F1, KLF4) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone a, in vitro differentiated to NSC, 2 replicates CD34-4FiPSCa-NSC, Human four factors (POU5F1, KLF4, SOX2, CMYC) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone a, in vitro differentiated to NSC, 2 replicates CD34-4FiPSCb-NSC, Human four factors (POU5F1, KLF4, SOX2, CMYC) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone b, in vitro differentiated to NSC, 2 replicates Fib-4FiPSCa-NSC, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone a, in vitro differentiated to NSC, 2 replicates Fib-4FiPSCb-NSC, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone b, in vitro differentiated to NSC, 2 replicates Fib-4FiPSCc-NSC, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone c, in vitro differentiated to NSC, 1 replicate H1-ESC-NSC, Human H1 embryonic stem cell (ESC), in vitro differentiated to NSC, 2 replicates HUES6-ESC-NSC, Human HUES6 embryonic stem cell (ESC), in vitro differentiated to NSC, 1 replicate fNSC-1FiPSCa, Human one factor (POU5F1) fetal Neural Stem Cell (NSC) induced reprogrammed cell (iPSC) clone a, 1 replicate fNSC-1FiPSCb, Human one factor (POU5F1) fetal Neural Stem Cell (NSC) induced reprogrammed cell (iPSC) clone b, 1 replicate fNSC-2FiPSCa, Human two factors (POU5F1, KLF4) fetal Neural Stems Cell (NSC) induced reprogrammed cell (iPSC) clone a, 1 replicate CD34-2FiPSCa, Human two factors (POU5F1, KLF4) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone a, 1 replicate CD34-4FiPSCa, Human four factors (POU5F1, KLF4, SOX2, CMYC) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone a, 1 replicate CD34-4FiPSCb, Human four factors (POU5F1, KLF4, SOX2, CMYC) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone b, 1 replicate Fib-4FiPSCa, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone a, 1 replicate Fib-4FiPSCb, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone b, 1 replicate H1-ESC, Human H1 embryonic stem cell (ESC), 3 replicates HUESC6-ESC, Human HUESC6 embryonic stem cell (ESC) , 2 replicates Fib-4FiPSCa-NSC-In Vivo, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone a, in vivo differentiated to NSC, 2 replicates fNSC-1FiPSCa-NSC-In Vivo, Human one factor (POU5F1) fetal Neural Stem Cell (NSC) induced reprogrammed cell (iPSC) clone a, in vivo differentiated to NSC, 4 replicates
2014-09-12 | E-GEOD-55107 | biostudies-arrayexpress
Project description:mRNA-seq analyses for the differentially expressed genes (DEGs) between Parkinsons's disease patient (holding Lin28 R192G hetero mutation)-iPSC (Pt-iPSC) derived neural stem cell vs Lin28 R192G hetero mutation corrected-iPSC (Corrected Pt-iPSC) derived neural stem cell. To gain further molecular insight into the observed Lin28R192G mutation in Parkinsons's disease (PD) patient iPSC-derived neural stem cell (NSC) functions, we performed mRNA-sequencing (RNA-seq) analysis of the PD patient (holding Lin28 R192G hetero mutation)-iPSC (Pt-iPSC) derived NSC and Lin28 R192G hetero mutation corrected-iPSC (Corrected Pt-iPSC) derived NSC.
2019-08-20 | GSE135995 | GEO
Project description:MYCN and HDAC2 jointly repress the transcription of tumor suppressive micro RNA miR-183 in neuroblastoma. Enforced miR-183 expression induces neuroblastoma cell death and inhibits anchorage-independent colony formation and subcutaneous xenograft growth in mice. We here aimed to unravel the miR-183 signaling network and elucidated the role of MYCN mediated transcriptional activation of members of the minichromosome maintenance (MCM) family protein family involving miR-183 . The hexamer protein complex formed by MCM proteins is involved in the initiation and elongation of eukaryotic genome replication, thereby contributing to genomic integrity. Analysis of miR-183 versus negative control transfected neuroblastoma cells identified 85 differentially expressed proteins in a label-free mass spectrometric approach. Six members of the MCM family were found to be lower expressed upon enforced miR-183 expression, and subsequent annotation category enrichment analysis revealed a 14-fold enrichment in the protein module category “MCM”. Down-regulation was confirmed by western blot analysis. MicroRNA target prediction software studies revealed that miR-183 was predicted to directly target several MCMs.
2016-06-01 | PXD003596 | Pride
Project description:Studies of human embryonic stem cells (hESCs) commonly describe the non-functional p53-p21 axis of the G1/S checkpoint pathway with subsequent relevance for cell cycle regulation and the DNA damage response (DDR). Importantly, p21 mRNA is clearly present and upregulated after the DDR in hESCs but p21 protein is not detectable. In this article, we provide evidence that expression of p21 protein is directly regulated by the microRNA pathway under standard culture conditions and after DNA damage. The DDR in hESCs leads to upregulation of tens of microRNAs, including hESC-specific microRNAs such as those of the miR-302 family, miR-371-372 family, or C19MC microRNA cluster. Most importantly, we show that the hESC-enriched microRNA family miR-302 (miR-302a, miR-302b, miR-302c, and miR-302d) directly contributes to regulation of p21 expression in hESCs and thus demonstrate a novel function for miR-302s in hESCS. The described mechanism elucidates the role of microRNAs in regulation of important molecular pathway governing the G1/S transition checkpoint before as well as after DNA damage.
2015-01-01 | GSE36615 | GEO
Project description:The integration of cell metabolism with signalling pathways, transcription factor networks and epigenetic mediators is critical in coordinating molecular and cellular events during embryogenesis. Induced pluripotent stem cells (IPSCs) are an established model for embryogenesis, germ layer specification and cell lineage differentiation, advancing the study of human embryonic development and the translation of innovations in drug discovery, disease modelling and cell-based therapies. The metabolic regulation of IPSC pluripotency is mediated by balancing glycolysis and oxidative phosphorylation, but there is a paucity of data regarding the influence of individual metabolite changes during cell lineage differentiation. We used <sup>1</sup>H NMR metabolite fingerprinting and footprinting to monitor metabolite levels as IPSCs are directed in a three-stage protocol through primitive streak/mesendoderm, mesoderm and chondrogenic populations. Metabolite changes were associated with central metabolism, with aerobic glycolysis predominant in IPSC, elevated oxidative phosphorylation during differentiation and fatty acid oxidation and ketone body use in chondrogenic cells. Metabolites were also implicated in the epigenetic regulation of pluripotency, cell signalling and biosynthetic pathways. Our results show that <sup>1</sup>H NMR metabolomics is an effective tool for monitoring metabolite changes during the differentiation of pluripotent cells with implications on optimising media and environmental parameters for the study of embryogenesis and translational applications.
2023-05-24 | MTBLS4854 | MetaboLights
Project description:Our purpose was to investigate genes and molecular mechanisms involved in patients with Leber congenital amaurosis (LCA). Fibroblasts from two unrelated clinically-identified patients (Coriell) were reprogrammed to pluripotency by retroviral transduction. These human induced Pluripotent Stem Cells (hiPSCs) were differentiated into neural stem cells (NSC) that mimicked the neural tube stage and retinal pigmented epithelial (RPE) cells that could be targeted by the disease. A genome wide transcriptome analysis was performed with Affymetrix Exon Array GeneChipM-BM-., comparing LCA-hiPSCs derivatives to controls. The aim was to identify differentially expressed genes which may be associated with early developmental defect before the establishment of mature retinal circuitry. We analyzed iPSC-derived neural stem cells from LCA patient's fibroblast (n=2) and iPSC-derived neural stem cells from healthy people fibroblast (n=2). A total of 21 samples were analyzed : 9 NSC derived from iPSC LCA and 12 NSC derived from wild-type iPSC.
2015-01-21 | E-GEOD-43924 | biostudies-arrayexpress
Project description:Maintaining a full differentiation potential along self-renewal ability is a major property of stem cells during development and regeneration. miR-203 is a microRNA previously involved in skin differentiation and tumor suppression. We show here that transient expression of miR-203 enhances the potential of embryonic (ESC) and induced pluripotent stem cells (iPSC) in contributing to multiple lineages without decreasing their self-renewal properties. In fact, miR-203 significantly improves the efficiency of ES/iPS cells in the generation of quimeras and tetraploid complementation assays, in addition to improving the generation of complex teratomas and embryo-like structures in vivo. These effects are mediated by the direct miR-203-dependent repression of de novo DNA methyltransferases Dnmt3a and Dnmt3b, leading to genome-wide demethylation of pluripotent cells. Transient exposure to miR-203 improves functional differentiation and maturation of pluripotent cells into cardiomyocytes in a Dnmt3a/b-dependent manner, suggesting the possible therapeutic uses of this microRNA in regenerative medicine.
2020-07-05 | GSE86899 | GEO
Project description:A common experimental setup to investigate the function of a microRNA is a â??gain-of-functionâ?? approach, in which the microRNA of interest is overexpressed to analyze its impact on a given process. However, a major drawback of this technique is that microRNAs, when expressed significantly above endogenous levels, may target genes that are not affected under physiological conditions. To circumvent this problem, we have generated a retroviral microRNA-knockdown library encompassing the majority of the microRNA families expressed throughout lymphocyte development. MicroRNA knockdown is mediated by sponges, mRNAs that possess multiple binding sites for a certain microRNA family in their 3â??-UTR region. MiRNA sponges function as competitive inhibitors that sequester all specific microRNA/RISC complexes, thereby de-repressing the endogenous target genes. Using this library in pre-B cells as a model system, we have characterized all microRNAs expressed in early B cells development according to their impact on processes such as apoptosis and differentiation. In doing so, we show that functional knockdown of the known tumor-suppressive miR-15 family has only slight effects under cellular steady-state conditions. Upon withdrawal of the growth factor IL-7, however, loss of miR-15 function almost completely inhibits pre-B to immature B cell differentiation, partially protects from apoptosis and enables prolonged proliferation. In correspondence with that, knockdown of the miR-15 family confers a competitive advantage in suboptimal IL-7 concentrations, indicating that levels of miR-15 determine the sensitivity of cells towards growth factor receptor signaling. Moreover, IL-7 withdrawal seems to increase the activity of endogenous miR-15 family members in pre-B cells. Using microarray analysis, we have identified cyclin E1 as a direct and cyclin D3 as an indirect putative target gene of the miR-15 family in B cell precursors. Exogenous expression of cyclin E1 or cyclin D3 recapitulates the miR-15 loss-of-function phenotype in B cell precursors, validating their biological importance. Together, this suggests that the miR-15 family functions as a central element in a positive feedback loop that reinforces cell-cycle arrest at low growth factor concentrations, which is considered a prerequisite for light chain gene recombination and differentiation. Gene expression in cells expressing a scrambled sponge as a control vector or a sponge designed to sequester all members of the miR-15 family (miR-15a, miR-15b, miR-16, miR-497, miR-195, miR-322) was measured either in the presence or 36 hours after withdrawal of the growth factor IL-7. Two independent experiments were performed, using independently generated cell clones for each repetition.
2016-07-03 | E-GEOD-72767 | biostudies-arrayexpress
Project description:Activated T cells inhibit neurogenesis in adult animal brain and cultured human fetal neural stem cells (NSC). However, the role of inhibition of neurogenesis in human neuroinflammatory diseases is still uncertain because of the difficulty in obtaining adult NSC from patients. Recent developments in cell reprogramming suggest that NSC may be derived directly from adult fibroblasts. We generated NSC from adult human peripheral CD34+ cells by transfecting the cells with Sendai virus constructs containing Sox-2, Oct3/4, C-MyC and Klf-4. The derived NSC could be differentiated to astroglia and action potential firing neurons. Co-culturing NSC with activated autologous T cells or treatment with recombinant granzyme B caused inhibition of neurogenesis as indicated by decreased NSC proliferation and neuronal differentiation. Thus, we have established a unique autologous in vitro model to study the pathophysiology of neuroinflammatory diseases that has potential for usage in personalized medicine. 11 Human samples from 7 sources representing 4 different cell types: 2 CD34 (CD34+ cells purified from adult peripheral blood), 3 iNS (induced Neural Stem Cells derived directly from CD34+ cells), 2 iNS derived from iPSC (Neural Stem cells differentiated from induced Pluripotent Stem Cells from CD34+ cells), 4 NPC (human primary cultured neural progenitor cells)
2014-01-01 | E-GEOD-44532 | biostudies-arrayexpress
Project description:The highly conserved MicroRNA-9 (miR-9) family consists of three members. We discovered that miR-9-1 deletion reduced mature miR-9 expression, causing 43% of the mice to display smaller size and postweaning lethality. MiR-9-1-deficient mice with growth defects experienced severe lymphopenia, but other blood cells were unaffected. The lymphopenia wasn’t due to defects in hematopoietic progenitors, as mutant bone marrow (BM) cells underwent normal lymphopoiesis after transplantation into wild-type recipients. Additionally, miR-9-1-deficient mice exhibited impaired osteoblastic bone formation, as mutant mesenchymal stem cells (MSCs) failed to differentiate into osteoblastic cells (OBs). RNA sequencing revealed reduced expression of master transcription factors for osteoblastic differentiation, Runt-related transcription factor 2 (Runx2) and Osterix (Osx), and genes related to collagen formation, extracellular matrix organization, and cell adhesion, in miR-9-1-deficient MSCs. Follistatin (Fst), an antagonist of bone morphogenetic proteins (BMPs), was found to be a direct target of miR-9-1. Its deficiency led to the up-regulation of Fst, inhibiting BMP signaling in MSCs, and reducing IL-7 and IGF-1. Thus, miR-9-1 controls osteoblastic regulation of lymphopoiesis by targeting the Fst/BMP/Smad signaling axis.
2023-08-31 | GSE239376 | GEO