Expression profile in bone marrow Nestin-GFP cells
ABSTRACT: Expression profile analysis in steady-state bone marrow-derived GFP+ cells obtained from transgenic mice in which GFP expression is regulated under the nestin gene promoter Overall design: Three replicates
INSTRUMENT(S): [MoEx-1_0-st] Affymetrix Mouse Exon 1.0 ST Array [probe set (exon) version]
Project description:Expression profile analysis in steady-state bone marrow-derived GFP+ cells obtained from transgenic mice in which GFP expression is regulated under the nestin gene promoter Three replicates
Project description:Cell cycle quiescence is a critical feature contributing to haematopoietic stem cell (HSC) maintenance. Although various candidate stromal cells have been identified as potential HSC niches, the spatial localization of quiescent HSC in the bone marrow (BM) remains unclear. Here, using a novel approach that combines whole-mount confocal immunofluorescence imaging technique and computational modelling to analyse significant tridimensional associations among vascular structures, stromal cells and HSCs, we show that quiescent HSCs associate specifically with small arterioles that are preferentially found in endosteal BM. These arterioles are ensheathed exclusively by rare Nestin-GFP-peri/NG2+ pericytes, distinct from sinusoid-associated Nestin-GFP-retic/LepR+ cells. The present RNA-seq study sought to obtain a comprehensive understanding of the differences between the two distinct HSC cellular niches. mRNA profiles of sorted Nestin-GFP-peri and -GFP-retic bone marrow stromal cells were generated from pooled mice in triplicate by Illumina HiSeq 2000 sequencing.
Project description:Genetic comparison between periosteal skeletal stem cells and bone marrow skeletal stem cells in mice Overall design: Microarray was performed on periosteal derived skeletal stem cells and bone marrow derived skeletal stem cells in Mx1-cre; Ocn-GFP and Mx1-cre; Nestin-GFP mice, respectively. We used Affymetrix array.
Project description:The HSC niche factor SCF is required for HSC maintenance. Using an Scf-GFP knockin mouse, we have identified a perivascular cell type in the bone marrow expressing high level of Scf. To characterize the novel Scf-GFP+ cells from the bone marrow, we performed microarray analysis on these cells. Total RNA were isolated from 3 independent, freshly aliquots of FACS sorted 5,000 SCF-GFP+ cells or whole bone marrow cells isolated from young adult mice. Purified RNA was amplified using the WT-Ovation™ Pico RNA Amplification system (NuGEN Technologies). Sense strand cDNA was generated using WT-Ovation™ Exon Module (NuGEN), then fragmented and labeled using the FL-Ovation™ cDNA Biotin Module V2 (NuGEN). 2.5µg of labeled cDNA were hybridized to Affymetrix Mouse Gene ST 1.0 microarrays.
Project description:Although endothelial cells (ECs) have been shown to contribute to HSC maintenance in bone marrow (BM), differential contributions of EC subtypes remain unknown, owing to the lack of methods to separate with high purity arterial (AEC) from sinusoidal (SEC) endothelial cells. We show that combination of podoplanin (PDPN) and Sca-1 expression distinguishes AEC from SEC where Sca1brightPDPN—CD45—Ter119— cells exhibit an arterial gene signature and PDPN+Sca1dimCD45—Ter119— marks sinusoids. PDPN can be substituted for antibodies against the adhesion molecules ICAM1 or E-selectin that also mark SEC. We performed functional analysis of these different types of endothelial cells and found that SCF secreated from AECs regulate HSCs. Overall design: We sorted live, singlet, CD45-Ter119-CD31+PDPN-Sca-1brightNestin-GFPdim artery endothelial cells (AECs), and live, singlet, CD45-Ter119-CD31+PDPN-Sca-1bright Nestin-GFPbright AECs and live, singlet, CD45-Ter119-CD31+PDPN+Sca-1dim sinusoidal endotehlial cells(SECs) from digested bone marrow cells from Nestin-GFP. RNA was extracted, amplification and sequenced on Hiseq 2500 in Single Cell Genomics and Epigenomics core facility at Albert Einstein College of Medicine.
Project description:H1299 cells were stably transfected with the Oct4 promoter/GFP or Nestin promoter/GFP reporter vectors. By FACS, 106 cells expressing high levels of GFP were isolated and placed into cell culture for twenty-four hours. Total RNA was used. Bone morphogenetic proteins (BMP) are aberrantly expressed in most lung carcinomas. BMPs mediate cell fate decisions and self-renewal of stem cells. Inhibition of BMP signaling decreases the growth and induces cell death of lung cancer cells lines. It is not known whether the BMP signaling cascade is growth promoting in lung cancer cells expressing the stem cell markers Oct4 and/or nestin. Lung cancer cells expressing Oct4 or nestin were isolated from lung cancer cell lines by stably transfecting the Oct4 promoter or Nestin promoter expression vectors that activate the green fluorescent protein reporter. Our studies support that lung cancer cells activating the Oct4 or nestin promoter are different cell populations. Microarray and quantitative RT-PCR demonstrated that the expression levels of specific stem cell markers were different between the isolated Oct4 and nestin cells. Both the Oct4 and nestin populations were more tumorigenic that controls but histologically they were quite different. The isolated Oct4 and nestin cells also responded differently to inhibition of BMP signaling. Blockade of BMP signaling with the BMP receptor antagonist DMH2 caused significant growth inhibition in both the Oct4 and nestin cell populations but only increased cell death in the nestin population. DMH2 also induced the expression of nestin in the Oct4 population but not in the nestin cells. We also show that BMP signaling is an important regulator of the inhibitor differentiation proteins Id1 and Id3 in Oct4 and nestin cell populations Lung cancer cells expressing Oct4 or nestin were isolated and transfected with Oct4 or Nestin promoter expression vectors that activate the green fluorescent protein reporter. Microarray and quantitative RT-PCR were performed to study the differentially expressed genes. BMPs mediate cell fate decisions and self-renewal of stem cells were investigated.
Project description:Analysis of Nestin-GFP+ pericytes flow sorted from 3-day-old mouse cutaneous adipose tissue, comparing controls with wild type PDGFRa, and mutants with increased PDGFRa signaling driven by a Cre/lox-inducible D842V knockin mutation in the PDGFRa kinase domain. The control cells have adipogenic properties in vitro or when transplanted subcutaneously into recipient mice. The D842V mutant cells show altered behavior in the same assays, with poor adipogenic differentiation but a propensity to transition into profibrotic cells that secrete collagen Overall design: 3 Nes-GFP+ cells samples; 3 Nes-GFP;Nes-Cre;PDGFRα+/[S]D842V samples
Project description:Thiele2013 - Bone marrow hematopoietic cells
The model of bone marrow hematopoietic cells metabolism is derived from the community-driven global reconstruction of human metabolism (version 2.02, MODEL1109130000
This model is described in the article:
A community-driven global reconstruction of human metabolism.
Thiele I, et al
Multiple models of human metabolism have been reconstructed, but each represents only a subset of our knowledge. Here we describe Recon 2, a community-driven,
consensus 'metabolic reconstruction', which is the most comprehensive representation of human metabolism that is applicable to computational modeling. Compared
with its predecessors, the reconstruction has improved topological and functional features, including ~2x more reactions and ~1.7x more unique metabolites. Using
Recon 2 we predicted changes in metabolite biomarkers for 49 inborn errors of metabolism with 77% accuracy when compared to experimental data. Mapping metabolomic
data and drug information onto Recon 2 demonstrates its potential for integrating and analyzing diverse data types. Using protein expression data, we automatically
generated a compendium of 65 cell type-specific models, providing a basis for manual curation or investigation of cell-specific metabolic properties. Recon 2 will
facilitate many future biomedical studies and is freely available at http://humanmetabolism.org/.
This model is hosted on BioModels Database
and identified by: MODEL1310110030
To cite BioModels Database, please use: BioModels Database: An enhanced,
curated and annotated resource for published quantitative kinetic models
To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer
to CC0 Public Domain Dedication
for more information.
Project description:Bone marrow Hdc-GFP+/hi and Hdc-GFP-/loCD11b+Gr1+ cells were isolated from bones from histidine decarboxylase (Hdc) green fluorescent protein (Hdc-GFP) mice Hdc-GFP+/hiCD11b+Gr1+ cells and Hdc-GFP-/loCD11b+Gr1+ cells were sorted by combinations of GFP and myeloid cell surface markers CD11b and Gr1 and their differential mRNA expression compared with Affymetrix microarrays. Overall design: a. Hdc-GFP-/loCD11b+Gr1+cells (n=3) b. Hdc-GFP+/hiCD11b+Gr1+ cells (n=3)
Project description:The intermediate filament protein Nestin serves as a biomarker for stem cells and has been used to identify subsets of cancer stem-like cells. However, the mechanistic contributions of Nestin to cancer pathogenesis are not understood. Here we report that Nestin binds the hedgehog pathway transcription factor Gli3 to mediate the development of medulloblastomas of the hedgehog subtype. In a mouse model system, Nestin levels increased progressively during medulloblastoma formation resulting in enhanced tumor growth. Conversely, loss of Nestin dramatically inhibited proliferation and promoted differentiation. Mechanistic investigations revealed that the tumor-promoting effects of Nestin were mediated by binding to Gli3, a zinc finger transcription factor that negatively regulates hedgehog signaling. Nestin binding to Gli3 blocked Gli3 phosphorylation and its subsequent proteolytic processing, thereby abrogating its ability to negatively regulate the hedgehog pathway. Our findings show how Nestin drives hedgehog pathway-driven cancers and uncover in Gli3 a therapeutic target to treat these malignancies. Nestin+ and Nestin- GNPs (granule neuron precursors) were purified from Nestin-CFP/Math1-Cre/Ptch1-loxp cerebella at postnatal day 4 by FACs, and total RNA from these two cell populations were extracted, and then labeled and hybridized to Affymetrix Mouse Genome 430 2.0 arrays.