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MARIS: Method for Analyzing RNA following Intracellular Sorting [array]

ABSTRACT: We've developed a new Method to Analyze RNA following Intracellular Sorting (MARIS) allowing us to carry out gene expression studies on cells sorted based on intracellular immunoflourescence. The purpose of this study is to determine the degree of bias that MARIS introduces on gene expression. We report Illumina microarray gene expression data from human embryonic stem cells differentiated to a stage in which insulin-expressing cells are present. Gene expression data using RNA isolated from live cells is compared to gene expression data using RNA isolated from MARIS processed cells (fixed, permeabilized, antibody stained and mock sorted) to determine the degree of correlation in gene expression between these two biologically identical samples. Human embryonic stem cells are differentiated to a stage in which insulin-expressing cells are present and split into two biologically identical samples. RNA is immediately isolated from one sample using the RNAeasy protocol (live sample). RNA is isolated from the second sample following MARIS (processed sample) with all cells collected after the sort in order to keep the cell type composition between the live and processed samples the same.

ORGANISM(S): Homo sapiens

SUBMITTER: Sinisa Hrvatin

PROVIDER: E-GEOD-53941 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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MARIS: Method for Analyzing RNA following Intracellular Sorting [RNA-seq]

Project description:We've developed a new Method to Analyze RNA following Intracellular Sorting (MARIS) allowing us to carry out gene expression studies on cells sorted based on intracellular immunoflourescence. The purpose of this study is to determine the degree of bias that MARIS introduces on gene expression. We report RNA-seq gene expression data from human embryonic stem cells differentiated to a stage in which insulin-expressing cells are present. Gene expression data using RNA isolated from live cells is compared to gene expression data using RNA isolated from MARIS processed cells (fixed, permeabilized, antibody stained and mock sorted) to determine the degree of correlation in gene expression between these two biologically identical samples. Human embryonic stem cells are differentiated to a stage in which insulin-expressing cells are present and split into two biologically identical samples. RNA is immediately isolated from one sample using the RNeasy protocol (live sample). RNA is isolated from the second sample following MARIS (processed sample) with all cells collected after the sort in order to keep the cell type composition between the live and processed samples the same.

2014-01-17 | E-GEOD-54178 | biostudies-arrayexpress

Differentiated human stem cells resemble fetal, not adult, Î² cells

Project description:Human pluripotent stem cells (hPSCs) have the potential to generate any human cell type, and one widely recognized goal is to make pancreatic Î² cells. To this end, comparisons between differentiated cell types produced in vitro and their in vivo counterparts are essential to validate hPSC-derived cells. Genome-wide transcriptional analysis of sorted insulin-expressing (INS(+)) cells derived from three independent hPSC lines, human fetal pancreata, and adult human islets points to two major conclusions: (i) Different hPSC lines produce highly similar INS(+) cells and (ii) hPSC-derived INS(+) (hPSC-INS(+)) cells more closely resemble human fetal Î² cells than adult Î² cells. This study provides a direct comparison of transcriptional programs between pure hPSC-INS(+) cells and true Î² cells and provides a catalog of genes whose manipulation may convert hPSC-INS(+) cells into functional Î² cells RNA is isolated and processed using MARIS from the following samples: H1 human embryonic stem cells (hESCs) in duplicate, HUES8 hESCs in duplicate, human induced pluripotent stem cells (hiPSCs) in duplicate, H1 cells differentiated to a stage in which insulin-expressing cells are present (stage 6) in duplicate, HUES8 cells differentiated to stage 6 in duplicate, hiPSCs differentiated to stage 6, insulin-expressing cells sorted from H1 cells differentiated to stage 6 in duplicate, insulin-expressing cells sorted from HUES8 cells differentiated to stage 6 in duplicate, insulin-expressing cells sorted from hiPSCs differentiated to stage 6 in duplicate, human week 16 fetal pancreata in duplicate, insulin-expressing cells sorted from human week 16 fetal pancreata in triplicate, adult human pancreatic islets in triplicate, and insulin-expressing cells sorted from adult human pancreatic islets in triplicate.

2014-03-25 | E-GEOD-56130 | biostudies-arrayexpress

MARIS: Method for Analyzing RNA following Intracellular Sorting

Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series

2014-01-17 | E-GEOD-54179 | biostudies-arrayexpress

Generation of functional human pancreatic beta cells in vitro

Project description:The generation of insulin-producing pancreatic cells from stem cells in vitro would provide an unprecedented cell source for drug discovery and cell transplantation therapy in diabetes. However, insulin-producing cells previously generated from human pluripotent stem cells (hPSC) lack many functional characteristics of bona fide β cells. Here we report a scalable differentiation protocol that can generate hundreds of millions of glucose-responsive β cells from hPSC in vitro. These stem cell derived cells (SC) express markers found in mature β cells, flux Ca2+ in response to glucose, package insulin into secretory granules and secrete quantities of insulin comparable to adult β cells in response to multiple sequential glucose challenges in vitro. Furthermore, these cells secrete human insulin into the serum of mice shortly after transplantation in a glucose-regulated manner, and transplantation of these cells ameliorates hyperglycemia in diabetic mice. Differentiated cells were sorted and processed for RNA isolation using the MARIS protocol published previously (PMID: 24516164.) Human embryonic stem cell (hESC) line HUES8 was differentiated into SC-beta cells. Two biological replicates were analyzed. Those data were normalized together with and compared to existing, previously published data from Hrvatin et al. ( (PMID: 24516164) from human islet -derived insulin+ cells, undifferentiated HUES8 hES cells, and insulin+ cells derived from HUES8 cells according to previously published protocols.

2014-09-25 | E-GEOD-61714 | biostudies-arrayexpress

Generation of Stem Cell-Derived Î² Cells from Type 1 Diabetic Patients

Project description:We recently reported the scalable in vitro production of functional stem cell-derived Î² cells. Here we extend this approach to generate SC-Î² cells from Type 1 diabetic patients (T1D), a cell type that is destroyed during disease progression and has not been possible to extensively study. These cells express Î² cell markers, respond to glucose both in vitro and in vivo, prevent alloxan-induced diabetes in mice, and respond to anti-diabetic drugs. Furthermore, we use an in vitro disease model to demonstrate the cells respond to different forms of Î² cell stress. Using these assays, we find no major differences in T1D SC-Î² cells compared to SC-Î² cells derived from non-diabetic patients (ND). These results show that T1D SC-Î² cells can be used for the treatment of diabetes, drug screening, and the study of Î² cell biology. Differentiated cells were sorted and processed for RNA isolation using the MARIS protocol published previously (PMID: 24516164.) Human induced pluripotent stem cell (hiPSC) line were differentiated into SC-beta cells or dysfunctional, polyhormonal cells (PH). Four biological replicates were assessed with differentiation to both SC-beta and PH cells. Those data were normalized together with and compared to existing, previously published data from Hrvatin et al. (PMID: 24516164) and Pagliuca et al. (PMID: 25303535) from human islet-derived insulin+ cells, undifferentiated HUES8 hES cells, SC-beta cells derived from HUES8 and PH cells derived from HUES8 according to previously published protocols.

2016-04-01 | E-GEOD-70901 | biostudies-arrayexpress

MARIS: Method for Analyzing RNA following Intracellular Sorting [array]

2014-01-13 | GSE53941 | GEO

MARIS: Method for Analyzing RNA following Intracellular Sorting [RNA-seq]

2014-01-17 | GSE54178 | GEO

High-flow chromatography for reproducible and high-throughput quantification of proteomes

Project description:This study evaluates liquid-chromatography operated at 50 ul/min using 1 mm diameter columns coupled online to tandem mass spectrometry (LC-MS/MS) for proteome analysis. Data from >1,000 human cell line, tissue, body fluid, affinity- and phospho-proteomes demonstrate excellent chromatographic (<0.3 % CV) and quantitative (<7.5% CV) reproducibility. Combined with sample multiplexing by tandem mass tags, the system allows profiling of 11 proteomes in 16 hours to a depth of 8,000 proteins.

2019-12-09 | PXD015087 | Pride

miR-132 mediates renal fibrosis: Genome-wide analysis of gene expression in foxd1-derivative interstital cells derived from fibrotic kidneys from mice treated with antagomir-132 or scramblemir

Project description:Chronic kidney disease is associated with progressive renal fibrosis, where perivascular cells give rise to the majority of Î±-SMA positive myofibroblasts. We sought to identify pericytic miRNAs that could serve as a target to decrease myofibroblast formation. We induced kidney fibrosis in FoxD1-GC;Z/Red-mice by unilateral ureteral obstruction (UUO) followed by FACS sorting of dsRed-positive FoxD1-derivative cells and miRNA profiling. MiR-132 selectively increased 21-fold during pericyte-to-myofibroblast formation whereas miR-132 was only 2.5-fold up in total kidney lysates (both in UUO and ischemia-reperfusion injury). MiR-132 silencing in UUO decreased collagen deposition (35%) and tubular apoptosis. Immunohistochemistry, western blot and qRT-PCR confirmed a similar decrease in interstitial Î±-SMA+ cells. Pathway analysis identified a rate-limiting role for miR-132 in myofibroblast proliferation that was confirmed in vitro. Indeed, antagomir-132 treated mice displayed a reduction in the number of proliferating, ki67+ interstitial myofibroblasts. Interestingly, this was selective for the interstitial compartment and did not impair the reparative proliferation of tubular epithelial cells, as evidenced by an increase in ki67+ epithelial cells, as well as increased (p-)RB1, Cyclin-A and decreased RASA1, p21 levels in kidney lysates. Taken together, silencing miR-132 counteracts the progression of renal fibrosis by selectively decreasing myofibroblast proliferation and could potentially serve as a novel antifibrotic therapy. Total RNA obtained from FACS sorted mouse renal FoxD1-derivatve interstitial cells from mice that were treated with antagomir-132 or scramblemir and underwent UUO (n=4)

2016-02-16 | E-GEOD-76548 | biostudies-arrayexpress

SNP analysis of human induced pluripotent stem cells

Project description:Human induced pluripotent stem cells (hIPSCs) represent a unique opportunity for regenerative medicine since they offer the prospect of generating unlimited quantities of cells for autologous transplantation as a novel treatment for a broad range of disorders. However, the use of hIPSCs in the context of genetically inherited human disease will require correction of disease-causing mutations in a manner that is fully compatible with clinical applications. We analyzed hiPSC line and genetically modified derivatives using high-density SNP array to investigate genomic instability associated reprogramming and genetic modification. Primary iPSC lines derived from patients with alpha-1 antitrypsin deficiency were generated. This genetic disorder is caused by homozygous mutation (Glu342Lys) in the SERPINA1 gene. We carried out mutation correction by 2 steps: zinc-finger nuclease-stimuated gene targeting and piggyBac trasnsposon-mediated selection cassette elimination. Parental fibroblast lines, primary iPSC lines and homozygously targeted iPSC lines were subjected to SNP genotyping using Illumina CytoSNP-12 BeadChiP.

2011-08-31 | E-GEOD-31035 | biostudies-arrayexpress

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