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Isolation and Characterisation of Renal Precursor Cells Derived from Human Embryonic Stem Cells

ABSTRACT: HES4 hESC were cultured in serum media and maintained on a layer of mouse embryonic fibroblast feeder cells at a density of 6 x 104 cells/cm2. For differentiation: hESC were differentiated for a total of 14 days. Differentiation was induced by passaging 4 human ES cell pieces onto 12 well plates seeded with 0.67 x 10E4 cells/cm2. Cells were maintained in media containing 20% FCS for 2 days before media containing 5% FCS was used. Reduced serum media was changed every second day for the remaining 12 days ESC cells were taken at time zero and RNA was isolated (hESC_undiff). Cells differentiated by the above procedure at day 14 were isolated and RNA extracted (Diff). The remaining cells were FACs sorted with antibodies to Podocalyxin, CD24 and GCTM-2. Two populations were isolated, 1: Positive for podocalyxin and CD24 and low level of expression of GCTM-2 (POD+CD24+GCTM2Low) and 2: Positive for podocalyxin and CD24 and negative for GCTM-2 (POD+CD24+GCTM2Neg). The experiment was repeated in triplicate. The final aim was to determine the gene expression enrichment in cells with the marker profile (Podocalyxin+, CD24+, GCTM-2-neg) which is predicted to be enriched for kidney precursors. Keywords: cell type comparison Three consecutive passages of HES4 cells were grown in standard conditions, RNA derived or differentiated for 14 days subject to FACs sorting, collection and RNA isolated. Microarray was performed on a total of 12 samples, (3 replicates of 4 populations).

ORGANISM(S): Homo sapiens

SUBMITTER: Gabriel Kolle

PROVIDER: E-GEOD-15257 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Isolation and Characterisation of Renal Precursor Cells Derived from Human Embryonic Stem Cells

Project description:HES4 hESC were cultured in serum media and maintained on a layer of mouse embryonic fibroblast feeder cells at a density of 6 x 104 cells/cm2. For differentiation: hESC were differentiated for a total of 14 days. Differentiation was induced by passaging 4 human ES cell pieces onto 12 well plates seeded with 0.67 x 10E4 cells/cm2. Cells were maintained in media containing 20% FCS for 2 days before media containing 5% FCS was used. Reduced serum media was changed every second day for the remaining 12 days ESC cells were taken at time zero and RNA was isolated (hESC_undiff). Cells differentiated by the above procedure at day 14 were isolated and RNA extracted (Diff). The remaining cells were FACs sorted with antibodies to Podocalyxin, CD24 and GCTM-2. Two populations were isolated, 1: Positive for podocalyxin and CD24 and low level of expression of GCTM-2 (POD+CD24+GCTM2Low) and 2: Positive for podocalyxin and CD24 and negative for GCTM-2 (POD+CD24+GCTM2Neg). The experiment was repeated in triplicate. The final aim was to determine the gene expression enrichment in cells with the marker profile (Podocalyxin+, CD24+, GCTM-2-neg) which is predicted to be enriched for kidney precursors. Keywords: cell type comparison

2010-03-16 | GSE15257 | GEO

Transcriptomic analysis of undifferentiated human embryonic stem cells (hESCs) and day-25 differentiated cortical neuronal progenitor cells from 5 isogenic hESC lines with low and high levels of alpha-synuclein expression

Project description:Purpose: To compare the cortical neuroonal differentiation capacity of clonal isogenic hESC lines with different levels of alpha-synuclein (aSyn) expression. Methods 1: Shef4 hESCs was used as a parental line to create an allelic series of clonal transgenic hESC lines expressing a human SNCA (encoding aSyn) contruct. Clonal transgenic lines with high (S8, S37) and low (S9, S34) aSyn expression were established and characterized. Methods 2: hESCs were cultured on Laminin-521 (BioLamina) in StemMACS iPS-Brew XF self-renewal media (Miltenyi) prior to lifting for isolation of total RNA. Methods 3: Cortical neuronal progenitor cells were differentiated from hESCs on Laminin-111 coated (Biolamina) 24-well plates at an initial plating density of 80,000 cells/cm2. Differentiation commenced in neural induction media (NIM) consisting of 50% DMEM/F12 (ThermoFisher Scientific), 50% Neurobasal Media (ThermoFisher Scientific), B27 supplement with Retinoic Acid (ThermoFisher Scientific), N2 supplement (ThermoFisher Scientific) and 2 mM L-Glutamine (ThermoFisher Scientific), 10μM SB431542 (Tocris) and 100 nM LDN-193189 (Miltenyl Biotec). From day 4 onwards, the base media was changed to 50% NIM, 25% DMEM/F12 and 25% Neurobasal Media. SB431542 (10 μM) and LDN-193189 (100 nM) were present for the first 12 days of differentiation. Cells were lifted and re-plated at day 12 and day 17 with Collagenase Type IV (Life Technologies). At day 25, differentiated cells were lifted for total RNA isolation.

2022-02-04 | GSE195877 | GEO

Gene expression profile of hESC/hIPC-derived keratinocytes

Project description:To induce the differentiation, undifferentiated human pluripotent stem cells (both hESC andh iPSC; T0 time point) were transferred into 20% O2 atmosphere environment and treated with mTESR1 basal media supplemented with 1 μM ATRA (Sigma-Aldrich) and 25 ng/ml BMP4 (R&D) for 7 days (Induction). To select for the cells that acquired early ectodermal fate, cells were harvested and re-plated onto freshly prepared 3D human dermal fibroblast ECM at a density of 5-10x10^3 cells per cm2 and grown in DMEM:Ham F12 (3:1) (Life Technologies) supplemented with 1 μM ATRA and 25 ng/ml BMP4 for a further 7 days (Selection). To enrich for putative epidermal progenitors, rapid-adhesion to type IV collagen-coated dishes was used, and the rapidly-adhering cells were cultured in DK SFM supplemented with 1 μM ATRA for 7 days (Enrichment). After that, the cells were cultured in EpiLife medium (Life Technologies) for a further 7 days (Expansion) before final harvest (T3 time point) and analysis. We analyzed here gene expression profiles of undifferentiated hESC/hiPSC (T0), hESC/hIPC-derived keratinocytes (T3) and primary normal human keratinocytes from skin biopsy (NHK). We found that hESC/hIPC-derived keratinocytes are similar to NHK.

2015-04-01 | GSE55898 | GEO

Gene expression profile of hESC/hIPC-derived keratinocytes

2015-04-01 | E-GEOD-55898 | biostudies-arrayexpress

Effects of plasticizers on human preadipocytes (SGBS) using global proteomics

Project description:SGBS cells were used as a model system to investigate the cellular mode of action of a variety of plasticizers in human adipocytes. In brief, cells were cultured at 5% CO2 and 37°C in 95% humidity. Cells of generation 40 and passage 3 after thawing were grown to confluence with DMEM/F12 containing 33 µM biotin, 17 µM pantothenate, 100 U/l penicillin, and 0.1 mg/l streptomycin (basal medium) supplemented with 10% FCS (Gibco, Carlsbad, CA, USA). According to the standard differentiation protocol, differentiation was initiated (day 0) after changing to serum-free basal medium supplemented with 0.1 μM cortisol, 0.01 mg/ml apo-transferrin, 0.2 nM triiodothyronine, and 20 nM human insulin (differentiation medium) with the addition of 2 µM rosiglitazone, 25 nM dexamethasone, and 200 µM 3-isobutyl-1-methylxanthine for the first four days. The positive control was differentiated using the standard protocol with rosiglitazone. For plasticizer treatments, differentiation was conducted without rosiglitazone and cells were continuously exposed from day 0 – day 16 to the plasticizers or their transformation products. As for plasticizer treatments, the negative control was differentiated with rosiglitazone-free medium containing equivalent amounts of solvent (0.01 MeOH, v/v) for 16 days, resulting in minimal differentiation. All media were renewed every second day to mimic continuous exposure.

2022-06-20 | PXD031041 | Pride

Derivation of a CDX2+/p63+ human cytotrophoblast stem cell from pluripotent stem cells in defined conditions

Project description:We show that WA09 human embryonic stem cells (hESC) cultured in minimal media and treated with BMP4 for 4 days differentiate towards a cytotrophoblast (CTB)-like state.

2019-11-28 | GSE65654 | GEO

Transcriptomic profiling of myogenic newt A1 cells undergoing senescence induction, differentiation or serum and senescence-induced dedifferentiation

Project description:To investigate the molecular basis of senescence-induced dedifferentiation, we performed RNAseq on control proliferating cells (DMSO only) and etoposide-induced senescent cells to profile senescence induction. Additionally, we performed RNAseq on purified populations of differentiated myotubes derived from untreated A1 cells to investigate differentiation, as well as myotubes subsequently induced to dedifferentiate, either by serum exposure (10% FCS) or by exposure to 48 hour conditioned media from senescent cells (including proliferating cell conditioned media controls). We then performed gene expression profiling analysis using data obtained from RNA-seq of all 6 populations in triplicate.

2023-06-21 | GSE211798 | GEO

Gene expression profiles of control and EWS-FLI1-transduced neural crest stem cells

Project description:Expression profiles were generated from hESC-derived neural crest stem cells following transduction with GFP control vector or EWS-FLI1 vector. Expression was analyzed in stem cell conditions 5 days after transduction (undifferentiated conditions) and after 6 weeks in differentiation media (differentiation conditions). Changes in gene expression over time were compared between control and EWS-FLI1 expresssing cells. Total RNA was extracted from 3 replicates for each of 4 conditions (undifferentiated control, undifferentiated EWS-FLI1, differentiated control and differentiated EWS-FLI1. Samples were analyzed by Affymetrix exon arrays using standard procedures.

2015-05-15 | E-GEOD-68898 | biostudies-arrayexpress

Analysis of microRNA transcriptomic changes in ethanol-exposed human embryonic stem cell (hESC)-derived cortical interneurons using small RNA sequencing

Project description:Purpose: The goal of this study to use ethanol-exposed human embryonic stem cell (hESC)-derived neural cells as models to investigate microRNA expression changes in the brains of subjects with alcohol use disorder (AUD). Methods: hESCs were differentiated into neural cells (mainly cortical interneurons), which were then cultured in media with or without ethanol (50-100 mM) for 7 days (by duplicate experiments). Total RNAs were extracted from hESC-derived neural cells (with or without ethanol exposure) for small RNA sequencing. The sequence reads were processed using the Comprehensive Analysis Pipeline for miRNA Sequencing Data (CAP-miRseq) workflow. Ethanol-induced miRNA transcriptomic changes were analyzed by the Limma-Voom method. Results: A 7-day ethanol exposure led to differential expression of six miRNAs (absolute FC>2.0 & P<0.05) in hESC-derived cortical interneurons. Three miRNAs were upregulated (>2-fold increase & P<0.05), while three other miRNAs were downregulated (> 2-fold decrease & P < 0.05) due to ethanol exposure. Conclusions: The hESC-derived neural cell model study can partially validate miRNA transcriptomic changes in postmortem brains of subjects with alcohol use disorder.

2021-10-13 | GSE181050 | GEO

Analysis of mRNA transcriptomic changes in ethanol-exposed human embryonic stem cell (hESC)-derived cortical interneurons using ribosome depletion RNA sequencing

Project description:Purpose: The goal of this study to use ethanol-exposed human embryonic stem cell (hESC)-derived neural cells as models to investigate mRNA expression changes in the brains of subjects with alcohol use disorder (AUD). Methods: hESCs were differentiated into neural cells (mainly cortical interneurons), which were then cultured in media with or without ethanol (50-100 mM) for 7 days (by duplicate experiments). Total RNAs were extracted from hESC-derived neural cells (with or without ethanol exposure) for mRNA sequencing. The sequence reads were processed using the RNA-seq processing pipeline Pipeliner [Front Genet. 2019; 10:614] workflow. Ethanol-induced mRNA transcriptomic changes were analyzed by the Limma-Voom method. Results: When the significance level was set at FC>2.0 & P<0.01, 19 coding genes showed differential expression, and all of them were downregulated after a 7-day ethanol exposure. Conclusions: The hESC-derived neural cell model study can partially validate mRNA transcriptomic changes in postmortem brains of subjects with alcohol use disorder.

2021-10-13 | GSE181049 | GEO

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