Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptomic profiling of human neuromesodermal-like cells and spinal cord progenitors derived in vitro from H9 ES cell line

ABSTRACT: Human ES (H9) cells were directed towards a neuromesodermal progenitor-like cell state and these cells were then subsequently differentiated towards a neural cell fate. Human ES cells (H9) were differentiated into neuromesodermal progenitor-like cells by culturing in Neurobasal/1x N2/1x B27 medium (N2/B27) supplemented with 20 ng/ml bFgf and 3 μM CHIR99021 for 3 days and exposure to dual SMAD inhibition (dSMADi) (Noggin 50 ng/ml and the TGFb receptor type 1 inhibitor SB431542 10 μM) during day 3 (D3). Transcriptome analysis was then carried out following a selection procedure to enrich for NMP-like cells (sD3/NMP-like). This involved use of a hES (H9) cell line engineered with CRISPR-Cas9 to express GFP under the control of the endogenous Nkx1.2 promoter. At the end of day 3 cells were selected for high GFP expression, as high Nkx1.2 transcription is characteristic of NMP cell populations in mouse and chick embryos. These cells were then lysed and RNA extracted for RNASeq. Humans ES cells (H9) differentiated into NMP-like cells as above (without selection) were also allowed to develop further on day 4 (in the presence of dSMADi and Retinoic acid (RA) 100nM, in N2/B27) and then in RA alone until end of day 8 (D8). These cells were then lysed and RNA extracted for RNASeq.

INSTRUMENT(S): NextSeq 500

ORGANISM(S): Homo sapiens

SUBMITTER: Kate Storey

PROVIDER: E-MTAB-6680 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:in the present study, we evaluated whether microbiota modulation is able to restore hepatic steatosis induced by n-3 PUFA depletion in mice. For this purpose, mice were fed during three months with a n-3 PUFA-depleted diet (presenting a high n-6/n-3 PUFA ratio), and then supplemented with fructooligosaccharides (FOS, 0.25g/day/mice), a prebiotic, during the last ten days of the experiment (DEF/FOS). In the same time, some n-3 PUFA-depleted mice were returned on a control diet during the last 10 days of treatment (DEF/CT) to compare the effect of FOS supplementation to a restored intake in n-3 PUFA. Microarray analyses were performed to identify the molecular targets modified by FOS supplementation in the liver of n-3 PUFA depleted mice. These mice were compared to control mice (fed a control diet during the 112 days of experiment) and to n-3 PUFA-depleted mice (fed a n-3 PUFA-depleted diet during the 112 days of experiment) for which the results have been previously published (Pachikian B.D. et al. PLoS One. 2011;6(8):e23365, accession number GSE26986) Male C57Bl/6J mice (9 weeks old; Charles River, Brussels, Belgium) were housed in groups of 4 mice per cage at 22Â°C in a 12 h light/dark cycle and given free access to diet and water. After an acclimatisation period of 1 week, mice were fed a control (CT) (D08041805, Research Diets, New Brunswick, USA) or an n-3 PUFA-depleted diet (DEF) (D08041806, Research Diets, New Brunswick, USA) for 112 days ad libitum. The CT diet contained the following (percent w/w): casein 20, total carbohydrate 72.4 (including corn starch 44.2, sucrose 10, maltodextrin 13.2, cellulose 5), soybean oil 5, mineral mix 3.5 and vitamin mix 1. The depletion was induced by replacing the soybean oil with sunflower oil, which exhibited a higher n-6/n-3 PUFA ratio. The n-6/n-3 PUFA ratio was 6.9 and 127.2 for the CT diet and the DEF diet, respectively. Ten days before the end of the experiment, DEF mice were divided into three groups: DEF, DEF/FOS and DEF/CT mice. The DEF mice were still fed with the n-3 PUFA depleted diet (DEF). The DEF/FOS mice were still fed the DEF diet and were supplemented for 10 days with fructooligosaccharides (DEF/FOS). FOS (Beneo P95 gift from Orafti; Tienen, Belgium) was added to tap water in a concentration adequate to reach an intake of 0.25g of FOS per day. For DEF/CT mice, the DEF diet was replaced by the CT diet during the last 10 days of treatment. At the end of the study period, mice fed the CT (n=4), DEF (n=7), DEF/CT (n=8) or DEF/FOS diet (n=8) were anaesthetised (ketamine/xylazine i.p., 100 and 10 mg/kg of body weight, respectively). The liver tissue was immediately clamped in liquid N2 and kept at -80Â°C until analysis. All mice experiments were approved by the local animal ethics committee and the housing conditions were as specified by the Belgian Law of April 6, 2010 on the protection of laboratory animals (agreement nÂ° LA 1230314).

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Transcriptomic profiling of human neuromesodermal-like cells and spinal cord progenitors derived in vitro from H9 ES cell line

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