Unknown,Transcriptomics,Genomics,Proteomics

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Combined analysis of single cell RNA-Seq and ATAC-Seq data reveals regulatory toggles operating in native and iPS-derived retina

ABSTRACT: To improve the comparison of the developmental trajectories of native retina (NaR) and iPSC-derived 3D retinal aggregates(3D-RA) , we performed scRNA-Seq of murine NaR and 3D-RA at four matched stages of development: embryonic day (E)13 vs differentiation day (DD)13, postnatal day (P)0 vs DD21, P5 vs DD25 and P9 vs DD29. NaR were dissected from two to 11 C57Bl6 mice (of both sexes) per stage. Mouse 3D-RA were generated from the iPSC Nrl-GFP line. Cells from NaR and OV were dissociated and subjected to droplet-based scRNA-Seq using a 10X Genomics Chromium platform. We obtained sequence information for 21,249 cells from NaR and 16,842 cells from 3D-RA, distributed evenly amongst developmental stages. We generated an average of 74,808 reads per cell, corresponding to 5,940 unique molecular identifiers (UMIs) and 2,471 genes per cell.

INSTRUMENT(S): NextSeq 500

ORGANISM(S): Mus musculus

SUBMITTER: Haruko Takeda

PROVIDER: E-MTAB-9395 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The development of more complex but reliable systems for compound testing in a pharmaceutical context is a challenging task to date. Three-dimensional (3D), organ mimetic cell culture is aiming to become an alternative to common two-dimensional (2D) cell culture or animal testing in that field. We developed a biocompatible 3D cell culture environment for a hepatocellular carcinoma (HCC) model that enables cellular maintenance in a polycarbonate scaffold structure. Albumin, regarded as a differentiation marker, was elevated in statically 3D cultivated HepG2 cells. Expression of HCC tumor marker alpha-fetoprotein (AFP) was reduced compared to immunofluorescence stainings of 2D cultivated cells. Remarkably, expression of cytokeratin and pathophysiologically relevant beta-1 integrin (ITGB1) was found enhanced in nonperfused 3D cell culture. Changes in gene expression induced by the 3D cultivation environment were investigated using Ingenuity Pathway Analysis (IPA). Our findings revealed involvement of the insulin growth factor (IGF) signaling pathway in upregulation of matrix metalloproteinases (MMP) and ITGB1. The experimental data indicate a more differentiated state in 3D cultivated HepG2 cells than in the respective 2D experiments. Hence, scaffold-supported 3D cultivation of HepG2 cells may lead to a gain of information valuable for both drug testing and cancer research. HepG2 cells were cultivated for five days under 2D and 3D statical and perfused conditions. Cultivation was started with 0.25x10^6 cells in 2D and with 1x10^6 vital cells for the 3D experiments. The day of seeding was defined as d0. The groups were classified as follows: 2D, i.e., monolayer cultures, 3D, i.e., statical 3D cultures and BR, which denotes perfused 3D culture of HepG2 cells. The perfusable bioreactor system was operated using a peristaltic pump. It houses the MatriGrid, a polycarbonate-based microporous cellular support. For 3D static cultivation, cell-inoculated MatriGrids were placed in wells of a 24-well plate. Microarray experiments of three 2D (i.e., control), three 3D statically and three actively perfused 3D cultivations were performed at SIRS-Lab GmbH (SIRS-Lab GmbH, Jena, Germany) according to the manufacturer's instructions (Illumina, San Diego, CA). Altogether, 9 RNA samples of hepatocyte cultures and an internal control RNA were hybridized on two HumanHT-12 v4 Expression BeadChips.

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Combined analysis of single cell RNA-Seq and ATAC-Seq data reveals regulatory toggles operating in native and iPS-derived retina

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