An optimized embryonic stem cell model for consistent gene expression and developmental studies

Abstract

SummaryIn vitro differentiation of embryonic stem(ES) cells results in generation of tissue-specific somaticc ells and mayr epresent a powerful tool forg eneral understandingo fc ellular differentiation and development in vivo.Culturing of most ES cell lines requires murinee mbryonicf ibroblasts (MEF),w hich mayi nfluence adventitiously the genetic differentiation programofES cells.We compared the expression profile of keydevelopmental genesinthe MEF-independentCGR8EScellline and in the MEFdependentD 3E Sc elll ine.Using neomycin-resistant MEFs we demonstrated that MEFs areable to contaminate theD3EScells even after removing the MEFs.Subsequently, optimal differentiation conditions were establishedf or thed ifferentiation of CGR8 ES cells into various germ layerc ells.Detailed gene expression studies in differentiatingCGR8cells were donebyR TKeywords CGR8 embryonic stemc ells,g enee xpression,m icroarrays, development PCR analysis and by microarrayanalysis demonstrating ageneral trendofthe assessedgenes to be expressedeitherin3days-or 10-days oldembryoid bodies(EBs)whencompared to undifferentiatedE Sc ells.S ubsetsw ithin the various functional gene classesweredefinedthat arespecificallyup-or down-regulated in concert. Interestingly, the presentr esults demonstratet hat developmental processes towardgermlayer formation areirreversible andmostlyindependent of the cultureconditions. Notably, apoptotic and mitochondrial ribosomal genesweredownand up-regulated in 10-days oldEBs, respectively, whereas compared to the 3-days old EBs whereas theactivity of the extracellular signal-regulatedkinase(ERK)1/2 decreasedwith progressivedevelopment.This article defines aplatformfor ES cell differentiation and gene expression studies.

Gene Expression Signatures Defining Fundamental Biological Processes in Pluripotent, Early, and Late Differentiated Embryonic Stem Cells

“…CGR8 ESCs (ECACC 95011018) were cultured without feeder cells in Glasgow minimum essential medium supplemented with 10% fetal bovine serum (FBS), 2 mM lglutamine, 100 U/mL LIF, and 50 mM b-mercaptoethanol (ME) in 0.2% gelatine-coated flasks as previously described [10]. CGR8 ESCs (passage No.…”

Subcellular distribution of choline acetyltransferase by immunogold electron microscopy in non-neuronal cells: Placenta, airways and murine embryonic stem cells

“…The murine embryonic stem cell line CGR8 was a generous gift from Dr. J. Hescheler, University of Cologne, Germany (Gissel et al, 2005). Undifferentiated cells were cultured under standard conditions in gelatinized flasks with Glasgow's buffered minimal essential medium (Gibco, Germany), supplemented with 100 U/ml leukaemia inhibitory factor (LIF; Sigma Aldrich, Germany) to maintain pluripotency.…”

Proteomic comparison of spherical aggregates and adherent cells of cardiac stem cells

“…3 we selected four spots, 8,9,36 commercial antibodies are available for quantification on Westernblot filters. Fig.…”

“…For regenerative medicine, an understanding of the regulatory molecular mechanism for cell differentiation and self-replenish is crucial. Towards achieving this goal, gene expression analyses with transcription profiling (transcriptome) and micro-arrays have been conducted including neurospheres [7], mouse and human embryonic stem cells [8,9,10] and left atrium derived stem cells [11]. These mRNA analyses, however, have limitations because several investigations revealed that protein expression levels correlated poorly with mRNA level due to the multiple regulatory factors including mRNA stability, rate of translation and protein degradation [12,13].…”