An optimized embryonic stem cell model for consistent gene expression and developmental studies: a fundamental study.
In vitro differentiation of embryonic stem (ES) cells results in generation of tissue-specific somatic cells and may represent a powerful tool for general understanding of cellular differentiation and development in vivo. Culturing of most ES cell lines requires murine embryonic fibroblasts (MEF), which may influence adventitiously the genetic differentiation program of ES cells. We compared the expression profile of key developmental genes in the MEF-independent CGR8 ES cell line and in the MEF-dependent D3 ES cell line. Using neomycin-resistant MEFs we demonstrated that MEFs are able to contaminate the D3 ES cells even after removing the MEFs. Subsequently, optimal differentiation conditions were established for the differentiation of CGR8 ES cells into various germ layer cells. Detailed gene expression studies in differentiating CGR8 cells were done by RT-PCR analysis and by microarray analysis demonstrating a general trend of the assessed genes to be expressed either in 3 days- or 10-days old embryoid bodies (EBs) when compared to undifferentiated ES cells. Subsets within the various functional gene classes were defined that are specifically up- or down-regulated in concert. Interestingly, the present results demonstrate that developmental processes toward germ layer formation are irreversible and mostly independent of the culture conditions. Notably, apoptotic and mitochondrial ribosomal genes were down- and up-regulated in 10-days old EBs, respectively, whereas compared to the 3-days old EBs whereas the activity of the extracellular signal-regulated kinase (ERK) 1/2 decreased with progressive development. This article defines a platform for ES cell differentiation and gene expression studies.
Gissel
Cornelia
C
Center of Physiology and Pathophysiology, Institute of Neurophysiology and Centre of Molecular Medicine, University of Cologne (CMMC), Germany.
Voolstra
Chris
C
Doss
Michael Xavier
MX
Koehler
Christoph I
CI
Winkler
Johannes
J
Hescheler
Jürgen
J
Sachinidis
Agapios
A
D000818:Animals; D017209:Apoptosis / Q000235:genetics; D001755:Blastocyst / Q000166:cytology; D055396:Bone Morphogenetic Protein 2; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins / Q000235:genetics; D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D005819:Genetic Markers; D008297:Male; D008565:Membrane Proteins / Q000235:genetics; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D023421:Models, Animal; D005810:Multigene Family; D050814:Octamer Transcription Factor-3 / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D010766:Phosphorylation; D013234:Stem Cells / Q000166:cytology / Q000502:physiology*; D016212:Transforming Growth Factor beta / Q000235:genetics
C525719:Bmp2 protein, mouse; C525810:Bmp4 protein, mouse; D055396:Bone Morphogenetic Protein 2; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins; D005819:Genetic Markers; D008565:Membrane Proteins; D050814:Octamer Transcription Factor-3; D016212:Transforming Growth Factor beta; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3