Biochemical and Biophysical Research Communications
Transcriptional profiling of initial differentiation events in human embryonic stem cells
Section snippets
Materials and methods
Human ES cell culture. BG01 human ES cells [13] were maintained on mitomycin C inactivated mouse embryonic feeder (MEF) layers in DMEM/F12, 20% Knockout Serum Replacer, 2 mM l-glutamine, 0.1 mM MEM non-essential amino acids, 50 U/ml penicillin, 50 μg/ml streptomycin (all from Gibco/Invitrogen), 1000 U/ml hLIF (Chemicon), 0.1 mM βME (Sigma), and 4 ng/ml bFGF (Sigma). Cells were passaged with 0.05% trypsin–EDTA (Invitrogen) every three days and re-plated on fresh feeder layers. For microarray analysis,
MEDII treatment altered adherent hESC colony morphology
After treatment with MEDII, adherent hESCs were flatter than their non-treated counterparts and appeared to have less intercellular space (Figs. 1A and B). However, MEDII-treated hESCs continued to grow in colonies and their morphology was distinctly different from that of spontaneously differentiated cells seen routinely in the laboratory (Figs. 1C and D). We have also observed that MEDII-treated hESCs were more resistant to dissociation than untreated hESC cell colonies and may have formed
Discussion
Knowledge gained from understanding the signaling events occurring in the embryo will likely lead to improved in vitro differentiation strategies for ES cells. This is the first report that describes early positive modulation of genes involved in vertebrate signaling pathway for mesoderm formation (INHBE1/NODAL/TGFB1) in hESCs. In this study, we have utilized an in vitro model of early ICM differentiation to hESCs exposed to MEDII medium. The treated cells expressed both pluripotent gene and
Acknowledgments
The authors thank the Ovarian Cancer Institute for use of their Affymetrix microarray equipment. We also thank Mrs. Deb Weiler for feeder cell preparation and Mrs. Deanne Tibbetts for generation of MEDII medium. We also thank Ben Bolstad, Terry Speed, and Russ Wolfinger for valuable advice on analyzing Affymetrix array data. This work was supported in part by BresaGen Inc. and partial funding from the Georgia Tech/Emory Center (GTEC) for the Engineering of Living Tissues, an ERC Program of the
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The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint first authors.