Transcriptional profiling of initial differentiation events in human embryonic stem cells

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Abstract

Currently, there are no differentiation strategies for human embryonic stem cells (hESCs) that efficiently produce one specific cell type, possibly because of lack of understanding of the genes that control signaling events prior to overt differentiation. sed HepG2 cell conditioned medium (MEDII), which induces early differentiation in mouse ES cells while retaining pluripotent markers, to query gene expression in hESCs. Treatment of adherent hESCs with 50% MEDII medium effected differentiation to a cell type with gene expression similar to primitive streak stage cells of mouse embryos. MEDII treatment up-regulates TDGF1 (Cripto), a gene essential for anterior–posterior axis and mesoderm formation in mouse embryos and a key component of the TGFB1/NODAL signaling pathway. LEFTYA, an antagonist of NODAL/TDGF1 signaling expressed in anterior visceral endoderm, is down-regulated with MEDII treatment, as is FST, an inhibitor of mesoderm induction via the related INHBE1 pathway. In summary, the TGFB1/NODAL pathway is important for primitive-streak and mesoderm formation and in using MEDII, we present a means for generating an in vitro cell population that maintains pluripotent gene expression (POU5F1, NANOG) and SSEA-4 markers while regulating genes in the TGFB1/NODAL pathway, which may lead to more uniform formation of mesoderm in vitro.

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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.

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