title: Activin a efficiently specifies definitive endoderm from human embryonic stem cells only when phosphatidylinositol 3-kinase signaling is suppressed.
abstract: Human ESCs (hESCs) respond to signals that determine their pluripotency, proliferation, survival, and differentiation status. In this report, we demonstrate that phosphatidylinositol 3-kinase (PI3K) antagonizes the ability of hESCs to differentiate in response to transforming growth factor beta family members such as Activin A and Nodal. Inhibition of PI3K signaling efficiently promotes differentiation of hESCs into mesendoderm and then definitive endoderm (DE) by allowing them to be specified by Activin/Nodal signals present in hESC cultures. Under conditions where hESCs are grown in mouse embryo fibroblast-conditioned medium under feeder-free conditions, approximately 70%-80% are converted into DE following 5 days of treatment with inhibitors of the PI3K pathway, such as LY 294002 and AKT1-II. Microarray and quantitative polymerase chain reaction-based gene expression profiling demonstrates that definitive endoderm formation under these conditions closely parallels that following specification with elevated Activin A and low fetal calf serum (FCS)/knockout serum replacement (KSR). Reduced insulin/insulin-like growth factor (IGF) signaling was found to be critical for cell fate commitment into DE. Levels of insulin/IGF present in FCS/KSR, normally used to promote self-renewal of hESCs, antagonized differentiation. In summary, we show that generation of hESC-DE requires two conditions: signaling by Activin/Nodal family members and release from inhibitory signals generated by PI3K through insulin/IGF. These findings have important implications for our understanding of hESC self-renewal and early cell fate decisions.
authors.affiliation: Department of Biochemistry and Molecular Biology and Paul D. Coverdell Center for Biomedical and Health Sciences, University of Georgia, Athens, Georgia 30602, USA.
authors.forename: Amanda B
authors.initials: AB
authors.lastname: McLean
authors.forename: Kevin A
authors.initials: KA
authors.lastname: D'Amour
authors.forename: Karen L
authors.initials: KL
authors.lastname: Jones
authors.forename: Malini
authors.initials: M
authors.lastname: Krishnamoorthy
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Kulik
authors.forename: David M
authors.initials: DM
authors.lastname: Reynolds
authors.forename: Alan M
authors.initials: AM
authors.lastname: Sheppard
authors.forename: Huiqing
authors.initials: H
authors.lastname: Liu
authors.forename: Ying
authors.initials: Y
authors.lastname: Xu
authors.forename: Emmanuel E
authors.initials: EE
authors.lastname: Baetge
authors.forename: Stephen
authors.initials: S
authors.lastname: Dalton
mesh_terms: D028341:Activins / Q000502:physiology*; D000818:Animals; D002454:Cell Differentiation / Q000502:physiology*; D017077:Culture Media, Conditioned; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000502:physiology*; D004707:Endoderm / Q000166:cytology / Q000502:physiology*; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D006801:Humans; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D015398:Signal Transduction; D013370:Subrenal Capsule Assay; D014158:Transcription, Genetic
chemical_list: D028341:Activins; D017077:Culture Media, Conditioned; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; C422405:activin A