title: Self-renewal of human embryonic stem cells requires insulin-like growth factor-1 receptor and ERBB2 receptor signaling.
abstract: Despite progress in developing defined conditions for human embryonic stem cell (hESC) cultures, little is known about the cell-surface receptors that are activated under conditions supportive of hESC self-renewal. A simultaneous interrogation of 42 receptor tyrosine kinases (RTKs) in hESCs following stimulation with mouse embryonic fibroblast (MEF) conditioned medium (CM) revealed rapid and prominent tyrosine phosphorylation of insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R); less prominent tyrosine phosphorylation of epidermal growth factor receptor (EGFR) family members, including ERBB2 and ERBB3; and trace phosphorylation of fibroblast growth factor receptors. Intense IGF1R and IR phosphorylation occurred in the absence of MEF conditioning (NCM) and was attributable to high concentrations of insulin in the proprietary KnockOut Serum Replacer (KSR). Inhibition of IGF1R using a blocking antibody or lentivirus-delivered shRNA reduced hESC self-renewal and promoted differentiation, while disruption of ERBB2 signaling with the selective inhibitor AG825 severely inhibited hESC proliferation and promoted apoptosis. A simple defined medium containing an IGF1 analog, heregulin-1beta (a ligand for ERBB2/ERBB3), fibroblast growth factor-2 (FGF2), and activin A supported long-term growth of multiple hESC lines. These studies identify previously unappreciated RTKs that support hESC proliferation and self-renewal, and provide a rationally designed medium for the growth and maintenance of pluripotent hESCs.
authors.affiliation: Division of Hematology, Department of Medicine, Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, 98195, USA.
authors.forename: Linlin
authors.initials: L
authors.lastname: Wang
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Schulz
authors.forename: Eric S
authors.initials: ES
authors.lastname: Sherrer
authors.forename: Derek S
authors.initials: DS
authors.lastname: Dauphin
authors.forename: Soojung
authors.initials: S
authors.lastname: Shin
authors.forename: Angelique M
authors.initials: AM
authors.lastname: Nelson
authors.forename: Carol B
authors.initials: CB
authors.lastname: Ware
authors.forename: Mei
authors.initials: M
authors.lastname: Zhan
authors.forename: Chao-Zhong
authors.initials: CZ
authors.lastname: Song
authors.forename: Xiaoji
authors.initials: X
authors.lastname: Chen
authors.forename: Sandii N
authors.initials: SN
authors.lastname: Brimble
authors.forename: Amanda
authors.initials: A
authors.lastname: McLean
authors.forename: Maria J
authors.initials: MJ
authors.lastname: Galeano
authors.forename: Elizabeth W
authors.initials: EW
authors.lastname: Uhl
authors.forename: Kevin A
authors.initials: KA
authors.lastname: D'Amour
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Chesnut
authors.forename: Mahendra S
authors.initials: MS
authors.lastname: Rao
authors.forename: C Anthony
authors.initials: CA
authors.lastname: Blau
authors.forename: Allan J
authors.initials: AJ
authors.lastname: Robins
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D052160:Benzothiazoles / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D017077:Culture Media, Conditioned; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006801:Humans; D051379:Mice; D020890:Neuregulin-1 / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020893:Receptor, ErbB-3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D017527:Receptor, IGF Type 2 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011972:Receptor, Insulin / Q000037:antagonists & inhibitors / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D020032:Tyrphostins / Q000494:pharmacology
chemical_list: D000911:Antibodies, Monoclonal; D052160:Benzothiazoles; D017077:Culture Media, Conditioned; D016222:Fibroblast Growth Factor 2; D020890:Neuregulin-1; D018719:Erb-b2 Receptor Tyrosine Kinases; D020893:Receptor, ErbB-3; D017527:Receptor, IGF Type 2; D011972:Receptor, Insulin; D020032:Tyrphostins; C098214:tyrphostin AG825; C087230:heregulin beta1; C508053:ERBB2 protein, human; C524056:Erbb2 protein, mouse