Self-renewal of human embryonic stem cells requires insulin-like growth factor-1 receptor and ERBB2 receptor signaling.
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.
Wang
Linlin
L
Division of Hematology, Department of Medicine, Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, 98195, USA.
Schulz
Thomas C
TC
Sherrer
Eric S
ES
Dauphin
Derek S
DS
Shin
Soojung
S
Nelson
Angelique M
AM
Ware
Carol B
CB
Zhan
Mei
M
Song
Chao-Zhong
CZ
Chen
Xiaoji
X
Brimble
Sandii N
SN
McLean
Amanda
A
Galeano
Maria J
MJ
Uhl
Elizabeth W
EW
D'Amour
Kevin A
KA
Chesnut
Jonathan D
JD
Rao
Mahendra S
MS
Blau
C Anthony
CA
Robins
Allan J
AJ
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
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