Therefore, global sensitivity analysis was used to cluster cell-signaling steps based on intrapathway interactions and identified biologically relevant control nodes of Stat3 activation. In Silico Investigation of Desensitization to Ligand Stimulation Desensitization, which is the inability to respond to ligand restimulation , may significantly limit cytokine-driven in vitro stem cell propagation . LYN antibody developed and validated a computational model of signal transducer and activator of transcription-3 (Stat3) pathway kinetics, a signaling network involved in embryonic stem cell (ESC) self-renewal. Our analysis identified novel pathway responses; for example, overexpression of the receptor glycoprotein-130 results in reduced pathway activation and increased ESC differentiation. We used a systematic in silico screen to identify novel targets and protein interactions involved in Stat3 activation. Our analysis demonstrates that signaling activation and desensitization (the inability to respond to ligand restimulation) is regulated by balancing the activation state of a T863 distributed set of parameters including nuclear export of Stat3, nuclear phosphatase activity, inhibition by suppressor of cytokine signaling, and receptor trafficking. This knowledge was used to devise a temporally modulated ligand delivery strategy that maximizes signaling activation and leads to enhanced ESC self-renewal. Author Summary Directing stem cell fate requires knowledge of how intracellular signaling pathways integrate environmental stimuli to make decisions to stay as stem cells (self-renew) or to differentiate into specific functional cell types. We developed and validated a computational model of signal transducer and activator of transcription-3 (Stat3) pathway kinetics, a signaling network involved in mouse embryonic stem cell (ESC) self-renewal. Our analysis demonstrates that stem cell fate control is regulated by a distributed set of parameters that positively and negatively regulate Stat3 activation. We further demonstrate that we can take advantage of differences in the timing of signaling pathway activation and inhibition to design T863 a strategy to deliver self-renewal stimuli to stem cells in a more efficient manner. Ultimately, the use of stem cells in biotechnological applications will require an in-depth understanding of how cells integrate diverse environmental stimuli to make cell fate decisions. Introduction Self-renewal is one of the defining characteristics of embryonic stem cells (ESCs) . This fate choice is influenced by ligandCreceptor-mediated activation of intracellular signaling pathways. Significant work is being done to understand the signaling proteins and pathways that control self-renewal of ESCs, and an emerging picture is that these pathways influence self-renewal in a context-dependent and temporally modulated manner [2C4]. One such pathway is the Jak/Stat3 (Janus kinase / signal transducer and activator of transcription-3) pathway . Activation of Stat3 by phosphorylation at Tyr-705 results in induction of genetic programs that are sufficient for maintenance of self-renewal in mouse ESCs [6C8]. Understanding how Stat3 activation is controlled may be useful for controlling ESC self-renewal. Stat3 is activated by a variety of ligands from the interlukin-6 (IL-6)Ctype family . In mouse ESCs, Stat3 activation results from binding of leukemia inhibitor factor (LIF) to the LIF receptor and glycoprotein-130 (GP130), forming a heterodimeric receptor complex [10,11]. Jak-mediated Src homology-2 (SH2)Cdomain phosphorylation of receptors leads to Stat3 recruitment to the T863 receptor complex , and its Tyr-705 phosphorylation and subsequent nuclear accumulation [13C25]. This pathway is under control of three main inhibitors, protein inhibitor of activated Stat3 (PIAS3), Src-2 homology containing phosphotyrosine phosphatase (SHP2), and suppressor of cytokine signaling (SOCS3). PIAS3 and SHP2 function to lessen Stat3 availability receptor and  activation [21,24C26], respectively, and SOCS3, which is normally under transcription control of Stat3, inactivates turned on receptors by binding to GP130 [26,27]. Activation of Stat3 is influenced by an assortment.