Abstract
cYes, a member of the Src family of non-receptor tyrosine kinases, is highly expressed in mouse and human embryonic stem (ES) cells. We demonstrate that cYes kinase activity is regulated by leukemia inhibitory factor (LIF) and serum and is down-regulated when cells differentiate. Moreover, selective chemical inhibition of Src family kinases decreases growth and expression of stem cell genes that mark the undifferentiated state, including Oct3/4, alkaline phosphatase, fibroblast growth factor 4, and Nanog. A synergistic effect on differentiation is observed when ES cells are cultured with an Src family inhibitor and low levels of retinoic acid. Src family kinase inhibition does not interfere with LIF-induced JAK/STAT3 (Janus-associated tyrosine kinases/signal transducer and activator of transcription 3) or p42/p44 MAPK (mitogen-activated protein kinase) phosphorylation. Together the results suggest that the activation of the Src family is important for maintaining mouse and human ES in an undifferentiated state and may represent a third, independent pathway, downstream of LIF in mouse ES cells.
Highlights
Embryonic stem (ES)1 cells are derived directly from the inner cell mass of a blastocyst
Expression Levels and Kinase Activity of cYes in Mouse and Human embryonic stem (ES) Cells—To determine whether cYes protein expression or activity was regulated in ES cells under different conditions, Western blotting, and in vitro kinase assays were performed on undifferentiated mouse and human ES cells as well as on 10-day-old mouse and human embryoid bodies (EBs)
The decrease in Oct3/4 expression in response to differentiation is slower and less robust than that observed for alkaline phosphatase staining, immunohistochemistry and subsequent analysis by flow cytometry for Oct3/4 revealed a dose-dependent decrease in the number of Oct3/4ϩ cells in response to SU6656 (Fig. 3D), a result consistent with the notion that inhibition of Src family kinases leads to ES cell differentiation
Summary
Embryonic stem (ES)1 cells are derived directly from the inner cell mass of a blastocyst. Together the results suggest that the activation of the Src family is important for maintaining mouse and human ES in an undifferentiated state and may represent a third, independent pathway, downstream of LIF in mouse ES cells.
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