The intracellular signaling mechanism for the switch between self-renewal and differentiation of ES cells is a central and unanswered question in stem cell biology, and the initiation of ESC differentiation is apparently determined by multiple pathways working in concert. This complexity requires a modulator(s) that fine-tunes signals coordinately in negative or positive fashion, although little is known in this regard. We demonstrate here that Shp2 acts as a negative or positive regulator of signaling events controlling self-renewal and differentiation of mESCs and hESCs.
In this study, we have taken three different experimental approaches to determine Shp2 functions in human and mouse ES cell differentiation. First, we established homozygous mutant mES cell lines with a targeted deletion of exon 4 at the Shp2
locus. Shp2-deficient mES cells displayed dramatically impaired capacity of differentiation into all three germ layer cell lineages, accompanied by improved self-renewal potential. These results clearly define a biological function of Shp2 in control of mouse ES cell pluripotency and differentiation. Similar results were obtained from Shp2 mutant (exon 3−/−
) mES cells in our previous experiments 
, supporting the notion that targeted deletion of exon 3 at the Ptpn11/Shp2
locus created a loss of function mutant Shp2 molecule. Second, to extend the functional analysis of Shp2 from mouse to human ES cells, we have successfully used the siRNA approach to knockdown Shp2 expression in hESCs, unveiling a similar role of Shp2 in promoting human ES cell differentiation. Thus, the Shp2 function in promoting ES cell differentiation is conserved between mESCs and hESCs, despite a body of literature documenting differences existing between the two types of ES cells 
. Third, a small molecule inhibitor of Shp2 enzyme has been identified that partially inhibits mouse and human ES cell differentiation at low dosages. This chemical biology experiment not only renders strong support to our gene knockout and knockdown experiments, but also suggests that development of specific Shp2 inhibitors can provide useful experimental tools for amplification of hESCs in vitro for molecular analysis and biological characterization.
Our results suggest different molecular mechanisms underlying the concerved function of Shp2 in regulation of mouse and human ES cell differentiation. In mouse ESCs, Shp2 participates in modulation of LIF signals by promoting the Erk pathway and suppressing the Stat3 signal. Upon LIF stimulation, Shp2 is physically recruited to gp130, the signaling component of LIF receptor, and acts to regulate the LIF signal 
. Bi-directional regulation of Erk and Stat3 pathways by Shp2 appears to be conserved in embryonic and adult stem cells as well as in differentiated cell types. In previous work, we have found opposite effects of Shp2 deletion on Erk and Stat3 activation in coordinated regulation of neural stem cell proliferation and differentiation into neuronal/glial cell lineages during brain development 
, and also in control of energy balance by leptin in hypothalamic neurons of adult brain 
. In addition, we detected a similar fashion for Shp2 regulation of Erk and Stat3 activation in hepatocytes during liver regeneration and epithelial cells in mammary gland development and involution 
. Both the decreased Erk activity and enhanced Stat3 signals may lead to suppression of mES cell differentiation and improved self-renewal. Attenuation of Erk signal may also be responsible for the decreased proliferation rate observed in Shp2−/−
mESCs and Shp2 knockdown hESCs as well as in mESCs/hESCs treated with Shp2 inhibitors. Activation of the Stat3 pathway may lead to different physiological consequences in various cell types. The LIF-Stat3 signaling appears to be required for self-renewal in mESCs 
, while activation of Stat3 promotes glial cell differentiation from neural stem/progenitor cells during cell fate specification in the brain 
. On the other hand, Stat3 is required for timely initiation of physiological epithelial cell apoptosis during mammary gland involution 
. Consistent with the literature 
, we did not detect Stat3 activation signals in hESCs under a variety of growth factor/cytokine stimulation, supporting the theory that the Stat3 pathway is not required for hESC self-renewal.
One interesting observation made in this study is the bi-directional regulation of BMP4-Smad signaling by Shp2 in mouse and human ES cells. Genetic ablation of Shp2 in mouse ES cells resulted in enhanced p-Smad1/5/8 signals in response to BMP4 stimulation and, consistently, increased expression of Id1
, target genes downstream of the BMP4-Smad pathway, was detected in Shp2−/−
mESCs compared to wild-type cells. In contrast, Shp2 knockdown in hESCs leads to impaired p-Smad1/5/8 levels following BMP4 treatment. Consistent to this observation is the decreased induction of ID2
gene expression by BMP4 in Shp2 knockdown hESCs. These results suggest a cell context-dependent manner for Shp2 regulation of the BMP4-Smad pathway in hESCs and mESCs. Interestingly, this bi-directional modulation of Smad signaling can lead to the same consequence of differentiation suppression in human and mouse ES cells. BMP4-Smad signaling has been shown to work cooperatively with the LIF-Stat3 pathway in supporting mouse ES cell self-renewal 
. However, BMP4 can induce trophoblast differentiation in human ES cells , and Noggin, a BMP antagonist, has been shown to support hESC self-renewal in concert with bFGF 
. It is unclear yet how Shp2 regulates the BMP4-Smad pathway, which may involve direct and indirect mechanisms. For example, Shp2 may influence Smad signaling strength via regulation of Erk and Stat pathways, since cross-talks between Erk, Stat and Smad pathways have been reported previously by several groups 
Deletion of Shp2 also resulted in reduced mESC proliferation, suggesting a role of Shp2 in mitogenic signaling. Consistently, we observed reduced proliferation rate of hESCs when Shp2 expression is downregulated by siRNA, and supplement of Shp2 inhibitor DCA leads to reduced cell proliferation of both hESCs and mESCs. However, the reduced differentiation capacity of Shp2-deficient ES cells is unlikely due to decreased proliferation rate. In contrast, the primary and secondary EB formation assay detected dramatically increased self-renewing proliferation of Shp2−/− mES cells, as compared to wt cells. Furthermore, molecular signaling analysis strongly suggests that Shp2 ablation suppressed pathways favoring differentiation, while enhancing signals leading to self-renewal. The cross-talk and balance of proliferation, self-renewal and differentiation signals modulated/coordinated by Shp2 contribute to the switch between pluripotency and differentiation of ES cells.
We have identified a potent and selective Shp2 inhibitor by screening a small chemical library. The specificity of Shp2 inhibition by DCA has been tested and confirmed by a number of experiments in this study: a) inhibition of purified Shp2 was examined against a list of other PTPs; b) DCA was shown to suppress growth factor-stimulated Erk activation; c) DCA was found to have no effect on Shp2−/−
cells. As expected, the inhibitors can suppress growth factor-stimulated Erk activation and also cell proliferation. Importantly, DCA exhibits similar inhibitory effects on mouse and human ES cell differentiation. In recent experiments, several groups have shown similar effects with Mek inhibitor and GSK3 inhibitor on mouse or human ES cells 
. One concern with small molecule inhibitors is often associated with their toxicity that limits their value in their use for long-term hESC culture. We have used an inhibitor cocktail and our results show that in combination, these molecules can be used at much lower concentrations to reach a similar biological effect. Further chemical modification of the isolated small molecule in order to increase specificity and reduce toxicity will increase the application value.
In summary, multiple pathways, including Erk, Stat3 and Smad, have been shown to participate in cellular decisions for ESC self-renewal or differentiation; this study identifies Shp2 as a critical player orchestrating these pathways in programming initial differentiation of human and mouse ES cells.