SYNOPSIS OF PROPOSAL: This proposal seeks to establish a role of SHP2 in hESC function. The preliminary data indicate that knockdown of SHP2 expression using siRNA or pharmaceutical inhibition of SHP2 activity in hESCs leads to impaired differentiation and enhanced self-renewal. The hypothesis is that by elucidating the SHP2 action, the investigators will be able to sort out the common and distinct signaling events between hESCs and mESCs in the control of self-renewal versus differentiation for better understanding of the molecular basis of “stemness”. Areas of focus are to determine the impact of SHP2 deficiency on genetic control of hESC pluripotency; to investigate the effect of SHP2 deficiency on epigenetic control of hESC identity; to elucidate the link of intracellular signals to genetic/epigenetic control of hESC pluripotency; and to isolate/develop reagents that maintain hESC pluripotency. IMPACT AND SIGNIFICANCE: This investigator seeks to use a single-gene approach focusing on the SHP2 phosphatase and its intracellular signaling partners in mouse and human ESC cells, and determine its role in hESC pluripotency. The impact/significance of this study is that it is true, as claimed by the PI, that there is relatively little information on cytoplasmic signaling molecules and signal transduction cascades (which are so well defined in fibroblast and in cancer cells) that talk to the key nuclear regulators such as NANOG, OCT4 and SOX2. Thus, the definition of these cascades and their role in maintaining or instructing pluripotency would be quite useful. However, this moderate enthusiasm is tempered by the fact that it is extremely well established that molecules like SHP2 are highly promiscuous and talk to many, many different signal transduction pathways suggesting that their manipulation, either genetically or pharmacologically, may not be useful in deriving or maintaining specific lineages of hESCs. Shp2 is a signal transduction molecule expressed in many cells of the body and is downstream of many growth factors. An understanding of this pathway and its regulation is of fundamental interest, however the study proposed is focused strictly on this molecule and its potential role in ES cell differentiation. The PI has previously shown that in mESC null for shp2, differentiation is slowed as is the growth rate. Here extensive studies are proposed based on the hypothesis that shp2 is a key player in maintaining the undifferentiated state, and that this would be most helpful for future large scale use of hESCs. Extensive data gathering with state of the art technology are proposed but unfortunately without much rationale or indication of how the data will be interpreted. While this molecule has a role that is likely to be downstream, the more innovative and interesting potential outcome is the identification of small molecules that may be used transiently to sustain the undifferentiated status of hESC as they are expanded. However, there are several big “ifs” to this outcome, including whether this inhibition could be reversible, and if proliferation could be maintained during this inhibition. The PI was the first to show an effect of Shp2 deletion on mouse and now human ES cell self renewal and differentiation, and now proposes to use this as a hook to undertake an extensive investigation of human ES cell self renewal. To the extent that the proposed work could lead to an improved understanding of hESC self renewal it may have high level of significance. The major concern is that the entire approach depends on demonstrating that Shp2 is a valid target for promoting ES cell self renewal and preventing differentiation. Unless this is definitively established, one questions the entire basis of the application. For example, experiments in which Shp2 is conditionally knocked down (for example using a tet regulatable siRNA) and showing that shp2 knock-down cells are capable of extensive self renewal in the absence of CM, THEN showing that a normal pattern of differentiation is restored with restoration of shp2 function would have generated a considerably higher degree of enthusiasm. This would ideally be demonstrated both in mouse ES cells (where one could show that restoration of Shp2 function may permit germ line transmission) and human ES cells (where restoration of Shp2 could restore teratoma forming ability). Unless data of this type exists, there is cause to question whether the extensive studies proposed here are justified. QUALITY OF THE RESEARCH PLAN: This is a very descriptive study of SHP2 function in hESCs. It seems to mirror many of the previous studies done by this PI. The key piece of preliminary data seems to be that SHP2 in mouse ES cells increases self renewal and suppresses differentiation. This is interpreted by the PI as SHP2 functioning in a key role in mESC pluripotency. The PI will determine gene expression profiles in wild type and knockout mESC cells and apparently in hESC cells as well. These experiments are fraught with difficulties as a huge number of genes are modified by these relatively far upstream signal transduction cascade molecules. The proposal continues with highly descriptive information including: analysis of global CpG methylation and the role of SHP2 function in the genetic or epigenetic control of hESC pluripotency. In fact, for one aim there is no specific experiment described at all other than integrating an expression data and epigenetic data set. The proposal provides an ambitious array of high technology experiments without clear rationales or interpretations. A team of collaborators has been enlisted to provide expertise for all the high tech experiments. While differences in regulation of differentiation and self renewal between mESCs and hESCs are important for our understanding and future use of these cells, the experiments proposed are mainly in mESCs and seem more to be included for their “state of the art” technology than for clear hypothesis testing. It is striking that the extensive databases from Ivanova et al (Aug 2006) on the effects of knocking down known self renewal genes in mESCs were not used to generate hypotheses. In that study Shp2/PTPN11 mRNA expression was decreased when shRNA for Oct4, Sox2 and Esrrb were used but not when Nanog, Tcl1, Tbx3 nor dppa4 were knocked down. Overall, a considerable body of data will be generated on mESCs and less on hESCs, but the impact of this is not apparent. However, the final goal is aimed at developing small molecule inhibitors for SHP2, and to test one identified compound DCA on hESCS to maintain hESCs in an undifferentiated state while expanding them without long term effects; this aim has important potential for the scale up of hESCs. However, this is pretty much highly speculative, and it is questionable whether it is appropriate to pursue at this time. In summary, the experimental plan is, essentially, a huge amount of descriptive experiments on a global cytoplasmic signal transduction molecule, which very likely mediates many signals. Whether this is at all specific to hESC function or maintenance is unclear. There was not enough preliminary data to evaluate this and it is simply premature at this time to know whether this is a promising new direction. In addition, the application is not very responsive to the RFA as the laboratory is not doing mature and ongoing studies of hESCs. This may be more appropriate for a SEED grant. STRENGTHS: The experience of the investigator in signal transduction, and the importance of defining potential pharmacological targets in the signal transduction apparatus for manipulating hESC function are strengths. A talented team of collaborators has been enlisted to provide expertise for all the high tech experiments. The PI has large base of knowledge and reagents for the study of shp2/PTPN11 and has previously reported that its loss results in delayed differentiation and decreased growth rate. The investigator's track record of productivity is a strength, and he and is a leading authority in Shp2 signaling in ES cells. A head-to-head comparison of mESCs and hESCs in the same lab is important, but the shp2 null mESC have a more complete loss of shp2 than the knock down hESCs, thus complicating the interpretations. The small molecule screen for inhibitors of shp2 has already identified one potential molecule that is to be tested in hESC to see whether there is a reversible maintenance of a undifferentiated state while scaling up expansion. The apparent consistency of Shp2's effects across both mouse and human ES cells is attractive, and the proposed extensive use of local and national resources and expertise is a strength. Also, the environment is outstanding. WEAKNESSES: As mentioned above, it is striking that the extensive databases from Ivanova et al (Aug 2006) on the effects of knocking down known self renewal genes in mESCs were not used to generate hypotheses. In that study Shp2/PTPN11 mRNA expression was decreased when shRNA for Oct4, Sox2 and Esrrb were used but not when Nanog, Tcl1, Tbx3 nor dppa4 were knocked down. These data suggest the shp2 is downstream of some but not all the known renewal genes. These data imply a great complexity since the PI reports data that Nanog, Oct4 and Sox2 are changed when shp2 is absent in mESCs. It is unclear how effective the knockdown of shp2 in hESCs is. There is a suggestion that with nucleofection a 90% efficiency is achieved but no preliminary data on the efficacy of knock down is provided. It is promising that a Lentiviral vector for shRNA is being tested. Many of the high-tech experiments are proposed but without much detail of rationale or interpretations on how they will be used to address the hypothesis that shp2 is regulating hESC Pluripotency. Earlier work of the PI and figure 1 of Preliminary data show loss of shp2 in mESCs had much reduced growth rate (50-75%) particularly under high serum concentrations (15%). How does this reduction in proliferation balance with the expected amplification of undifferentiated hESCs if shp2 is inhibited with a small molecule? The lack of experience of the PI in hESC function is a concern, and the preliminary data on the role of SHP2 in mESC function is of marginal quality; the figures in the Preliminary data are of exceptionally poor quality. One questions the extent to which the apparent similarity in Shp2's effects in mouse and human ES cells is mechanistically similar since STAT3 activation is not sufficient for hESC self renewal. The single-gene approach proposed is a weakness, and combined with the unclear specific relevance of SHP2 to mESC/hESC pluripotency maintenance or function, there is cause for concern. It is very likely that signaling by SHP2 will be highly promiscuous. More focus on validating Shp2 as a true "self renewal" target is needed. DISCUSSION: Reviewers and discussants agreed that the central premise of this proposal was unfounded given the lack of convincing preliminary data to support the idea that SHP2 is involved in self-renewal. Recommended were careful knock-down and add-back experiments to establish the role of SHP2, especially in light of its predicted signalling promiscuity. Also agreed upon was a sense that the proposal was propelled more by an historical interest in a single gene than by hypothesis-driven science. The discussants felt that the proposal suffered from a lack of three important elements: experimental support for the central idea, clear relevance to hESCs and hypothesis-driven science.