SYNOPSIS: Rho GTPases are known to influence cytoskeletal organization and cell-cell interaction in a variety of tissues, and modulation of Rho signaling has been recently shown to modify the differentiation of MSCs in vitro. In this proposal, the PI seeks to study the effect of Rho activation on hESC biology, hypothesizing that Rho mediated signals induce differentiation of a subfraction of adherent hESC in a nodule, thus creating molecular and functional heterogeneity among the cell population. To test this, the PI proposes to add a pharmacologic inhibitor (Y-27632)of Rock (downstream member of the Rho signaling pathway) or siRNA specific for Rho A,B, or C or Rock I, or II, or C3 3xoenzyme, and then study hESC morphology, gene expression and function in vitro and in vivo.
IMPACT AND SIGNIFICANCE: Embryonic stem cells are hetergeneous in their phenotypic and functional properties, and it is important to understand the basis for this heterogeneity. The PI’s major goal of identifying the mechanisms for this molecular heterogeneity in clonally replicating hESCs, if achieved, will provide fundamentally very important insights for this field of research. The work based on the hypothesis that the Rho family of GTPases are central to this problem, and that manipulation of Rho signaling will lead to less heterogeneity is both innovative and potentially significant. If the hypothesis is further validated by the research, then it would provide a ready approach to improving the cloning efficiency of hESC cultures, and/or replacing requirements for cytokines and feeder layers and significant gain will have been made towards using ESCs therapeutically.
QUALITY OF THE RESEARCH PLAN: The research plan is very clearly and logically laid out and overall appears reasonable. The work flow is good, in that it begins from gene expression and moves directly to studies of inhibition of molecular function. Success of the research depends on the design and validation of specific inhibitors, and on the quality of the readout assays.
There is some concern regarding the efficacy and specificity of the pharmacological inhibitor of Rock activity (Y-27632). Two fold higher concentrations of drug are required for hESCs relative to mESCs, and the effect on kinase activity (judged from Fig 2k) appears quite modest. Another inhibitor (H1152) is reported to have less of an effect, however details of these experiments are not provided. Overall, the proposal would have generated more enthusiasm with the reviewers had it focused on more extensively examining the consequences of Rock inhibition using genetic methods (e.g. ko in mouse ESCs, lentivirus siRNAs in mouse and human ESCs, or modulating Rock levels or siRNA levels using regulatable systems). If Rock were validated as a target using these or similar approaches, then more enthusiasm could be mustered for identifying pharmacological inhibitors as described in the second aim.
The section entitled: "hurdles and alternative methods" is superficial. While it contemplates the likely possiblity that introducing siRNAs into hESCs will be difficult because of the inefficiency of transducing hESCs, it cites a paper using ecotropic retroviruses to infect hESCs engineered to express an ecotropic envelope (reference #66) as an alternative approach. This paper is unrelated to what the authors would need to do in delivering siRNAs into hESCs.
Another concern is whether the amount of work justifies the length of time the PI plans to spend on these experiments. The time taken will relate directly to the intent to repeat the experiments in 12 different ESC lines. The merit in investigating the same issue in so many cell lines is not addressed in the proposal.
Overall, it is likely that some data will emerge, although whether it will support the PI's hypothesis in a clear and useful way seems more problematic.
STRENGTHS: The PI has excellent cell biological skills. He has focused on a well-defined goal and has written a logical, concise research plan that has a reasonable probability of answering the questions that he has posed about the mechanism of molecular heterogeneity in replicating stem cells. The problem of heterogeneity within hESC cultures is real and important, and the contribution of cell-cell contact on hESC function is worthy of exploration. Rho GTPases clearly effect stem cell function and the proposal to modulate Rho pathway signaling in ESCs is novel. Emphasis on demonstrating the reversibilty of Rho inhibition is another strength of this proposal.
WEAKNESSES: The effects of Rho blockade are undoubtedly complex, and may be difficult to unravel. This approach would be more convincing if the PI had, at this point, validated ways to specifically, quantitativly control, Rho or Rock isoform levels in hESC (or even in another target cell), and had developed quantitative assays to measure hESC self-renewal (e.g. LDA chimera assays, either in vivo or in vitro). Without such first steps, this project will lack analytical power.
The PI proposes to address enhancing or inhibiting Rho signaling pathway function and to determine the phenotypic consequences and Rho signaling pathway component responsible for the phenotype. Subsequently, the PI will determine the effect of alteration of Rho signaling on hESC heterogeneity and pluripotency. None of the proposed siRNA reagents have been designed, let alone tested or validated. Since these are often not specific, at present the PI has very few reagents in hand.
The in vitro assays of ESC function following alteration of Rho signaling are not very clearly laid out. For example, it is proposed to monitor the effect of Rho inhibition on the ability of ESCs to make embryoid bodies and to form 3 germ layers in vitro. Perhaps a more interesting approach would be to track the frequency of clonogenic cells within the ESC population. For example, about 10% of mouse ES cells are capable of extensive growth in culture using limiting dilution assays. Would Rock inhibition increase the frequency of cells that have clonogenic capacity?
For the preliminary data, it would be helpful if all results were presented quantitatively as well as qualitatively. Statistical measurements are not provided, raising some question of how firmly the effects of Rho inhibition on mESC function are established. The effects of Rho inhibition on reducing heterogeneity versus a potential effect on promoting self-renewal (as suggested by Figure 4) are not very well distinguished. The data purporting to show that Rho blockade increases self-renewal is not quantitated, so it is hard to know how strong it is or is not. What is really needed are quantitative assays of function.
The preliminary data would be dramatically strengthened by a demonstration that pharmacological inhibition of Rho signaling still allows mouse ESC to contribute to chimeras and to the germline. While studies of this type are proposed in the current application, it would be reassuring to know that this is the case before undertaking the extensive exploration proposed here.
The rationale of repeating the same series of experiments in 12 or perhaps more different stem cell lines is not provided in the application. How much new will be learned by repeating the same experiments in this many lines?
The PI is a newly appointed, unfunded (by peer-review) Assistant Professor who plans on recruiting a team of two graduate students, two post-docs, and a research technician. He plans to mentor and direct all five individuals. This large group of junior people may consume far more time that the PI realizes and actually be a detriment to his research progress. The PI should consider staging increases in the experimental group over time rather than starting immediately with 5 junior people.
Parenthetically, the PI states that it will be essential to achieve homogeneity in hESC cultures prior to clinical application, however one reviewer thinks this point is arguable. First, it is unlikely that hESC cultures will ever be completely homogeneous, and second, clinical applications using hematopoietic stem cells have not required the use of homogeneous cell populations
DISCUSSION: The reviewers’ noted that the focus of this proposal, rather than on a new way to stimulate self renewal in hESC, was on reducing heterogeneity in hESC which is an issue and would be desirable to do. The question proposed is could hESC heterogenity be due to cell-cell contact; if so then if cell-cell contact is inhibited, there should be a positive impact on hESC heterogeneity. This is a neat idea and is the positive and laudable aspect of this proposal. Recent published studies in adult stem cells have shown that actin tethering, thus Rho GTPase signaling, impacts some stem cell functions. The PI has shown in unpublished data with murine ESC (muESC), that following addition of a chemical inhibitor of Rho to culture, Lif is not required for 6-7 passages at least, then, when the culture is released from Rho inhibition, the culture still was mostly pluipotent. This contrasts with cultures not treated with inhibitor which in the absence of Lif differentiate. The hypothesis is that the Rho inhibitor maintains the culture in the self renewal state; this is the hypothesis that the PI proposes to test with hESC. The problem is that the PI has not really shown with the muESC work that Rho inhibition results in self renewal as the results are more qualitative than quantitative. There is some concern of a possible artifact. The preliminary data would have been stronger if the PI had already done embryo chimera studies in the mouse.
The application proposes to look at rather narrowly focused aims but with 12 different lines - why so many? It seemed like a lot of grant to support the scope of the work. There was also concern that the PI is a newly independent investigator and may not be ready to run a lab of a size that the project as proposed will entail.