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RC1-00139-1: Investigation of the mechanism that initiates the molecular heterogeneity in human embryonic stem cells

Recommendation: Not recommended for funding 

Public Abstract (provided by applicant)

Human embryonic stem cells (hESCs) can give rise to virtually all types of specialized adult cells in human body. Thus they are thought to be the potential source of the cell-transplantation therapy for the treatment of diseases such as Parkinson’s disease, myocardial infarction, and diabetes mellitus. While hESC research has made a considerable progress in developing methods to generate specialized cell types including neurons, cardiomyocytes, and insulin-secreting cells, studies to identify the key mechanisms that allow hESCs to maintain such an enormous differentiation capacity (pluripotency) have just begun. Because obtaining the pure undifferentiated cell population establishes the basis of hESCs-based therapeutic approach, these studies are essential to find the way to grow hESCs without losing their pluripotency. Moreover, as the recent findings have shown that hESCs grown under the current culture condition are not homogenous, but rather mosaic of different populations which may contain cells that have less differentiation capacity, it is critical to understand how this heterogeneity has occurred during the propagation process of hESCs.

The goal of the proposed study is to determine the core mechanism that generates mixed populations in hESCs which prevents them from keeping their differentiation capacity throughout the propagation process. Our preliminary data have indicated that a specific signaling pathway (like a hormone that mediates various biological information) may be responsible for making inhomogeneous populations in hESCs. Based on this finding, we will focus on investigating the role of the signaling pathway in hESCs through a series of molecular experiments, and developing the novel technology which enables us to grow hESCs as a pure population of undifferentiated cells that would further standardize the hESCs-based therapeutic strategy.

Statement of Benefit to California (provided by applicant)

Our research will focus on identifying the mechanism that regulates the multiple differentiation capacity (pluripotency) in hESCs. With the knowledge obtained through the project, we will concentrate on developing a novel technology that will allow us to culture uniformly pluripotent hESCs which is not possible under the current culture protocols. The establishment of such a new method would impact virtually all hESCs-based application programs as it involves a common basic process to expand hESCs before turning into any type of adult cells for the therapeutic purposes. It is therefore predictable that the new methodology will be promptly translated as an intellectual property to be commercialized, and would substantially activate the biotechnology field in the State of California. More importantly, the new methodology will be provided to the Institutes in California at the highest priority where the method will accelerate the process to apply the hESCs-based transplantation approach for the clinical settings that would further contribute to the enhancement of the medical environment for California citizens.

Review

SYNOPSIS: In this proposal, the Principal Investigator (PI) seeks to study the effect of activation of a signaling pathway on human embryonic stem cell (hESC) biology, hypothesizing that these signals induce differentiation of a sub-fraction of adherent hESCs in a nodule, thus creating molecular and functional heterogeneity among the hESC population. To test this, the PI proposes to add a pharmacologic inhibiter of the signaling pathway, or siRNA specific for different components of the signaling pathway, and then study hESC morphology, gene expression and function in vitro and in vivo.

IMPACT AND SIGNIFICANCE: Embryonic stem cells are heterogeneous 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 a specific signaling pathway is central to this problem and that manipulation of this signaling pathway 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 to replacing requirements for cytokines and feeder layers and significant gain will have been made towards using hESCs 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 the signaling pathway, based on the preliminary data provided. Overall, the proposal would have generated more enthusiasm with the reviewers had it focused more extensively on examining the consequences of pathway inhibition using genetic methods. If the signalling pathway component 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 possibility that introducing siRNAs into hESCs will be difficult because of the inefficiency of transducing hESCs, it cites as an alternative approach a reference that 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 hESC 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. The PI has focused on a well-defined goal and has written a logical, concise research plan that has a reasonable probability of answering the questions 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 the proposed mechanism in generating heterogenity on hESC function is worthy of exploration. The specific signaling pathway clearly affects stem cell function and the proposal to modulate it in ESCs is novel. Emphasis on demonstrating the reversibility of pathway inhibition is another strength of this proposal.

WEAKNESSES: The effects of blockade of this signaling pathway 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, quantitatively control, pathway component 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 signaling pathway function and to determine the phenotypic consequences and signaling pathway component responsible for the phenotype. Subsequently, the PI will determine the effect of alteration of pathway 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. Also, the in vitro assays of ESC function following alteration of pathway signaling are not very clearly laid out.

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 pathway inhibition on murine ESC function are established. The effects of pathway inhibition on reducing heterogeneity versus a potential effect on promoting self-renewal are not very well distinguished. The data purporting to show that pathway blockade increases self-renewal is not quantified, 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 pathway signaling still allows murine ESC to contribute to chimeras and to the germline. 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 independent investigator who plans on recruiting a team to conduct the research. The PI plans to mentor and direct this new team. This large group of junior people may consume far more time that the PI realizes and actually be a detriment to research progress. The PI should consider staging increases in the experimental group over time rather than starting immediately with so many 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 the action of a specific signaling pathway; if so, then if that pathway 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. There is some recent published data and some preliminary data that is supportive. The hypothesis is that an inhibitor of this pathway 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 murine ESC work that pathway inhibition results in self renewal as the results are more qualitative than quantitative and 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 hESC lines – why so many? It seemed like a lot of grant application 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.

The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:

• Brivanlou, Ali
• Lansing, Sherry