SYNOPSIS: Dr. Hong Wu is Professor of Molecular and Medical Pharmacology at UCLA. He proposes to study the role of PTEN/PI3K in regulating self-renewal and the neurogenic potential of hESC-derived cells, as well as 10 other molecular pathways. They will identify factors that promote survival and integration of transplanted NSCs in post-stroke brain and in atrophic neural circuits. Dr. Wu had earlier found that PTEN/PI3K plays an important role in the ability of hNSC to integrate into neural circuits. Transient inactivation of the PTEN/PI3K pathway boosts stem cell populations and promotes their engraftment potential.
IMPACT AND SIGNIFICANCE: This is a very important study that is original and potentially highly translational. The finding that the PTEN/PI3K pathway regulates the balance between self-renewal and the neurogenic potential of NSC cells needs to be investigated in hESC. Understanding the signaling factors that control self-renewal of NSCs is an important research goal, and PTEN plays a significant role in this process as demonstrated by PI’s group more than 6 years ago in mice. The investigators plan here to test PTEN knockdown and other factors in self renewal effects, as well as in an in vivo brain injury model. These studies will likely confirm/extend the earlier findings in mouse to a human neural stem cell system and provide fundamental insights into PTEN and additional pathways as related to neural stem cell renewal. While there was only limited discussion on why improving NSC self-renewal is critical for the application of neural stem cells, understanding the basic mechanisms of stem cell self-renewal is of obvious importance. That being said, the impact of such findings for practical use in hESC or NSC biology is less clear. NSC self-renewal is not considered a main limiting factor in hESC or hNSC technology or its applications. The use of PTEN null cell lines also will cause safety concerns. Those limitations aside, the applicant is a remarkably well-supported investigator with several NIH grants and many collaborations, and there are a number of interesting and innovative approaches in this proposal that will likely yield relevant data for the field.
QUALITY OF THE RESEARCH PLAN: The application is well-written, well-structured and logically designed, and it follows up on a 2001 study by the investigators published in Science showing that the PTEN mouse knockout shows increased brain neuron proliferation. More recent preliminary data suggests that PTEN favors olfactory neuron recovery after ablation as well as engraftment on transplantation. In addition to investigating the PTEN/PI3K pathway, the PI plans to investigate 10 other pathways. The PI also plans to assess the cells and their ability to integrate into the brains of post-stroke mice and atrophic circuits.
The main concern in the research plan is the lack of detail on the description of the hESC-derived hNSC to be used in this study. The PI describes variable results in preliminary data and it is unclear which system is going to be used in the current study. In addition, with respect to each aim of the research plan, there is often limited detail regarding precisely what the applicants will do. In the first aim the applicants will use RNAi or small molecule inhibitors in hESCs but they provide little detail or explanation about precisely what they will do with these cells. The second aim is to test several other pathways, chosen fairly arbitrarily, for interaction and "function" in hESCs, but again what they actually plan to do is somewhat murky. The third aim is to transplant knockdown hESCs into mouse brains with experimental strokes. Here the rationale for using a xenograft model is unclear. Mouse ES cells without the attendant immunological issues or compatibility issues might be better suited for this work. The fourth aim is to test the transplanted cells in their integration into neural circuits in the olfactory bulb. Except for the xenograft issue, this is a more solid aim given the PI’s track record in this area.
STRENGHTS: The applicant is a productive and accomplished scientist who worked in the prostate cancer field but has branched out into the neuroscience and now human embryonic stem cell fields. This excellent investigator has expertise in NSC self-renewal and PTEN signaling. Trained initially with an MD from Beijing University, the PI obtained his PhD from Harvard (1984-1991), did postdoctoral training at MIT (1991-96), and joined UCLA where he is now Professor. He holds multiple NIH grants. The discovery that the PTEN/PI3K pathway plays a role in neurogenesis is interesting and very important, and although the mouse data on PTEN and neural self-renewal are now dated, the work needs to be taken to human cells. The experiments are feasible, and the applicant has carried out similar experiments before with other cells. A number of innovative approaches are proposed including the genetic/toxic elimination of the grafted NSCs using diphtheria toxin, and the use of a mutant mouse model (DNMT-KO) that seems to have a selective adult, SVZ-specific effect in adult neurogenesis.
WEAKNESSES: While two reviewers feel that this is a well-written proposal, one reviewer feels that the grant is disorganized in that preliminary data is included in the Specific Aims and seeded into the Research Design and Methods, thus exceeding the page limit for that section and compromising an already page-limited research strategy section. Overall, reviewers agreed that the key limitation of this study is its relative lack of mechanistic depth, which may ultimately limit the work's utility. There is no acknowledgment of the complexities of NSC biology with regard to developmental stage, regional identity, and growth conditions. In addition, there is a lack of detail for Aim 3. It is unclear which cells are going to be used, and what the hypothesis and assays are regarding the possible mechanism of repair. The use of the xenograft model is also confusing. The proposal is overambitious with regard to number of pathways to pursue. A large number of additional candidate pathways already have been identified, but the applicant proposes to yet again screen for additional pathways instead of focusing the efforts on a selected set of candidates.
DISCUSSION: This proposal represents an approach to boosting neurogenic potential by studying “your-favorite-gene”, in this case, PTEN. The applicants propose to look at transient inhibition of PTEN or PI3K function of hESC-derived NSC in vitro and in vivo; to assess other key factors/pathways for importance in NSC function; and to assess how well hESC-derived NSC (treated or not) integrate and function into model. Overall reviewers are enthusiastic about the proposal, the expertise of the team, and the primary data generated over the years. In previous work in mouse, the PI found that PTEN/PI3K plays important role in neurogenesis. An innovative aspect of this work is the mouse model (GFAP-Cre-DNMT-CKO) that is submitted but not published. Another innovative aspect is the use of the diptheria toxin approach to eliminating introduced stem cell derived population.
The key weakness of the proposal is that the practical advantages for using hESC from a therapeutic standpoint are not well justified. In the models of stroke and repair there are complexities regarding regional issues in cell fate that are not discussed in the application. The PI does not discuss how the cells would integrate, and there is a limited discussion of the potential outcomes. Another factor not considered by the investigators is that the conditional DNA methytransferase knockout model may have a dual/competing effect on tumorigenesis, which is promoted in early stages and inhibited in later stages. This is a complicating factor not considered by the PI.
That the PI proposes to study additional pathways beyond the 6-7 that are already available was considered another weakness. One reviewer liked the idea of doing specific knock down in the PTEN pathway for self renewal, but commented that this was not a very systematic pathway-oriented approach to studying PTEN. Quite a bit is known about this pathway, but the PI has chosen to provide a somewhat arbitrary mechanistic description in looking at gene expression. Despite the PI’s original observation published in 2001 in Science, the PI has contributed only incrementally to the story with a 2006 PNAS paper. In contrast, a paper published in Nature showed that mTOR signaling could be used in distinguishing self-renewal capacity in stem cells, but PI did not acknowledge this work.
Reviewers suggest that, should future funds become available, the PI should give a better definition of the hNSC to be used for this study (e.g., developmental stage, growth conditions, regional identity markers). Aim 3 also requires better definition, including a clear description on the nature of the hNSC to be used as well as a discussion of the expected results. Reviewers suggest omitting the screen for additional pathways at the current stage and focusing on the key candidate pathways already identified.