ABILITY OF HUMAN ES CELL-DERIVED NEURAL PRECURSORS TO CONTRIBUTE TO AND REPAIR DAMAGED NEURAL CIRCUITS
This application seeks to improve our ability to use neural stem cells for brain and spinal cord repair. Human embryonic stem cells (hES) have the ability to produce many kinds of cells, including neural stem cells —cells that form the components of the brain and spinal cord. A fundamental problem is how hES cells maintain the ability to self-renew—to make more copies of themselves—while still retaining the capacity to become other cell types. Analogous to hES, an important question in utilizing hES-derived NSC is to determine how to maintain them in a state in which they can produce more stem cells while retaining the capacity to become (or differentiate to) the cell types needed for neural repair. For example, in stroke, one might want to use stem cells to replace lost neurons. However, for reasons that are unclear, neural stem cells transplanted into the brain often lose their ability to produce neurons. If we have a means to enhance this ability, we could ultimately benefit stroke patients. In our previous work, we have shown that mouse embryonic stem cells missing the gene, PTEN, have a much greater capacity to produce additional embryonic stem cells. Additionally, we have shown that mouse neural stem cells missing the PTEN gene also retain a greater self-renewal capacity as well as the ability to produce neurons than do normal neural stem cells, both in tissue culture as well as in the living animal. The protein that is made by the gene, PTEN, acts by inhibiting a molecular pathway, the PI3K/Akt pathway. In this proposal, we will discover whether this pathway is important for the self-renewal of hES as well as neural stem cells derived from hES. It is our belief that inactivating this pathway will inhibit the production of hES cells and neural stem cells, and that activating this pathway will enhance this production. We will then test whether promoting the pathway in hES-derived neural stem cells has meaningful consequences when we transplant the cells into the living (in vivo) mouse brain. First, we will test whether PTEN-deficient neural stem cells are better at replacing neurons than are normal neural stem cells following transplantation into the brains of mice that do not have the capacity to produce new neurons on their own. Next, we will determine if loss of PTEN function also enhances the ability of hES-derived neural stem cells to repair the brain following a stroke. In addition to the studies that specifically assess the PTEN/PI3K/Akt pathway, we will discover other genes and pathways that regulate the process of hES-derived neural stem cell self-renewal. High priority candidates from these studies will then be tested in our in vivo models. We believe the studies proposed here will have important implications on the ways that we use both transplanted as well as endogenous (the patients’ own) human neural stem cells in stroke and other disorders where replacement of brain or spinal cord cells is a therapeutic option.
Statement of Benefit to California:
Neurological disorders are the leading source of disability, and are not only devastating for individuals, but are an enormous social and financial burden that costs the State of California many billions of dollars per year. Currently there are no treatments that promote repair and recovery in the brain after any major neurological disorder, including stroke, degenerative disease and trauma. Research with stem cells derived from animals indicates that transplantation of stem cells can promote recovery of function in the brain after stroke and models of degenerative disease in experimental animals. This research suggests that human embryonic stem cell therapy may provide a way to promote repair and recovery in patients with brain disorders. Nevertheless, although the animal results look promising thus far, these animal stem cell studies have identified limitations in the procedure because most of the transplanted cells do not survive and integrate into the brain, and there is little understanding of the fate of the transplanted cells and the mechanisms that control this fate. The goals of this grant are: 1) to determine the precise molecular pathways that identify human embryonic stem cells at different stages of differentiation as they turn into neurons, or as they divide in a process of self-renewal; and 2) to identify the molecules that promote human embryonic stem cell survival, differentiation into different cell types including specific types of neurons, and integration into brain circuits to promote functional recovery after neurological conditions such as stroke or degenerative disease. The proposed studies will build on and draw from our work in mouse embryonic stems in which we have identified a key molecular pathway that promotes embryonic stem cell survival, differentiation and repair of the brain after stroke and other forms of cell death. This pathway is known as the “PI3K/Akt pathway”. The results from the studies proposed in this grant will identify molecules that promote human embryonic stem cell growth and survival and that may lead to repair of the human brain after various neurological disorders. If successful, the State of California and its citizens will benefit not only from the improvement of individual health and lifestyle afforded by the development of treatments for currently untreatable neurological conditions, but will benefit also financially as a whole from the economic impact of reduced costs for caring for disabled individuals, as well as from the development of novel medical technologies that will place California at the forefront of a new medical field with a global market.
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.