GOALS We propose to determine the effects of different forms of apoE on the development of induced pluripotent stem (iPS) cells into functional neurons. In Aim 1, iPS cells will be generated from skin cells of adult knock-in (KI) mice expressing different forms of human apoE and in humans with different apoE genotypes. In Aim 2, the development of the iPS cells into functional neurons in culture and in mouse brains will be compared. In Aim 3, the effects of different forms of apoE on the functional recovery of mice with acute brain injury treated with iPS cell–derived neural stem cells (NSCs) will be assessed. RATIONALE AND SIGNIFICANCE The central nervous system (CNS) has limited ability to regenerate and recover after injury. For this reason, recovery from acute and chronic neurological diseases, such as stroke and Alzheimer’s disease (AD), is often incomplete and disability results. Embryonic stem cells have great promise for treating or curing neurological diseases, but their therapeutic use is limited by ethical concerns and by rejection reactions after allogenic transplantation. The generation of iPS cells from somatic cells offers a way to potentially circumvent the ethical issues and to generate patient- and disease-specific stem cells for future therapy. In the CNS, apoE plays important roles in lipid homeostasis and in neuronal maintenance. However, apoE2, apoE3, and apoE4 differ in their ability to accomplish these tasks. ApoE4, the major genetic risk factor for AD, is associated with poor clinical outcome and more rapid progression or greater severity of head trauma, stroke, Parkinson’s disease, multiple sclerosis, and amyotrophic lateral sclerosis—all potential targets of stem cell therapy. This proposal builds on three novel findings in human apoE-KI mice. (1) NSCs express apoE. (2) ApoE plays a role in cell-fate determination (neuron vs astrocyte) of NSCs. (3) ApoE4 impairs the neuronal development of NSCs. Thus, we hypothesize that transplantation of iPS cells derived from apoE4 carriers (~20% of the general population and ~50% of AD patients) might not be beneficial or even detrimental for patients with neurological diseases. We propose in vitro and in vivo studies to assess the effects of different forms of apoE on the development of iPS cells into functional neurons and on the functional recovery of mice with acute brain injury treated with iPS cell-derived NSCs. These studies will shed light on the regulation of neuronal development of iPS cells and help to “optimize” future iPS cell therapy for neurological diseases. SPECIFIC AIMS Aim 1. To establish adult mouse and human iPS cell lines with different apoE genotypes. Aim 2. To determine the isoform-specific effects of apoE on the development of iPS cells into functional neurons in culture and in mouse brains. Aim 3. To assess the isoform-specific effects of apoE on the functional recovery of mice with acute (stroke) brain injury treated with iPS cell-derived NSCs.
CONTRIBUTION TO THE CALFORNIA ECONOMY: A major goal of regenerative medicine is to repair damaged cells or tissue. My research focuses on (1) understanding the role of neuronal regeneration in central nervous system function and (2) developing stem cell therapy for acute and chronic neurological diseases, including stroke and Alzheimer's disease. Stroke and Alzheimer's disease are the leading causes of disability and dementia and are the fastest growing form of neurological diseases in California, in the USA, and worldwide. My research could benefit the California economy by creating jobs in the biomedical sector. Ultimately, this study could help reduce the adverse impact of neurological diseases. Thereby, I hope to increase the productivity and enhance the quality of life for Californians. The results of my studies will also help develop new technology that could contribute to the California biotechnology industry. The studies will characterize multiple lines of induced pluripotent stem (iPS) cells carrying apoE3, a protein protective to the brain, or apoE4, which is detrimental to the brain and is associated with increased risk of Alzheimer’s disease and other neurodegenerative disorders. These cell lines could be valuable for biotechnology companies and researchers who are screening for drug compounds targeting different neurological diseases. CONTRIBUTION TO THE HEALTH OF CALFORNIANS: The most important contribution of the studies will be to improve the health of Californians. Diseases that are the target of regenerative medicine, such as stroke and Alzheimer’s disease, are major causes of mortality and morbidity, resulting in billions of dollars in healthcare costs and lost productivity. As we continue our efforts in medical research, we hope to one day unlock the secrets of brain development and repair. This knowledge will help medical researchers develop beneficial therapies beyond what is currently available and potentially improve the quality of life and life expectancy of patients with neurological diseases, such as stroke and Alzheimer’s disease.
The goal of this project is to determine the effects that different isoforms of apolipoprotein E (apoE) may have on the differentiation of induced pluripotent stem (iPS) cells into functional neurons. In Aim 1, the applicant proposes to establish adult mouse and human iPS cell lines with different apoE genotypes. In Aim 2, the applicant plans to determine the isoform-specific effects of apoE on the development of iPS cells into functional neurons in culture and in mouse brains. In Aim 3, the applicant proposes to assess the isoform-specific effects of apoE on the functional recovery on mice with acute (stroke) brain injury treated with iPS cell-derived neural stem cells (NSCs).
This is an application from a highly experienced researcher with considerable expertise in the field of apolipoprotein biology. Reviewers found this proposal clear and well written, and reviewers felt that the proposed experiments generally are quite feasible. However, reviewer enthusiasm was diminished by the lack of some important details and inadequate consideration of some potential obstacles.
The principal investigator (PI) presents a set of interesting preliminary findings including the observation that NSCs express ApoE and that ApoE plays a role in the fate choice governing glial vs neuronal differentiation. The applicant also presents some preliminary evidence that ApoE4 specifically impairs the neuronal development of NSCs. Furthermore; the use of iPSC technology in this application is of potential interest. However, the applicant does not make a strong case for the advantages of using iPSCs versus simply NSCs derived from the transgenic mice, or standard human embryonic stem cells (hESCs) that may already represent a number of different ApoE genotypes. One concern expressed by the review panel was the extent to which the iPS cells would function as embryonic stem cells (ESCs) and the extent to which these cells can be differentiated routinely and reproducibly into neural stem cells (NSCs) in the numbers and specificity required for the project. If the iPS cells do not function as anticipated, a clear alternative approach was not presented. An additional concern was the extent of retroviral manipulation necessary to generate iPS cells, and the extent to which this might interfere with or adversely influence normal ApoE function. Clearly, changes in the technology of generating iPS cells will impact this concern over time.
In terms of neural differentiation, the applicants only cite a single differentiation protocol and, to some reviewers, it was unclear if the applicant has evaluated different techniques using human embryonic stem cells to generate neuronal lineages in culture. Secondly, reviewers had some concerns about the in vivo electrophysiology studies, which represent an important in vivo analysis of the grafted cells in the last aim. The applicant has proposed a collaborator for this expertise, but the corresponding letter of collaboration is fairly generic and did not transmit enthusiasm for committing to the work. It was unclear to the review panel whether the local physiologists had sufficient experience and expertise to make and manipulate quality tissue preparations from adult mice. Lastly, the applicant will be assessing recovery in mice with acute (stroke) brain injury grafted with iPS cell-derived NSCs. However, the applicant did not discuss the many challenges and pitfalls in such studies, a discussion which would have increased enthusiasm for the proposal further. The reviewers also missed a clear exposition of the applicant’s thoughts on which cells were hypothetically responsible for mediating improvement in this model and how to reconcile the mechanistic considerations with ApoE genotype. Furthermore the PI does not discuss the various regional subytpes of neuron that are being produced using iPSC based neural induction protocols.
The PI is a physician scientist who has been at the host institution since 1995 and has been leading an independent lab there since 2004 at the time of promotion to Assistant Investigator. The applicant has published extensively in the field of apolipoprotein biology in a number of high impact journals. However, reviewers were concerned about the applicant’s lack of experience or expertise with stem cells, particularly given the highly technical nature of the proposed research. Although two senior researchers were proposed to act as mentors in stem cell biology, these scientists lack expertise in ESC differentiation and neural induction, which is critical to the present application. Furthermore the mentoring plan was found to be very minimal and some concern was expressed by reviewers as both mentors are very senior members of the scientific community, not full time at the host institution and therefore unlikely to be deeply involved in mentoring the PI.
In terms of institutional commitment, reviewers agreed that institutional support is very impressive and includes space, facilities and intellectual strengths.
A motion was made to recommend that this application be moved to Tier 2 - Recommended if funds are available. Reviewers felt that the research plan was not considered particularly innovative and contained too many potential pitfalls when compared with other Tier 1 applications. The motion to move this application to Tier 2 carried.