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Defining the Isoform-Specific Effects of Apolipoprotein E on the Development of iPS Cells into Functional Neurons in Vitro and in Vivo

Funding Type: 
New Faculty II
Grant Number: 
Principle Investigator: 
Funds requested: 
$2 847 600
Funding Recommendations: 
Recommended if funds allow
Grant approved: 
Public Abstract: 
Statement of Benefit to California: 
Review Summary: 
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.

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