Statement of Benefit to California:
SYNOPSIS: The goal of this proposal is to analyze the pathways of programmed cell death (PCD) that are active in human ES (hES) cells and their differentiated progeny. The PI's laboratory has established a set of five categories of PCD. Cell death-inducing stimuli will be used to establish which pathway of PCD is activated in undifferentiated hES cells, embryoid bodies, and derivative neural progeny. The hypothesis is that the type of PCD stimulated will change as a function of differentiation status. Moreover, one may expect that the quantitative aspects (i.e., sensitivity to amount of PCD-inducing stimulus) may also vary. The aims are (1) to test the sensitivity of undifferentiated stem cells to pro-death stimuli (intrinsic and extrinsic inducers of apoptosis, inducers of autophagy, PARP-mediated death, and death in response to the DNA damaging agent MNNG) (2) similarly test embryoid bodies for their cell death patterns in response to the various pro-death stimuli and (3) test neuronal stem cells derived from BG01 for sensitivity to the same repertoire of pro-death agents. Further choices of stimuli will be guided by large-scale (5000 proteins) Western blots that have already been performed during PCD. Cell death will generally be measured by using GFP/Annexin staining and flow cytometry. The proportions of viable, dying or dead hES cells will be measured by co-staining for SSEA4 expression. In the first Aim, these studies will be performed using 2 hES cell lines maintained as undifferentiated cells either with or without feeder cells. After stimulation the type of PCD will be established. SIGNIFICANCE AND INNOVATION: This is a well-written proposal focused on characterizing the major forms of cell death in hES cells as a function of differentiation. The proposed studies are highly significant in that they will provide important information to the hES cell field. When cultured, a significant proportion of hES cells die. This may limit the ability to expand these cells for large scale applications. Little is known regarding cell death-inducing mechanisms that are active or can be activated in hES cells. The PI cites 7 publications in total that have generally looked at the apoptotic pathway. For these studies, the PI describes 5 different pathways that can be utilized to achieve programmed cell death (PCD). The intention is to systematically analyze which of these is activated as a function of distinct sets of stimuli, which is well-described in the application. The 3 Aims in the proposal will analyze PCD in undifferentiated hES cells, embryoid bodies (focusing on TNF family members), and neural progenitors/stem cells. It is expected that a catalog of different pathways may be obtained that are preferentially activated (both qualitatively and quantitatively) depending on the differentiation status of the cells. The project represents a combined effort of a laboratory that specializes in PCD, and a laboratory that is focused on hES cells. Although the proposed experiments are not particularly innovative from a technical point of view, they are very important and appropriate to the questions addressed. The PI correctly states that this is a largely unexplored though very important area. Success in these studies should provide valuable information that will lead to improved culture conditions, as well as a wealth of basic information. STRENGTHS: This applicant proposes a study that should have been done long ago, but has not been systematically performed in the understandable haste to jump to translational studies. There are a number of strengths to this proposal, particularly the investigator’s clear vision of the study and his expertise. The PI has been instrumental in studying the subtleties of cell death pathways and this expertise is exactly what is needed to get a handle on hES cell apoptosis and non-apoptotic death patterns in vitro. In the first Aim the PI describes in adequate detail the types of inducer stimuli assay conditions, and importantly the experimental criteria that will be used to monitor PCD and identify which type of PCD is activated. The criteria to define which type of PCD is activated are fairly well-described. These include the use of inhibitors that are specific to distinct PCD pathways. All of these studies are feasible and will certainly lead to major insights. They appear relatively straightforward, and this is a major strength of the proposal. In the second Aim, cell death inducing stimuli will be applied to embryoid bodies. Particular emphasis will be placed on members of the TNF family. Preliminary microarray data suggest dramatic up-regulation of TNFR gene-products following hES cell differentiation. The Third Aim will use stimuli identified in Aim 1 to induce PCD in hES cell derived neural progenitors. The studies in the second and third Aims are well-described, and should produce valuable data. The PI presents a useful discussion of potential complications and possible solutions, which is also a strength of the proposal. An additional strength is the collaboration between 2 laboratories that bring complimentary sets of expertise to the overall project. WEAKNESSES: There are no major weaknesses and little to criticize in this proposal. However, apoptosis (and other cell death pathways) may be happening for good reasons, and the goal of completely blocking apoptosis as a way to improve scale-up of hES cells may have unintended downsides, which should be acknowledged. That said, several potentially complicating issues have been addressed by the PI. In particular, the ability to rigorously identify which pathway(s) of PCD are actually activated may be problematic. This would be a significant problem if more than one pathway is simultaneously activated. It could also be the case that weak activation of one pathway together with strong activation of another may not be sufficiently discriminatory; that is, there may be significant quantitative aspects that will obscure the resolution of the analysis. An additional complication may be that embryoid body cell type heterogeneity may not yield a high enough resolution to specifically identify one pathway over another. While these are significant complications, there is sufficient reason to believe that the joint expertise of the 2 laboratories will rise to any challenges as they occur. Moreover, simply identifying which PCD pathway exist and can be activated in hES cell will be a valuable contribution. DISCUSSION: This proposal seeks to test undifferentiated hESCs for their responses to stimuli so that a toolkit of "pro-death" reagents can be elucidated. Analysis of distinct death pathways triggered by the reagents will be done, first in hESCs and subsequently in embryoid bodies and neuronal cells. The PI is a clear thinker about multiple kinds of apoptosis and the discoverer of PARP-mediated cell death. There is a "between the lines" goal, which is to be able to block apoptosis in hopes of enabling more efficient expansion of given cell population. One reviewer noted that the applicant never acknowledged that apoptosis may be an essential process, and that inhibiting it for purposes of scaling up cells may be a bit naive.