The therapeutic potential of human embryonic stem cells is extraordinary. Without a doubt, regenerative medicines will save thousands of lives in the years to come. Before that day arrives, much needs to be learned from the cells themselves. The reasons that these cells hold so much promise are two-fold: (1) embryonic stem cells can renew themselves indefinitely (divide and divide and…) and (2) embryonic stem cells can be trained to become any cell type of the body (neurons, heart muscle, skin, liver, kidney…). However, it should be emphasized that these two points are only valid if the growth conditions are properly established. While we have made great strides in developing culture conditions that can support self-renewal of embryonic stem cells, we are a long way from mastering the conditions necessary for differentiating embryonic stem cells into every cell type of the body (of which there are about 200). Ultimately, if therapies based on stem cells are to be realized, these cells will have to be grown in massive quantities, with an unprecedented level of quality control to ensure that only one cell type can be found in the lot. Furthermore, the fate of stem cells is crucial to their use in new therapies—in other words, these cells must be kept alive and functional to have benefit to human patients.
However, one of the major challenges facing the growth of embryonic stem cells is the abundance of cell death that occurs. Cells typically die when their needs are not met (either lack of proper nutrients or growth factors) or when they face harsh conditions. If we could somehow block the cell death that occurs in these cultures or if we could change the conditions to remove the components that trigger cell death, we could achieve growth of hESCs of a greater scale. It turns out that when cells die, they do not do so passively. Instead, once given a “go” signal, cells utilize their own energy and cellular machinery to dismantle themselves, a process known as programmed cell death. There are at least five major forms of programmed cell death: apoptosis (the best described pathway), autophagic cell death, PARP-mediated cell death, paraptosis, and calcium-mediated programmed cell death. Each of these programmed cell death pathways are activated by different stresses. In the proposed research, we aim to determine which of the five major forms of programmed cell death occur in hESCs. Furthermore, we will evaluate how the repertoire of PCD pathways changes when hESCs change, or differentiate, into neurons. At the same time that we will be learning about the most appropriate conditions for growing hESCs, we will also be able to determine which conditions are ideal for cultivating neurons, which could ultimately be used in regenerative medicine therapies.
In passing Proposition 71, Californians have ushered in a new era of human embryonic stem cell research. However, before the therapeutic potential of human embryonic stem cells can be realized, several key issues relevant to programmed stem cell death must be addressed: (1) we must understand what insults trigger PCD in stem cells; (2) we must understand what programs of cell death are available to stem cells and stem cell-derived differentiated cells; (3) we must understand how to block cell death induction in hESCs and their derivatives; (4) we must address the technical hurdles of propagating these cells at an industrial scale. The goal of our research is to determine what measures might be taken to permit such a wide-scale expansion effort. Our laboratory has constructed a mechanistic taxonomy of cell death programs, and therefore has a unique ability to identify various novel forms of cell death that may occur in hESCs. Because improved methods of human embryonic stem cell propagation will stimulate research on human embryonic stem cells, the benefit of our research to Californians will be seen repeatedly and, ultimately, in the delivery of human embryonic stem cell-based therapies
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.