Experiments with human embryonic stem cells (hESCs) have clearly demonstrated their capacity to replicate continuously and maintain pluripotency. We hypothesize that the health of hESCs depends in part upon an increased ability to carefully control the health of their proteome. We have found that hESCs have an incredibly high level of proteasomal activity in comparison to their differentiated counterparts. Notably, hESCs exhibit a proteasome activity that is correlated with increased levels of one proteasome subunit, PSMD11, and increased assembly of the proteasome. FOXO4, an insulin/IGF-1 responsive transcription factor associated with stress resistance in invertebrates, regulates proteasome activity by modulating the expression of PSMD11 in hESCs. FOXO4 is also necessary for hESC differentiation into neuronal lineages. Our results establish a novel regulation of proteostasis in hESCs that links stress response pathways with hESCs function and identity. In this proposal, we take advantage of these findings to promote our understanding of exactly how stem cells ensure a careful regulation of the synthesis, folding, and degradation of their proteome. Moreover, we hypothesize that the activity and expression of the stress response pathways, including FOXO4, may be key determinants in our capacity to reprogram somatic cells. Understanding the mechanisms by which hESCs regulate their proteome will help us in our attempts to optimize and safeguard their use in therapies.
The number of Californians diagnosed with protein misfolding diseases is currently undergoing exponential growth: within the next 20 years, well over a million Californians are expected to be diagnosed with Alzheimer’s, for example. The cost of care and treatment for these individuals reaches into the 100’s of billions of dollars within California alone and could eventually undermine the economic and social stability of the state. Tragically, in such diseases, diagnosis usually occurs after wide spread neuronal death has already occurred. One of the more promising therapeutic options for patients with protein misfolding diseases is stem cell therapy, which hopes to replace lost neurons with ones generated from stem cells. However, we do not yet understand much of the basic biology of how stem cells maintain their health, including how they can maintain a control of the regulation of protein synthesis, folding, and degradation. This research is designed to address a basic and often overlooked question about stem cell health: what machinery does the stem cell employ to guarantee the health of its proteome, and what happens to stem cell pluripotency when this is lost? This research will provide fundamental insights into the mechanisms of protein homeostasis within the stem cell, findings that can be immediately applied by those searching for therapeutic options for these diseases.
This Track 1 (Fundamental Mechanisms) project explores mechanisms that allow stem cells to maintain pluripotency and self-renewal. A component of this maintenance is protection of the cellular proteome from changes that would alter the cell’s capacity to self renew or retain pluripotency. In support of such a mechanism, this group has previously shown that hESCs have higher levels of proteasomal activity than their differentiated counterparts and may be regulated by stress response pathways. The focus of this project is to elucidate further the contribution of stress response pathways in proteostasis of human pluripotent stem cells. Aim 1 of the proposal will assess stress response pathway contribution to proteostasis at a subcellular level. Aim 2 will apply in vivo sensors of stress responses towards the identification of stochastic determinants of hESC health In Aim 3, the PI will investigate the role of stress response pathways on iPSC reprogramming.
Significance and Innovation
- The grant focuses on an important issue which is the stress response pathways operating in human pluripotent stem cells that help retain cells in an undifferentiated state. The concept of enhanced proteostasis controlling stem cell fate of hESC is moderately novel, but highly significant.
- The project includes the generation of hESC lines stably expressing genetic reporters for different proteostasis processes by gene targeting. This is an elegant and innovative approach to follow real-time levels and dynamics of these processes at the single cell level.
- The PI provides strong preliminary data to support that hESCs have elevated levels of proteosomal activity, which is regulated by a transcription factor/proteosome subunit.
- The concept that a specific transcription factor may control stress response is risky to pursue. Although data show it is necessary, ithas not been established that the transcription factor pathway is sufficient to regulate these functions..
Feasibility and Experimental Design RFA
- The proposed approach seems appropriate and feasible for the questions asked, using state-of-the-art genetic and genomic technologies.
- The PI proposes individual knockouts of components of the transcription factor and/or stress response systems using gene targeting technologies and experiments in which the binding pattern changes when the cells differentiate which are seen as important studies in the experimental design.
- The research will be conducted in a well-respected research center that includes all the required facilities for the experiments outlined here.
- There were some concerns that some of the specific aims of the proposed studies lack specific details about how the proposed experiments would be conducted.
Principal Investigator (PI) and Research Team
- The principal investigator has extensive experience and a long track record of high-quality publications in the study of protein homeostasis, the main area of this grant proposal. The collaborating investigators are expertly qualified to fulfill their roles in the project. Levels of effort appear appropriate for successful completion of the project.
- It was pointed out that including additional collaborators in the areas of ESC biology and neurobiology would have strengthened the application.
- A letter of support appears to be absent from one of the key collaborators.
Responsiveness to the RFA
- This project is responsive to the RFA.