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.
Our project strives to understanding how changes in the health of the stem cell proteome, or its proteostasis, affect the physiology, mortality, and pluripotency of these unique cells. Previously, we discovered that stem cells exhibited a heightened state of proteostasis, as seen by dramatic increases in their capacity to degrade unwanted proteins. Human embryonic stem cells (hESCs) thus displayed a remarkable capacity to maintain their proteomes that was rapidly lost during differentiation. Subsequently, through the support of CIRM, we have embarked on an ambitious undertaking to understand how other subcellular pathways responsible for maintaining proteostasis play a role in stem cell health.
This year we are pleased to report progress on multiple fronts toward this end. We have found evidence for an important role of subcellular organelles in maintaining stem cell health. We have found a heightened sensitivity of these cells to specific stressors. We have made considerable progress in the establishment of endogenous fluorescent reporter lines such that we can monitor stress responses in hESCs in vivo. We have tested the effect of activation of a wide variety of stress response pathways on induced pluripotent stem cell (iPSC) reprogramming efficiency. Collectively, these results promise exciting, mechanistic gains toward an understanding of basic stem cell biology.
We began this line of research with a simple hypothesis: that because stem cells are subject to a pressure to constantly renew and remain pluripotent, they may maintain a heightened capacity to respond to and protect themselves from both intrinsic and environmental stressors in comparison to differentiated cells. Previously, we discovered that stem cells exhibited a heightened state of proteostasis, as seen by dramatic increases in their capacity to degrade unwanted proteins. Human embryonic stem cells (hESCs) thus displayed a remarkable capacity to maintain their proteomes that was rapidly lost during differentiation. Over the past two years, we have interrogated this hypothesis through the systematic dissection of both basal and stimulated individual stress response pathways, also known as unfolded protein responses (UPRs), within each of the individual subcellular compartments. To date, we have focused our efforts with an examination of two of the more distinctive and well-characterized stress responses within the mitochondria and ER.
This year we are pleased to report forward progress on many of the aims of our proposal. We find evidence for an important role of subcellular organelles in maintaining stem cell health, and have undertaken several types of genomic analyses to better understand the effect of dysfunctional and/or heightened UPR function on stem cell health. We have found a heightened sensitivity in hESCs to specific stressors, including an inability for hESCs to mount a heightened defense against ER stress. We have completed the construction of multiple fluorescent reporter lines such that we can monitor stress responses in hESCs in vivo, and have begun characterizing all of these in vivo. Finally, we have tested both loss-in-function and gain-in-function roles for the UPR in reprogramming. We find specific requirements of ER UPR transcription factors on the reprogramming efficiency of fibroblasts. Collectively, these results continue to promise exciting, mechanistic gains toward an understanding of basic stem cell biology.