Year 2

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.