Date of Award

2017

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Peter S. Klein

Abstract

Hematopoietic stem cells (HSCs) are able to self-renew and to differentiate into all blood cells. HSCs reside in a low-perfusion niche and depend on local signals to survive and to maintain the capacity for self-renewal. HSCs removed from the niche can survive if they receive hematopoietic cytokines, but they then lose the ability to self-renew. However, we showed previously that simultaneous inhibition of glycogen synthase kinase-3 (GSK-3) and mammalian target of rapamycin complex 1 (mTORC1) maintains HSC function ex vivo without the need for exogenous cytokines. As these experiments were initially done in heterogeneous cell populations, I then showed that purified HSCs can also be maintained under these conditions, demonstrating a direct effect of GSK-3 and mTORC1 inhibition on HSCs. Although Wnt/β-catenin signaling downstream of GSK-3 is required for this response, the downstream effectors of this network remained otherwise undefined. I therefore explored targets downstream of GSK-3 and mTORC1. I found that HSCs express a pro-autophagic gene signature and accumulate LC3 puncta only when both mTORC1 and GSK-3 are inhibited, identifying autophagy as a signature for a signaling network that maintains HSCs ex vivo. In contrast, I did not find evidence to support a role for other downstream targets of mTORC1, such as protein translation and mitochondrial biogenesis. I also report a significant reduction in total RNA content in cultured HSCs and describe a method to perform transcriptional profiling of these cells. Together, these findings provide new insight into the relative contributions of various mTORC1 outputs toward the maintenance of HSC function and build upon the growing body of literature implicating autophagy and tightly controlled protein synthesis as important modulators of diverse stem cell populations.

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