Human embryonic stem (hES) cells have received considerable attention with regards to their regenerative capacity. However, basic biological pathways including apoptosis in hES cells remain largely unexplored. We have started to investigate the apoptotic pathway in hES cells and have uncovered novel and fascinating mechanisms by which hES cells regulate cell death. We found that hES cells are highly sensitive to DNA damage, with all cells dying by 6 hours. A critical mediator of apoptosis in mammalian cells is Bax. In most cells, Bax is maintained in the cytosol in an inactive conformation and becomes activated only in response to apoptotic stimuli. Activated Bax then translocates to the mitochondria to induce cytochrome c release and caspase activation. Remarkably, we found that hES cells maintain Bax in its already active conformation. Surprisingly, active Bax was maintained at the Golgi rather than at the mitochondria, thus allowing hES cells to effectively minimize the risks associated with having pre-activated Bax. Our results show that after DNA damage, active Bax rapidly translocated from the Golgi to mitochondria by a p53-dependent mechanism. Thus, maintenance of Bax in its active form is a unique mechanism that can prime hES cells for rapid death, likely to prevent the propagation of mutations during the early critical stages of embryonic development. In this proposal, we will investigate this novel and unexpected mechanism by which apoptosis is regulated in hES cells. We will focus specifically on examining how Bax is maintained in an active state (Aim 1), determine how it localizes to the Golgi (Aim 2) and identify the molecular events triggered by DNA damage to induce the rapid translocation of active Bax from the Golgi to the mitochondria (Aim 3) in hES cells. These studies will undoubtedly uncover critical aspects of apoptosis regulation in cells and reveal key features of stem cell biology that can have significant impact for regenerative medicine.

Public Health Relevance

We have found that human embryonic stem (hES) cells are poised to undergo rapid cell death if exposed to DNA damage. This adaptation is likely critical to reduce the risk of accumulating genomic mutations and ensuring the integrity of the developing embryo. Our proposal focuses on understanding the novel molecular mechanism engaged by hES cells that primes them for rapid death. The results from this study will identify key features of stem cells biology that will be important not only for regenerative medicine but also for understanding early embryonic development.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM105612-02S1
Application #
8898490
Study Section
Program Officer
Maas, Stefan
Project Start
2013-04-01
Project End
2017-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
2
Fiscal Year
2014
Total Cost
$106,402
Indirect Cost
$36,401
Name
University of North Carolina Chapel Hill
Department
Physiology
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599