The goal of this grant is to test the hypothesis that hESCs and iPSCs acquire specific metabolic signatures important for stemness through a common means, possibly by an exposure to stabilized HIF activity. Recent data support this hypothesis;HIFs have shown to play a role in ESC fate and ESCs have shown to sustain highly unsaturated metabolome. However, it is not yet clear how ESCs acquire their metabolic state, how the state is maintained and further, how the metabolic state of differentiation is obtained. We have showed that hypoxia is involved specifically in the acquisition state of stemness since hypoxia alone can de-differentiate committed cells back to stem cell fate. The de-differentiated cells re-activate Oct4 promoter analyzed by Oct4- GFP. These hypoxia induced stem like cells also mimic hESCs by their colony morphology, long-term self- renewal capacity, capability to replicate both in hypoxia and normoxia, genome wide mRNA and miRNA profiles, cell surface marker, TRA1 and SSEA expression and capacity to form teratomas. Furthermore, we have observed a specific metabolic signature in ESCs. We will now test whether hypoxia induced transcription factor, HIF is sufficient to induce the ESC specific metabolic pattern. We will first analyze glycolytic and oxidative phosphorylation responses to hypoxia using a panel of stem cells with different stages of pluripotency (piSC, diSC, EpiSC, HiPSC) and differentiated cells, followed by detailed assessment of redox poise within the mitochondrial electron transport chain (ETC) and metabolic pathway flux. Secondly, we will define the contribution of HIF activity for induction of unique stemness signatures (metabolism, mtDNA copy number) using hypoxia de-differentiation paradigms (HiPSC). Paradoxically, hypoxia has shown to affect both stemness and differentiation, suggesting that HIF might be differentially regulated to obtain the desired cellular outcomes. Therefore, we will devise high throughput screens to identify molecules that specifically affect HIF- induced stemness by promoting or interfering with stem cell-associated metabolic pathways. These studies will beget a new level of understanding of the acquisition of stemness and cancer and as such are bound to result in new approaches both in re-generative and cancer medicine.

Public Health Relevance

The goal of this grant is to test the hypothesis that stem cells (in normal or cancer development) acquire a common characteristic metabolic signature through exposure to stabilized hypoxia-inducible factor (HIF) activity. Further, this metabolic signature may be determinative for stemness. Metabolites that are enriched in stem cells will be tested for their ability to reverse ESC differentiation in a high throughput format. These studies will spur improvements in regenerative medicine and potential therapies against cancer stem cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM097372-03
Application #
8442879
Study Section
Special Emphasis Panel (ZRG1-BDA-P (90))
Program Officer
Haynes, Susan R
Project Start
2011-04-01
Project End
2015-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
3
Fiscal Year
2013
Total Cost
$293,615
Indirect Cost
$110,265
Name
University of Washington
Department
Biochemistry
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Ware, Carol B; Nelson, Angelique M; Mecham, Brigham et al. (2014) Derivation of naive human embryonic stem cells. Proc Natl Acad Sci U S A 111:4484-9
Guan, Xuan; Mack, David L; Moreno, Claudia M et al. (2014) Dystrophin-deficient cardiomyocytes derived from human urine: new biologic reagents for drug discovery. Stem Cell Res 12:467-80
Mathieu, Julie; Zhou, Wenyu; Xing, Yalan et al. (2014) Hypoxia-inducible factors have distinct and stage-specific roles during reprogramming of human cells to pluripotency. Cell Stem Cell 14:592-605
Nguyen-Tran, Diem-Hang; Hait, Nitai C; Sperber, Henrik et al. (2014) Molecular mechanism of sphingosine-1-phosphate action in Duchenne muscular dystrophy. Dis Model Mech 7:41-54
Ware, Carol B (2014) Naive embryonic stem cells: the future of stem cell research? Regen Med 9:401-3
Sperber, Henrik; Beem, Alan; Shannon, Sandra et al. (2014) miRNA sensitivity to Drosha levels correlates with pre-miRNA secondary structure. RNA 20:621-31
Pantoja, Mario; Fischer, Karin A; Ieronimakis, Nicholas et al. (2013) Genetic elevation of sphingosine 1-phosphate suppresses dystrophic muscle phenotypes in Drosophila. Development 140:136-46
Kuppusamy, K T; Sperber, H; Ruohola-Baker, H (2013) MicroRNA regulation and role in stem cell maintenance, cardiac differentiation and hypertrophy. Curr Mol Med 13:757-64
Ieronimakis, Nicholas; Pantoja, Mario; Hays, Aislinn L et al. (2013) Increased sphingosine-1-phosphate improves muscle regeneration in acutely injured mdx mice. Skelet Muscle 3:20
Mathieu, Julie; Ruohola-Baker, Hannele (2013) Regulation of stem cell populations by microRNAs. Adv Exp Med Biol 786:329-51

Showing the most recent 10 out of 14 publications