The long-term goal of this research is to gain a better understanding and control over human embryonic stem cells (hESCs) by identifying and manipulating the key microRNAs required for hESC self-renewal and differentiation. MicroRNAs, small (21-23nt) non-coding RNA molecules that can regulate mRNA translation and stability by binding to the target mRNAs with incomplete complementarity, are required for normal stem cell function in mouse, Drosophila and plants. We have previously shown that microRNAs are required for germline stem cell (GSC) self-renewing divisions in Drosophila. Our preliminary results suggest that microRNAs are also critical for stem cell self-renewing division and differentiation in human ESCs, and the goal of this proposal is to identify the critical microRNAs and their mRNA targets in these cells. Towards this goal, we will analyze the phenotypes observed in hESCs lacking mature microRNAs due to gene knockdown (KD) of microRNA processing enzymes, Dicer or Drosha. We will identify up-regulated mRNAs in these KD-lines by genome wide mRNA profiling and determine whether they are responsible for the Dicer or Drosha phenotypes through loss-of-function (LOF), gain-of-function (GOF) and rescue experiments (Aim1). To identify the critical microRNAs that mediate this regulation we will carry out genome wide QPCR profiling of hESC microRNAs. The function of the hESC enriched microRNAs will be analyzed by LOF, GOF and rescue experiments (Aim2). The two aims will connect: The critical microRNAs should have the capacity to rescue dicer KD phenotypes (Aim 2.2) and may be sufficient to down-regulate the target genes identified in Aim 1.
2 (Aim 2. 3). This study should result in findings that allow better manipulation and more efficient differentiation of hESC which is of particular importance for regenerative medicine.

Public Health Relevance

This study will be the first analysis of the functions of mature miRNAs in hESCs. Identification of the miRNA function in hESCs holds the future for better control and manipulation of the hESCs in regenerative medicine. This is important due to the troubling fact that embryonic stem cells tend to form tumors when transplanted into adult tissue. microRNAs carry hope as molecules which can control stem cell self-renewing division.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM083867-04
Application #
8118467
Study Section
Development - 2 Study Section (DEV2)
Program Officer
Haynes, Susan R
Project Start
2008-09-15
Project End
2013-07-31
Budget Start
2011-08-01
Budget End
2013-07-31
Support Year
4
Fiscal Year
2011
Total Cost
$389,106
Indirect Cost
Name
University of Washington
Department
Biochemistry
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Artoni, Filippo; Kreipke, Rebecca E; Palmeira, Ondina et al. (2017) Loss of foxo rescues stem cell aging in Drosophila germ line. Elife 6:
Moody, James D; Levy, Shiri; Mathieu, Julie et al. (2017) First critical repressive H3K27me3 marks in embryonic stem cells identified using designed protein inhibitor. Proc Natl Acad Sci U S A 114:10125-10130
Mathieu, Julie; Ruohola-Baker, Hannele (2017) Metabolic remodeling during the loss and acquisition of pluripotency. Development 144:541-551
Kuppusamy, Kavitha T; Jones, Daniel C; Sperber, Henrik et al. (2015) Let-7 family of microRNA is required for maturation and adult-like metabolism in stem cell-derived cardiomyocytes. Proc Natl Acad Sci U S A 112:E2785-94
Sperber, Henrik; Mathieu, Julie; Wang, Yuliang et al. (2015) The metabolome regulates the epigenetic landscape during naive-to-primed human embryonic stem cell transition. Nat Cell Biol 17:1523-35
Xing, Yalan; Su, Tin Tin; Ruohola-Baker, Hannele (2015) Tie-mediated signal from apoptotic cells protects stem cells in Drosophila melanogaster. Nat Commun 6:7058
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
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
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
Sperber, Henrik; Beem, Alan; Shannon, Sandra et al. (2014) miRNA sensitivity to Drosha levels correlates with pre-miRNA secondary structure. RNA 20:621-31

Showing the most recent 10 out of 26 publications