Therapeutic applications of embryonic stem cells will depend on our ability to obtain large amounts of pluripotent stem cells and their predifferentiated progeny. Numerous efforts have been channeled into understanding of the molecular mechanism of stem cell self-renewal. Despite recent advancements, the mechanisms.of self-renewal and differentiation of Embryonic Stem (ES) cells are poorly understood. Our preliminary data and analysis of literature prompted us to formulate a hypothesis suggesting a link between the (i) intracellular redox state, (ii) members of Sirtuin protein family, (iii) acetylation status of individual regulatory proteins, and (iv) the self-renewal vs. differentiation decision of stem/progenitor cells. Here we propose the experiments, which begin testing our general hypothesis, namely we will establish the differences in protein acetylation profile between ES cells and their differentiated progeny, embryoid bodies and the role of individual Sirtuins in protein acetylation in ES cells. We propose that differential acetylation / deacetylation of specific protein targets is one of the key modulators of stem cell differentiation. In particular, we speculate that protein deacetylation mediated by the members of Sir2 family (mediated by the intracellular redox state) regulates the stem cell self-renewal vs. differentiation decision.
In Aim 1 we will establish the protein acetylation profile (acetylation signature) of undifferentiated mouse (ES) cells and their differentiated progeny embryoid bodies. We propose a novel mass spectroscopy approach to compare the acetylation of the entire cellular proteome in pluripotent ES cells and in their differentiated progeny, embryoid bodies.
In Aim 2 we will link the activity of individual members of mouse Sir2 family with the acetylation signature of mouse ES cells. We will use RNA interference technique, to systematically knock-down the members of mouse Sir2 family (individual and in combination) in ES cells and analyze the changes in acetylation profile of ES cells and their differentiation potential. The modulation of the intracellular redox state of embryonic and potentially somatic stem cells in order to manipulate their self-renewal status or to induce differentiation could be of great practical importance and constitute the future directions of research. Naturally, experiments with mouse ES cells will lay the foundation for the work to modulate self-renewal or induce differentiation of human ES cells. ? ?

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21HD047413-01A2
Application #
7031897
Study Section
Development - 1 Study Section (DEV)
Program Officer
Tasca, Richard J
Project Start
2006-06-06
Project End
2008-05-31
Budget Start
2006-06-06
Budget End
2007-05-31
Support Year
1
Fiscal Year
2006
Total Cost
$257,850
Indirect Cost
Name
Sanford-Burnham Medical Research Institute
Department
Type
DUNS #
020520466
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Cimadamore, Flavio; Curchoe, Carol Lynn; Alderson, Nazilla et al. (2009) Nicotinamide rescues human embryonic stem cell-derived neuroectoderm from parthanatic cell death. Stem Cells 27:1772-81