Induced pluripotent stem cells (iPSCs) are a potential source of material for cell replacement therapies. Thus, achieving maximal efficiency of reprogramming will be important for cellular medicine. In this submission we propose to test whether manipulating the mammalian glucose sensing pathway. The enzyme OGT is part of the glucose-sensing pathway and is necessary in pluripotent cells. OGT catalyzes the transfer of a sugar to target proteins, which regulates protein function. This transfer is the terminal step in the hexosamine signaling pathway (HSP). The HSP serves as a nutrient sensor, as the concentrations of the sugar donor used by OGT fluctuate with glucose levels. As a result, changes in intracellular glucose concentration cause alterations in modification of OGT target proteins. This nutrient-responsive signaling system modulates important cellular pathways, including the insulin-signaling cascade. Alterations in OGT activity are associated with diabetes mellitus and Alzheimer’s disease. Thus, our studies of role of OGT and the HSP in self-renewal and reprogramming may also shed light on the role of this pathway in other stem cells, such as neuronal and islet stem cells, whose depletion may contribute to these degenerative diseases. Understanding the role of OGT in ESCs and iPSCs will both increase our knowledge of the molecular mechanisms mammalian cells use to establish and maintain pluripotency and perhaps lead increase the efficiency of reprogramming.
Among ten leading death causes in California, five of them can directly benefit from cell-based tissue regeneration. These include heart disease, stroke, Alzheimer's disease, diabetes, and liver diseases. Currently, the economic burdens derived from these diseases are enormous. It is estimated from State of California, Department of Public Health that California taxpayers pay 48 billion dollars annually for cardiovascular diseases, 73 billion dollars excluding non-paid family care for Alzheimer's disease, and 116 billion dollars for diabetes-related diseases. Induced pluripotent stem cells (iPSCs) offer great promise as tools for regenerative medicine. However, the iPSC technology still has several shortcomings inhibiting its clinical application, one of which is the low efficiency in production iPSCs. The research outlined in this application has the potential to provide a method that substantially increases the efficiency of production of human induce iPSCs. If these studies lead to improvements in the production of iPSCs, facilitating their use in regenerative medicine, they will directly benefit the health of California citizens and reduce the economic burden presently borne by California taxpayers. This research may increase California's visibility in stem cells research and attract federal funding to sponsor future research. It may also enhance California's economic growth by stimulating the iPSC regenerative medicine industry for the treatment or cure of diseases.
This proposal is based on the applicant’s observation that the enzyme, O-linked N-acetylglucosamine transferase (OGT), which mediates a particular protein modification, is necessary for self-renewal of murine embryonic stem cells (mESC) and human induced pluripotent stem cells (hiPSC). OGT is part of a glucose sensing pathway and functions by catalyzing the addition of sugar residues to multiple specific proteins, leading to changes in these proteins’ activities. In this proposal, the applicant seeks to expand on these and other preliminary findings by determining the importance of OGT-mediated modification to the function of a key regulator of pluripotency in human cells (Aim 1), and by testing the effect of altered OGT expression on the efficiency of cellular reprogramming (Aim 2). For Aim 3, the applicant proposes to use mESC to elucidate the molecular mechanisms by which OGT-mediated modification affects the function of a pluripotency protein.
Significance and Innovation
- The proposed research addresses an important and understudied area of stem cell biology.
- The proposed studies are based on an interesting and novel premise. Although the proposed investigation is conceptually related to a study described in a recent publication, the proposal features novel goals including an emphasis on human stem cell biology and a focus on a different OGT target.
Feasibility and Experimental Design
- The rationale for each of the specific aims is scientifically sound and based on compelling preliminary data.
- The research plan is straightforward and well organized, with very logical and clearly designed experiments.
- Potential pitfalls and alternative plans have been adequately considered.
Principal Investigator (PI) and Research Team
- Although the focus of this application diverges somewhat from the PI’s primary expertise, reviewers were convinced that he/she possesses the necessary and appropriate skills to successfully conduct the proposed research.
- The research team and environment are excellent.
Responsiveness to the RFA
- The proposal shifts focus from human to murine cells in the third aim without appropriate justification. Reviewers therefore predicated their support on the recommendation that all studies be done comparatively between human and mouse.