Funding opportunities
The functional role of protein O-GlcNAcylation in hESC pluripotency and differentiation
Funding Type:
Basic Biology II
Grant Number:
RB2-01500
Funds Committed:
$1,354,090
Funding Recommendations:
Not recommended
Public Abstract:
Human embryonic stem cells can be changed into virtually any cell type in the adult body. Because of this unique capability, stem cells have the potential to cure a vast majority of existing human disorders. However, the mechanisms that govern the definition and function of stem cells has not been completely elucidated, and several hurdles exist and need to be overcome before stem cells can be used in the clinic. For example, the factors which govern the conversion of stem cells into a variety of tissue types such as liver, heart, and brain tissue - are not well understood. Our research employs a unique multidisciplinary approach to bridge this information gap. Proteins govern the daily life of cells by controlling when genes are activated, how cells communicate with one another, and several other critical processes. The action of proteins inside cells is commonly turned on and off by the appending to, or removal of, sugars from proteins. Though this control mechanism is well established in other areas of health and human disease, it has not been widely studied in the context of stem cell biology. The proposed research will examine how the sugars found on proteins impact processes such as the differentiation of stem cells into neurons, the generation of pluripotent stem cells, and how the genetic reprogramming of stem cells is actually carried out by cellular proteins. The results of these studies may lead to a greatly increased understanding of how stem cells retain their ability to be changed into other cell types, and also how the fate of stem cells is decided upon differentiation. Both are critical areas that need to be explored to enable modern regenerative medicine to realize its full potential as a tool for the treatment of human diseases.
Statement of Benefit to California:
Programs funded by CIRM and other state granting agencies will allow California to continue to be at the frontier of stem cell research for the development of new treatments to cure human diseases. Research such as ours will hopefully enable modern medicine to access exciting new areas such as spinal regeneration, and finding treatments for neurodegenerative disorders for which there is currently little hope for curing. Some illnesses which could be potentially impacted include multiple sclerosis, Alzheimer’s, Parkinson, and Batten diseases. Several hurdles exist, however, which need to be overcome before results from the exciting field of stem cell research can be used in the clinic. For example, the factors which govern conversion of stem cells into a variety of tissue types that may find uses in regenerative medicine such as the liver, heart, and brain, are not well understood. Our research employs a unique multidisciplinary approach to bridge this information gap. In particular, our research will examine how the sugars which are attached to proteins control processes such as the vast genetic reprogramming that accompanies the conversion of stem cells into mature tissues. Through initiatives like CIRM, California will continue to lead the nation in the discoveries resulting from multidisciplinary scientific research which will fuel tomorrow’s medical advances.
Review Summary:
EXECUTIVE SUMMARY
This proposal examines the role of protein modification, specifically the addition or removal of sugars, in three areas of stem cell biology: neuronal differentiation, reprogramming of induced pluripotent stem cells (iPSCs), and gene silencing. In Aim 1, the applicant proposes to comprehensively catalog proteins that are modified with a particular sugar during the differentiation of human embryonic stem cells (hESCs) into neurons. Aim 2 examines this sugar modification in the context of reprogramming mouse and human somatic cells into induced pluripotent stem cells (iPSCs). Finally, in Aim 3, the applicant proposes to identify sugar-modified proteins that bind and silence DNA in hESCs, as well as their DNA targets.
Reviewers found this proposal to be somewhat innovative, in that novel tools are being applied to hESC biology, but they had difficulty assessing its significance. The project is based on the idea that the sugar modification under investigation plays an important role in human stem cell biology, but to date there is little evidence supporting this assumption. Applying the applicant’s powerful technology to hESCs may yield exciting results, but it is difficult to determine at this early stage, so reviewers considered this a high-risk proposal.
Reviewers raised a number of concerns with the experimental design and preliminary data. With regard to Aim 1, they cautioned that recovery of hESCs following single-cell sorting is extremely low and will make analysis using this technique difficult. With regard to Aim 2, one reviewer noted that the technique proposed to label sugar-modified proteins appeared to alter the cellular profile of such proteins, so results might be altered by the analytical method. Additionally, it is not clear how deeply into the sugar-modified proteome this approach will be able to reach, as the technique employed in preliminary studies only identified 19 sugar-modified proteins. This result demonstrates basic feasibility but doesn’t raise confidence that a comprehensive set of proteins will be identified as proposed. Reviewers noted that only very limited preliminary data support the use of this technique in hESCs; additional proof-of-concept data supporting the importance of sugar modification in hESC biology would have strengthened the proposal. Reviewers found that some experiments in Aims 2 and 3 are proposed in mouse cells without convincing justification. They cautioned that differences in reprogramming mechanisms between mouse and human are likely to be significant, limiting the value of studies using murine iPSCs.
The reviewers praised the PI’s outstanding track record of productivity and publication. One reviewer stated that no other PI is more qualified to undertake the proposed experimental approaches. However, reviewers cited the lack of an identified collaborator with hESC or iPSC expertise as a weakness that seriously impacted the project’s feasibility.
Overall, while reviewers were enthusiastic about the PI and research team, they found the significance and potential impact of the proposal difficult to assess given the lack of adequate preliminary data supporting its rationale. Additionally, there were serious concerns about the proposal’s feasibility.
Conflicts:
