Funding opportunities

Cellular and Molecular Determinants of Pluripotency and Stability

Funding Type: 
Basic Biology I
Grant Number: 
RB1-01375
Funds requested: 
$1 364 484
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
hESCs are pluripotent cells that can be maintained indefinitely in culture. These characteristics have led to much enthusiasm for their potential use in cell-replacement therapies. One obstacle to realizing this potential is the incomplete knowledge of mechanisms involved regulating pluripotent fate decisions including survival, self-renewal and stability. While hESCs can be maintained in vitro, cells grown in continuous culture have been shown to develop aberrations associated with cancer in vivo. Therefore it is the goal of this work to determine the mechansims that regulate pluripotent stem cell growth without giving rise to abnormal stem cells with the potential for tumor formation.
Statement of Benefit to California: 
Pluripotent stem cells (PSCs) have the potential to revolutinize medicine for use in cell-based therapies. However, the translational use of hESCs will not be realized unless we understand how pluripotency is regulated in these cells. Defining how pluripotency is controlled could improve our ability to derive new cell lines in a more controlled conditions. This could enhance California as a recognized world leader in PSC research through the generation of a centralized and comprehensive resource of PSC lines with potential clinical use. This work will also create jobs to support this effort, and validate the decision of California voters who ensured the passage of Proposition 71. Our proposal to improve our understanding of PSC pluripotency and stability will make treatment strategies from hESCs one-step closer.
Review Summary: 
This proposal addresses the role of cell adhesion and polarity on the survival, pluripotency, and differentiation of human embryonic stem cells (hESCs). In Aim 1, the applicant proposes to determine the roles of adhesion and polarity proteins in establishing cell-cell contacts and symmetric division in hESCs. In Aim 2, the applicant will examine whether adhesion and/or polarity proteins regulate hESC self-renewal. Finally, in Aim 3, the applicant proposes to examine the role of adhesion and/or polarity proteins in hESC models of tumor formation in vivo. Reviewers agreed that this proposal addresses an important problem but felt that its potential impact is limited by flaws in its experimental design. Adhesion and polarity play essential roles in vivo in a variety of cellular events, including differentiation and tumor formation. Therefore, a basic understanding of the molecular processes involved in cell contact and polarity are very significant topics. Reviewers appreciated the proposal’s focus on mechanism, but did not find its approaches to be particularly innovative, noting that much of what is proposed has been demonstrated in other systems. Reviewers identified a number of problems with the research plan but focused on two issues in particular. The first concerned the preliminary data, which describes the identification of a small molecule inhibitor that enhances hESC survival. The role of polarity genes in hESCs is inferred indirectly as this inhibitor acts on a known protein regulator of cell polarity and also increases the expression of polarity genes. A key problem is that mouse embryonic fibroblast (MEF) feeder cells are present in the cultures. Therefore, it is unclear whether the inhibitor’s effect on hESC survival is a primary one, or secondary through effects on the MEFs. In addition, it is impossible to rule out a role for non-physiological cross-species interactions in these experiments. Additionally, the planned experiments do not differentiate between a growth effect of the inhibitor (which may not be specific to stem cells) and a true stabilization of the stem cell state. The second major problem concerned the feasibility of Aim 3, in which a tumor formation assay is used as an in vivo readout of the differentiation potential of hESCs. Reviewers felt that the proposed assay would be inadequate for accomplishing the experimental aim and suggested that this goal would be better-pursued using cancer stem cells. Reviewers noted significant ambiguity in the preliminary data supporting Aim 3 and felt that the issue of cell death was inadequately addressed throughout the proposal. For example, the lack of certain cell types, taken as evidence for differences in developmental capacity, may actually reflect decreased survival of these cell types. Finally, reviewers noted that the research plan failed to include discussion of potential pitfalls and alternative strategies. Reviewers described the applicant as a talented junior investigator but were concerned about a sparse publication record. They noted that the applicant has recruited two key collaborators with expertise in cell polarity/adhesion and genomics but would have preferred that the collaborators allocate more effort to the project. Reviewers praised the applicant’s expertise in hESC biology and found the team qualified to perform the aims described in the proposal. Overall, while reviewers appreciated the significance of the scientific problem addressed in this proposal, they raised a number of serious issues with the research plan that made them question its feasibility and potential impact.
Conflicts: 

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