Funding opportunities

The stem cell microenvironment in the maintenance of pluripotency and reprogramming

Funding Type: 
Basic Biology I
Grant Number: 
Principle Investigator: 
Funds requested: 
$1 440 822
Funding Recommendations: 
Grant approved: 
Public Abstract: 
Statement of Benefit to California: 
Review Summary: 
The goal of this effort is to elucidate the role of extrinsic factors in the human embryonic stem cell (hESC) microenvironment in the regulation of pluripotency. Specifically, the proposal is based on the fundamental observation that hESC cultures are heterogeneous as well as the applicant’s own research suggesting that only a subset of the hESC population, referred to as primordial embryonic stem cells (pESCs), fully expresses a pluripotent phenotype. In Aim 1, the applicant proposes to characterize this pESC population, as well as “less pluripotent” hESC populations, by gene and protein expression profiling. For the second Aim, the applicant will examine his/her hypothesis that the hESC microenvironment is crucial in determining cell fate by disrupting the pESC niche and attempting to reconstitute it with various combinations of hESCs as defined by the cell surface markers that were identified in Aim 1. Finally, the applicant proposes to identify key factors secreted by the stem cell niche to maintain pluripotency. In all three aims, induced pluripotent stem cells (iPSCs) will be studied in parallel to address similar questions about the role of the microenvironment in reprogramming and iPSC pluripotency. Reviewers agreed that this proposal addresses a major unsolved problem and could have a significant impact on the field of stem cell biology. The nature of the microenvironment and the identity of the component molecules that contribute to the extrinsic regulation of pluripotency are poorly understood. Greater knowledge of these extrinsic cues would lead to improved methodologies to maintain pluripotent stem cell lines and may also improve the efficiency of iPSC reprogramming. Reviewers found the proposal to be highly innovative, both in its experimental approaches and the problem addressed. Reviewers praised the research plan and its sound rationale, based on logical hypotheses explored through well-formulated specific aims. They appreciated the strong preliminary data, noting that it supports the proposal’s central hypothesis about hESC culture heterogeneity and the existence of pESCs. An important aspect of the preliminary data is that it demonstrates successful application of real-time PCR analysis to single ESCs defined by their spatial location or phenotype, as this technology is crucial to the success of the proposal. However, one reviewer would have been more confident if the applicant had included data to define the colony-forming efficiency of each population. Reviewers were particularly enthusiastic about Aim 1, focused on defining pESCs that are proposed to sit atop the ESC hierarchy. Powerful technologies for interrogating gene and protein expression will be used and these experiments should generate important data. One reviewer also appreciated that these techniques would also be applied to iPSCs, although another was uncertain as to how these studies would be designed. One reviewer raised concerns about the feasibility of Aim 3, noting that it represents a considerable amount of work given the broad range of factors and pathways that will be examined. In addition, there was some concern that changes in cell surface protein expression may be a consequence of perturbed signaling pathways and may not be coupled to changes in the pluripotency of the cell. Overall, reviewers considered the feasibility of the project to be high. Reviewers praised the applicant as a leader in the stem cell field with a long track record studying the biology of hESCs. They appreciated the enlistment of collaborators, both in academia and industry, with expertise in cutting edge technologies for single-cell gene profiling and live cell imaging. The reviewers judged the research team and resources to be excellent. Overall, reviewers were extremely enthusiastic about this proposal. They emphasized that it addresses a significant but underappreciated scientific problem using innovative technologies and that the logical research plan is supported by strong preliminary data.

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