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RS1-00189-1: Control of embryonic stem cell transcription by TGFb/Smad signaling

Recommendation: Not recommended for funding

Public Abstract (provided by applicant)

Statement of Benefit to California (provided by applicant)

Review

SYNOPSIS: The proposal investigates how signalling by the TGF-beta family of cytokines and their intracellular effectors, the Smads, regulates the expression and function of the key stem cell markers Sox2, Oct4, and Nanog (SON) with respect to both maintaining pluripotent stem cell activity (self-renewal), as well as directing differentiation. The Principal Investigator (PI) will manipulate Smad signaling by over-expression or knockdown and evaluate how this alters SON and affects self-renewal or differentiation. Co-infection with a reporter followed by equal mixing with uninfected cells will be used to follow the renewal of infected cells over several passages. Effects on differentiation and pluripotency (after EB formation) will be followed by qRT-PCR and immunocytochemistry.

INNOVATION AND SIGNIFICANCE: The general hypothesis is almost certain to be true – it seems very likely that TGF-beta family signaling, mediated at some level by Smads, will be a key component of regulating SON and in defining the stemness or differentiation state of human embryonic stem cells (hESC) and embroid body (EB) derivatives. The concepts are derived from what has already been established in a variety of developmental and cell lineage systems including mouse ESC. It is unclear how much will be added to our understanding of hESCs by these studies. The significance will be determined by revealing the level of specificity for signaling components. It might be that hESC do have some distinct target genes or use for SON compared to mouse, or different levels of redundancy. Innovation is not a strength of the proposal, beyond the attempt to translate key findings from mouse and other systems into the hESC system.

STRENGTHS: A major strength of the proposal is the PI, who is a recognized leader in the field of TGF-beta family signaling and has generated many of the key observations that have led to tremendous advances in our understanding of the molecular details for this and related signaling pathways, which impact perhaps every differentiation program in the embryo and adult. Another strength is the collaboration with the an expert in hESC.

Although descriptive, the experiments described to look at the effect of individual TGF-beta family cytokines on Smads and SON and studies to inhibit Smad(s) and determine the effect on SON seem feasible and likely to provide fundamental information about the state of TGF-beta family signaling components and their relationship to SON (albeit correlative rather than mechanistic).

WEAKNESSES: A major weakness of the proposal is that it lacks focus with respect to which differentiation pathways are being repressed or activated by which signaling pathways. It rather attempts to comprehensively consider all the ligands and signaling components and generalizes differentiation. Some of the proposed studies establish artificial reporter assays that may or may not reflect the normal regulation of SON genes. Likewise, the in vitro binding assays are unlikely to reflect the specific interactions that take place on these key regulatory nodes. There is little consideration for how forced expression of SON might disrupt the network or generate an artificial signaling environment. In this respect, the proposal seeks to translate tissue culture cell models that have had general success for establishing a paradigm into the more complicated scenario of an ESC, and this seems unlikely to be straight-forward.

DISCUSSION: There is no description of how the proposed work will lead to important changes in our understanding or use of hESC, this could have been better articulated. The reviewers noted that the application was incomplete in that several sections were left blank including the abstract, the public abstract, the statement of benefit to California and the budget justification.

The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:

• Feit, Marcie
• Lansing, Sherry
• Sheehy, Jeff