Statement of Benefit to California:
SYNOPSIS: In this proposal, which is highly responsive to the RFA, the goal is to improve culture conditions for hES cells by focusing on TGF-beta signaling components. Cells will be generated with a GFP cassette expressed under Oct4 control, and then cells will be screened for growth in defined media containing one or more of 22 different TGF-ligands that have been produced by recombinant methods. The "product" of this work will be culture conditions that maintain pluripotency of hES cells. SIGNIFICANCE AND INNOVATION: Improved culture conditions that maintain pluripotency of hES cells are a worthy goal. This application is novel for the production of 22 recombinant TGF-ligands for screening in vitro. While not particularly innovative, this project has the potential to generate extremely useful information for the field as a whole. This is the sort of fundamental research that is needed for ultimate progress in using hES cells, but the approach is not necessarily one that would usually be feasible outside of industry. STRENGTHS: The prior cloning and production of a large number of TGF-beta ligands is an important aspect of this proposal. The proposed screening, one that could yield a breakthrough in optimizing a defined culture condition that could become standard in the field, is straightforward and logical and thus likely to yield useful information. The project brings together outstanding expertise from a protein chemist/structural biologist (Choe) and a stem cell biologist (Belmonte). This concerted effort, rather than reliance on one or two components, is a strength of the proposal. Few groups could carry out such an ambitious “production factory” and there is even the possibility that novel factors could be engineered. WEAKNESSES: A potential weakness may relate to the focus on TGF-beta components. Nonetheless, there is sufficient prior data from mES cells to recommend consideration of these factors. A more minor weakness is that Oct4:GFP lines are already generated, and it was not made clear why the proposed line will be better. Reviewers recommend the use of the BAC-Oct4 reporter rather than targeting one of the Oct4 loci by homologous recombination. The latter approach will inactivate one Oct4 allele, and this may be detrimental to maintenance of the pluripotent state. DISCUSSION: This is a well thought out, well written, well focused proposal that very few people could execute; there is a huge number of combinations to test in this robotic screen to define optimal culture conditions. The approach is practical with straight-forward testing procedures, and the growth studies using 22 different ligands, already in-hand, is a real strength. Homologous recombination (HR) in hESC is an issue as reviewers questioned whether this group has the necessary experience with this technique since only 2 groups have reported success with HR in hES cells. A BAC-based approach may provide some information if the homologous integration approach fails. Information from this study is likely to affect the whole field.