$2 532 388
Statement of Benefit to California:
SYNOPSIS:The applicant, an experienced, highly productive developmental and stem cell biologist proposes to construct a fate map of the early human embryo. The specific aims of the proposal are 1. Molecular characterization of the 2-cell to late-blastocyst human embryo, using a battery of reagents to quantify expression of transcription factors, signaling molecules and cell surface antigens. 2. To determine the human embryonic stem cell(hESC) potential of newly derived hESC lines produced from single human blastomeres from various regions and times in development of the embryo IMPACT & SIGNIFICANCE:The Principal Investigator (PI) points out that much hESC research is based on the transfer of information from mouse ESCs to hESCs, and also points out the paucity of work directly on human embryos. Consequently, we know considerably more about the early mouse embryo than about the human embryo. This application is designed to fill some of this information void. This work has the potential to completely alter fundamental concepts about hESC fate and handling, as well as derivation. The goal of this project is to generate information about the early human embryo that will be useful, if not fundamental, to understanding the origin and basic properties of hESCs. This is a highly significant endeavor that has in the past been precluded by a Federal level moratorium of research involving human embryos. Lineage tracing has been conducted extensively in mammalian models and will now be thoughtfully and appropriately applied to the human. A major hypothesis under study is that targeted derivation of hESC lines from specific blastomeres will yield cells that are either more likely to be truly pluripotent or better able to assume specific fates. This line of research seeks to ask the most basic cell biologic/morphologic question for hESCs: when and where do they appear? Doing so could allow one to more effectively isolate pluripotent or partially committed hESC lines, which will provide an enormous resource to the community. The molecular characterization of embryonic development, coupled with efforts to derive cell lines from each stage, could give clues as to how lineage development is engaged and 'locked in.' QUALITY OF RESEARCH PLAN:The preliminary data highlights the broad experience of the group, one of the few really qualified and ready to study early human embryos, and the detailed methods developed for the proposal. The recent scientific findings and highlights from this group include the distinctive morphologic features of mouse vs. human blastocysts, and the importance of cell polarity in fate choice and differentiation patterns. The team, reagents, environment are uniquely suited to the proposal. One reviewer thought that this proposal is the most important proposal in the three cycles of review that this reviewer has been part of. The research plan is outstanding and will without doubt produce meaningful results within the 4 year timetable. Two specific aims are proposed. The first will characterize preimplantation stage human embryos with regards to various transcription factors, signaling molecules and cell surface antigens. Careful consideration has been devoted to the number and use of human embryos with the expectation that data from 7-10 embryos should provide significant results. The second will employ a recently published method for single blastomere isolation to derive hESC lines from different regions of the developing embryo. The PI and her team have cited preliminary results that increase the likelihood for success with this new technique of hESC derivation from isolated blastomeres. This is a very high quality research plan by a PI with tremendous expertise in precisely these sorts of studies (conducted in the murine system). STRENGTHS: The team, reagents, environment are uniquely suited to the proposal. The work is the most important proposal in the three cycles of CIRM review that one of the reviewer has seen. The PI is extremely strong and together with the team are outstanding, exceptionally well-funded, capable and experienced with impressive preliminary results. The proposal is hypothesis-driven and should produce a high yield of new fundamental information regarding the human embryo. A collaboration has been established with the senior author of the publication concerning the successful method of deriving hESCs from isolated blastomeres. The team has experience with the techniques required and feasibility issues are adequately addressed. The team also has experience with non-xenogenic culture systems. There is an appropriate use of the mouse as a model for the proposed studies. Finally, the studies are of basic biologic importance; experiments aiming to establish lines from each blastomere could be very informative. WEAKNESSES:The only criticism is that the PI is currently 121% committed on other grants, which will be 97% commitment when a few of them expire in 2007. Consequently, her 30% salary support request needs administrative attention. This is especially important since the PI has been cited as a collaborator, supporter, and general consultant on many of the SEED grants seen during the last cycle. Epigenetic regulation may be important during early development as imprint marks are established. The PI and her team may want to include strategies to evaluate this possibility. Although this is important work, it is extremely descriptive in nature. One could easily end up with a very limited answer to the question: when and where do hESCs first arise--and nothing more. No genes, no mechanisms. So the scope could end up being limited. If the experiments in the second aim demonstrate plasticity, i.e. that hESC lines can be derived at different stages, with variable or without fixed imprinting, then the studies will yield lots of new lines, but little new science. DISCUSSION: This proposal is from a highly experienced developmental biologist with a strong collaborator. This is an exceptionally well-written grant that is very different from the others in proposing a full molecular characterization of the 2-cell to blastocyst embryo, i.e. to construct a fate map of early human embryo. The PI has relationships with clinics that will afford access to the necessary tissue and has developed many reagents that will allow molecular characterization. The preliminary data presented are beautiful but exhaustive, almost too dense; the ability to extrapolate from previous work in mouse is useful. Concerns are mainly administrative: the major weakness is that it appears the PI is over-committed (i.e., over 100% time allocated) on other grants. One reviewer raised the concern that the project is purely a description of when and where hESCs arise from the embryo. At the end of the day, we may still know nothing about the genes and mechanisms involved. Another reviewer felt that the knowledge of the "lawn" on which hESCs grow will be inherently enhanced by this work. A discussant pointed out that there is an absence of knowledge of human embryogenesis; we know more about development of the worm, frog, mouse and bird that we do about ourselves, and therefore that this work must be done.