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RC1-00113-1: Constructing a fate map of the human embryo

Recommendation: Recommended for funding
Scientific Score: 88

First Year Funds Requested: $613,181.00
Total Funds Requested: $2,532,388.00

Public Abstract (provided by applicant)

The United States government does not fund research involving human embryos or cells that were grown from them after August 9, 2001. In addition, other restrictions have been imposed that make these types of experiments extremely difficult to do. For example, work cannot be conducted alongside research that is funded by government agencies, the typical mode in which academic research laboratories operate. In practical terms, this means that duplicate facilities must be created to do the large amount of research that is needed to turn human embryonic stem cells (hESCs) into robust experimental tools that will enable us to understand disease processes, the first step in curing them. These onerous regulations, unprecedented in our country, have stifled progress in this exciting new area of medical research. Thus, there is a great deal of basic work that remains to be accomplished. Our group is focusing on one particular area-the enigmatic process that occurs when an embryo-which would otherwise be discarded at the conclusion of an in vitro fertilization (IVF) treatment-is donated for research and grown in a laboratory. In certain cases, the cells that would have gone on to form specialized tissues such as blood cells, and major organs such as the heart and pancreas, continue to make copies of themselves. As first shown in 1998, the copies, termed hESC lines, may remember how to do their original job, i.e., differentiate into every type of cell in the human body. Scientists think that this is possible, because in many laboratory animals the equivalent populations retain this ability. Our group wants to optimize the methods that are used to make new hESC lines, because the techniques that are currently used are essentially random. Embryos are maintained in the laboratory until outgrowths-collections of cells that look very different from one another-appear. During this 2 to 3-week process, many of these cells die, but a subset start to make copies of themselves. Thus, much remains to be learned about the derivation process. For example, we do not know when, during this extended time period, the actual progenitor cell(s) arises, and it is unclear whether all the cells of the embryo are equally able to give rise to hESC lines. Thus, we propose to test the theory that there are better, more controlled ways to produce hESCs. Recently, our collaborators showed that it is possible to make lines from single cells that are removed from human embryos at a specific time. We want to use their method to determine if hESCs made from individual cells that are removed at different times from specific regions of the embryo are better equipped to generate all the cell types found in the body. Essentially, we want to harness and standardize the process of developing new lines. This work, which cannot be supported by the federal government, has important implications for devising hESC-based patient therapies.

Statement of Benefit to California (provided by applicant)

The people of California have gained in substantive ways from the biotechnology revolution, which was fueled by research done in the Bay Area beginning in the mid-1970s. The benefits to the state’s citizens that were provided by this sea change in the practice of science were summarized at the BIO meeting that was held in San Francisco in 2004. The economic rewards are clear. In 2000, it was estimated that nearly a quarter of a million Californians, including 50,000 biological scientists-11.5% of the nation’s total workforce-were employed by the biotechnology sector. These individuals worked for 2,500 biomedical companies and in the state’s public and private research institutions. Recent estimates suggest that, during this same time period, the biotechnology industry generated $7.8 billion in worldwide revenue and $6.4 billion in exports. The intellectual benefits are numerous, as talented scientists at all stages of their careers have joined California’s biotechnology community to be part of an exciting new industry that translates basic research into new patient therapies. The medical benefits are also clear, as these companies are targeting unmet medical needs in numerous areas, such as cardiovascular, autoimmune and respiratory diseases, cancer, and HIV/AIDS and other infectious diseases. Also during this time period, California’s research institutions received more National Institutes of Health (NIH) grant funding than any other state, totaling $2.3 billion in 2000. Thus, for the last 40 years, synergy between California’s private and public research enterprises has produced major medical advances that have improved the lives of millions of people here and around the world. Now we are on the brink of another scientific revolution that was sparked by the first report of methods for the isolation and propagation of human embryonic stem cells (hESC), which was published by Dr. James Thomson in 1998. However, in an unprecedented move, the United States government decided in 2001 to restrict work in this burgeoning new area by limiting research to hESC lines that were submitted to the Federal Registry by August 9 of that year. It is clear that only a small fraction of the lines that were registered are actually hESCs. Consequently, NIH-funded research is limited to cell lines that have been in culture for many years and that were generated using suboptimal methods. Thus, the field is at an impasse. To go forward, we need NIH-level funds to do the basic work that is needed to develop this exciting field, which many scientists envision will fuel research in the public and private sectors for decades to come. With the passage of Proposition 71 in 2004 and the creation of the Institute for Regenerative Medicine, California has stepped into the breach. As a result, the state will once again reap the economic, intellectual and medical benefits that an exciting new area of research creates.

Review

SYNOPSIS: The applicant, an experienced, highly productive developmental and stem cell biologist, proposes to construct a fate map of the early human embryo. The proposal will conduct a molecular characterization of the early human embryo including expression of transcription factors, signaling molecules and cell surface antigens. The Principal Investigator (PI) also proposes to determine the potential of human embryonic stem cells (hESCs) derived from single human blastomeres from various regions and times in development of the embryo.

IMPACT & SIGNIFICANCE: The PI points out that much hESC research is based on the transfer of information from mouse ESCs to hESCs. Currently there is a 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.

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 work has the potential to completely alter fundamental concepts about the derivation and handling of hESCs, as well as their fate. 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.

In summary, 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 team, reagents, and environment are uniquely suited to the proposal. One reviewer thought that this proposal is the most important proposal s/he has seen in the three cycles of review that this reviewer has taken part in. The research plan is outstanding and will without doubt produce meaningful results within the 4 year timetable. 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, and 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 reviewers has seen. The PI is extremely strong and the team 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. Established collaboration has been developed with the senior author 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. 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; the PI will be 97% committed when a few of them expire in 2007. Consequently, the 30% salary support requested by the PI needs administrative attention. This is especially important since the PI has been cited as a collaborator, supporter, and general consultant on many other grants applications submitted to the CIRM. Epigenetic regulation may be important during early development as imprint marks are established. The PI and 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.

DISCUSSION: This proposal is from a highly experienced developmental biologist with a strong collaborator. It is exceptionally well-written and very different from many others in proposing a full molecular characterization of several stages in young embryonic development, i.e. to construct a fate map of the early human embryo. The PI has access to the necessary tissue and has developed many reagents that will allow molecular characterization. The preliminary data presented are beautiful and exhaustive; 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 might 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 than we do about ourselves, and therefore that this work must be done.

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, Marcy
• Lansing, Sherry
• Sheehy, Jeff