The ultimate goal of the work described in this Comprehensive grant proposal is to develop methods to reliably derive new human embryonic stem cells (hESCs) without destroying the embryo, something that is required with current methods. This approach is designed to improve the efficiency of hESC derivation while significantly reducing the ethical objections to hESC research - the major objection to hESC research is that the current derivation method requires destruction of the human embryo. We propose to use the embryo micromanipulation techniques developed for the clinical technique which diagnoses embryos before the are implanted - pre-implantation genetic diagnosis (PGD) - to isolate single cells from developing embryos for generation of new hESC lines. During PGD, one or two cells are removed from the early embryo for genetic analysis; the resulting embryo retains its ability to develop into a normal human baby. While the proof-of-principal of this approach has recently been published, three major questions still need to be addressed, and all three will be addressed in the work described in this proposal: 1) How can the efficiency of establishing hESCs from single embryo cells be improved? Published methods demonstrate a very low efficiency of hESC derivation - less than 5% of attempts are successful; 2) Can it be shown that the single embryo cells can be used for both hESC derivation and PGD without compromising the clinical genetic diagnosis? Published literature suggests that single embryo cell culture before genetic analysis significantly reduces the clinical ability to detect certain genetic defects. 3) Are single embryo cell-derived hESCs similar to those derived from inner cell masses (ICMs)? The "gold-standard" for hESCs today is a hESC that was derived from the ICM during destruction of the human embryo. The results of our comprehensive analysis of the single embryo cell culture technique will allow us to determine whether single embryo cell hESC-derivation is a viable alternative to current methods that use whole embryos to derive new hESC lines. An important corollary to this research is that it may greatly improve our ability to derive hESCs from embryos with very rare genetic diseases, embryos themselves that are very rare. As this research involves the manipulation of human embryos, it cannot be funded by the federal government at the present time; thus, CIRM funding is critical to this research project.
Statement of Benefit to California:
Besides the considerable scientific obstacles that human embryonic stem cell (hESC) research has to overcome before it leads to a therapeutic reality, the ethical controversies associated with this research are likely to continue to haunt it. Even if a therapy is devised using hESCs that were derived during destruction of the human embryo, there will be some members of society who, in conscience, will be loath to or even refuse to use those therapies. It is thus morally incumbent upon scientists in this field to endeavor to devise methods to derive hESCs that do not incur the death of the embryo. This proposal describes work whose sole purpose is just that, to derive new hESCs without harming the embryo. The obvious benefits to the people of the State of California of this proposed research, therefore, are two-fold: 1) The major ethical objection to hESC research will be eliminated, and 2) Therapies resulting from use of these new cell lines will be palatable to a greater proportion of the citizens of California. In addition, the method will allow a greater efficiency of hESC derivation from embryos, especially those with very rare genetic diseases. These hESCs will be extremely useful in defining the detailed nature of these genetic diseases and in providing cells that may be used to search for new therapies. Finally, not only will the stem cell banking expertise of the Principal Investigator be used to supply these new hESCs to other qualified investigators, the new techniques themselves will be incorporated into the hESC culture training program that the Principal Investigator directs, allowing for other investigators to be trained in these new methods.
SYNOPSIS: The aim of the proposal is to develop methods to reliably derive new hESC lines without destroying the embryo. The applicants intend to use embryo micromanipulation techniques that have been developed for preimplantation genetic diagnosis and isolate blastomeres from developing embryos to generate new hESC lines. The approach is based on a recent Nature paper by the Lanza laboratory that reported positive results on human embryonic stem cell lines derived from single blastomeres, although another 2006 paper by Bongso’s laboratory reported negative results. The applicants will study how the efficiency of this technique can be improved, whether single blastomeres can be used both for hESC derivation and preimplantation genetic diagnosis without compromising the clinical genetic analysis, and finally, whether blastomere-derived hESC are similar to those derived from inner cell masses (ICM). IMPACT AND SIGNIFICANCE: The proposed research carries the objective of alleviating some of the ethical problems associated with hESC research through developing the ability to efficiently derive hESCs from individual biopsied embryonic blastomeres. Essentially, the PI proposes to obtain cells as a byproduct of the clinical technique of preimplantation genetic diagnosis in which one or two blastomeres are removed from the cleavage stage embryo and the isolated cells are subjected to genetic analysis. This way, the embryo can continue to develop in culture and, if the genetic analysis shows no evidence of abnormality, can be implanted to produce a pregnancy. Success with this approach that does not require the destruction of the embryo would likely eliminate ethical objections associated with the use of entire blastocysts and will address the issue of efficiency of hESC derivation. The significance to the general field of stem cell biology is limited since the focus is on improvements in blastomere culture conditions and not directly on studies that might increase our fundamental knowledge concerning hESCs. Also the innovative component is limited since the authors are using a recently published proof of principle publication as a springboard. QUALITY OF THE RESEARCH PLAN: The research plan is divided into 3 phases with the second two phases highly dependent on success in phase 1. Phase 1 proposes to develop improved culture conditions for growing isolated blastomeres. While the proof of principle for establishing hESC from single blastomeres has been reported in at least one publication, the efficiency is less than 5%. In order to improve the success rate, the applicants propose to co-culture blastomeres with mouse or human embryonic stem cell lines that have been genetically modified in order to permit eventual elimination of these cells from the culture. The investigators will study critical variables in early embryo culture and investigate trophic factor supplementation and medium modifications. In vivo characterization will involve the "gold standard" of generation of hESC-derived teratomas. The approach is inadequately focused and it is impossible to evaluate feasibility given the number of variables proposed for testing and that the plan uses low quality embryos as the blastomere source. Although this decision was driven by ethical principles, there remains a major concern over the relevance of optimized culture conditions based on studies with inferior blastomeres. The second phase of the proposed research addresses the clinical utility of deriving ESCs from blastomeres and concerns the potential co-use of blastomeres for PGD and ESC derivation. The third phase will compare blastomere-derived hESCs with blastocyst derived lines by global gene expression analysis, genotyping and global methylation analysis. Without a doubt, differences will be documented and the challenge is to relate those differences to the fundamental properties of ESCs. The actual experimental work will provide information of value, but it is not based on hypotheses nor can it be considered scientific in the general sense of the word. Rather, the work has more to do with technical development of methods for deriving hESCs from blastomeres rather than blastocysts. STRENGTHS: The possibility of deriving new hESC lines without harming the embryo is desirable and would facilitate laboratory work in this area of research. This all depends on whether the investigators are capable of repeating the positive study reported by the Lanza lab and, equally importantly, whether they can increase the efficiency from less than 5% to a practically useful percentage. If successful, a major ethical objection to hESC research would be eliminated. The assembled team is capable and experienced with hESC culture. The institution has strong collaborative ties with the Burnham Institute for Medical Research in San Diego, especially their Stem Cell Center, as well as with USC and the Coastal Fertility Medical Center, where solicitation of embryo donation, embryo culture, and blastomere extraction will take place. WEAKNESSES: While the investigator is experienced in extraction of blastomeres for preimplantation genetic diagnosis, there are as yet no preliminary data to support the notion that they are capable of growing cell lines from such cells so feasibility appears low. The Preliminary Data section is underdeveloped in general and inappropriately includes biographical information about the investigative team. This section currently focuses on a description of techniques available to the PI. Use of the published approach as a starting point should have been included in preliminary results. This section requires enrichment with actual data demonstrating initial success in some of the planned experiments. Another weakness of this proposal is that it is not hypothesis-driven and is more a plan to improve technology in the area of stem cell research rather than answer fundamental basic or clinical questions about hESCs. The proposal relies too much on a successful outcome of phase 1 which is hampered by the proposed use of low quality starting material. The rationale for parts of the experimental plan is not clearly developed. Also, since, by design, cells will be obtained in the course of prenatal diagnosis, there is likely to be a high incidence of blastomeres with genetic defects, which would not lend themselves to eventual clinical application. While hESC from embryos with specific genetic abnormalities might be of interest scientifically, it is not even known whether they could be expanded into cell lines. Although the proposal is focused on alleviation of ethical problems, the approach would still be subject to the criticism that if you can derive hESCs you likely could produce an embryo that could support a pregnancy. Finally, although commitments from several IVF programs are documented, the ability to obtain the large numbers of embryos required to support the proposed research may be limiting. The described work is certainly worthwhile, but it needs to be refocused and expanded into mechanistic and biologic, rather than purely technical, goals and hypotheses. This in turn will require additional collaborators who are more scientifically oriented and who have more extensive histories of scientific publications. DISCUSSION: The proposal is based on the attractive idea of improving the efficiency of deriving hESC lines from blastomeres without destroying the embryo. The technology has been demonstrated in one publication but was very inefficient. The PI is quite experienced and includes strong collaborators although one reviewer took issue with the strength of the overall collaborative team. There were concerns about the use of low quality embryos as the starting point for increasing the efficiency of hESC line derivation from blastomeres; about the team's lack of experience with the technique of deriving cells from blastomeres; about the lack of preliminary data and about the availability of getting sufficient embryos to do the work.