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.