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RL1-00679-1: Establishment of human embryonic stem cell lines using re-constructed human embryos derived from polyspermic eggs

Recommendation: Not recommended for funding

Public Abstract (provided by applicant)

Using human embryos produced from in vitro fertilization laboratories for generating embryonic stem cell lines is always a major ethical concern. There is a valuable resource neglected in this field, which is the eggs containing two sperms (polyspermy). This type of fertilized eggs, called tripronuclear zygotes, is routinely discarded in the in vitro fertilization laboratories. Approximately 7% of fertilized eggs contain more than one sperm, with tripronuclear zygotes being the most common polyspermic phenomenon. There is some evidence that tripronuclear zygotes could develop into normal embryos. More recently, a live birth was achieved by removing extra male pronucleus in a zygote. In pigs, the normal piglets were delivered by transferring the polyspermic eggs confirmed by microscope. The principal investigator for this proposal also reported in 2001 that polyspermy in pigs could be a physiological phenomenon if extra sperm did not affect the embryonic genome. Although there is a recent accomplishment of reprogramming adult cells into pluripotent stem cells by transfecting them with controlling genes, their pluripotency and safety remain concerns. Until the safety of reprogrammed cells is proven, it is important to continue to develop new lines of human embryonic stem cells from embryos. In this proposal, the extra male pronucleus from the tripronuclear zygotes will be removed by micromanipulation procedure and resulting zygotes will be cultured to the blastocyst stage when the inner cell mass can be used for establishing human embryonic stem cell lines. Once the technique is established, scientists will have many more options and numbers of embryonic stem cell lines for therapeutic applications and regenerative medicine. Future progress in using these cell lines for transplantation into humans will require many lines so that cells can be matched to recipients. Another important fact is that the abnormally fertilized polyspermic eggs are from large amount of diversity in our region, which will make the clinical applications more feasible in using newly established embryonic stem cell lines. To date, there is no report on utilizing these tripronuclear zygotes for generating new embryonic stem cell lines. Hence, there is an urgent need to preserve these tripronuclear eggs that are now being discarded. It is a valuable resource for generating human embryonic stem cell lines.

Statement of Benefit to California (provided by applicant)

Obtaining human embryonic stem cell lines is the prerequisite for developing therapeutic approaches. The proposed research is to use the eggs fertilized in vitro by two sperm . These eggs are routinely discarded in the in vitro fertilization laboratories. There is evidence that these eggs could develop normally after removing the extra sperm. These eggs are a valuable resource to generate human embryonic stem cell lines without the usual ethical concern of using normal human embryos. Although there is a recent accomplishment of reprogramming adult cells into pluripotent stem cells by transfecting them with four genes, their pluripotency and safety have not been proven and many researchers have concerns in this regard. Future progress in using human embryonic stem cell lines for transplantation into humans will require many lines so that cells can be HLA matched to recipients. The large amount of diversity in our region also makes the clinical applications more feasible. Therefore, the proposed project has a great potential of generating many new embryonic stem cell lines for clinical therapeutic applications.

Review

The overall goal of this project is to develop new human embryonic stem cell (hESC) lines from eggs that have been fertilized by multiple sperm (polyspermic eggs) during in vitro fertilization and are routinely discarded due to their aberrant genetic makeup. Supernumerary nuclei will be removed. In addition, the applicant proposes to separate cleavage stage embryos into individual blastomeres, and to attempt to derive cell lines from individual blastomeres.

Reviewers noted that polyspermic eggs are readily available and they represent an under-explored source for hESC derivation. Though this is an innovative method, it is also cumbersome, since by necessity the supernumerary nuclei will have to be removed. This may prove technically demanding and not reproducible. To date there have been no reports of hESC lines derived from this source of eggs. Thus, the utility of polyspermic eggs for generating hESC remains to be determined. Furthermore, it may prove difficult to determine with any certainty which nuclei are male. Consequently, some resulting hESC lines will be androgenotes (containing only sperm-derived chromosomes), an outcome that was not appropriately addressed in the application.

One additional feature of this proposal is to isolate individual blastomeres from 8-cell stage embryos and to transfer each of them to an empty zona pellucida, which will be obtained from eggs that fail to fertilize. These will then be cultured to the blastocyst stage, thus increasing the number of embryos for hESC derivation. One reviewer stated that s/he sees no scientific justification for adding complexity to this project by attempting to perform single blastomere hESC derivation.

The applicant states that once these cell lines are derived they will become available for clinical trials in the near future. The reviewers pointed out that these cells will not have been produced using good manufacturing practices (GMP) and will therefore not currently be acceptable for clinical applications. Furthermore, it is unlikely that the derived cell lines would serve any clinical utility as there are more feasible sources, excess frozen human embryos, available. Finally, the reviewers felt that although the research team has good reproductive medicine experience, their expertise with hESC technology appears to be limited.

Reviewer synopsis

The overall aim of this project is to develop new human embryonic stem cell lines using polyspermic eggs produced by IVF which are otherwise routinely discarded. In addition, the applicants propose to separate cleavage stage embryos into individual blastomeres, and attempt to derive cell lines from individual blastomeres.

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:

• None