Establishment of human embryonic stem cell lines using re-constructed human embryos derived from polyspermic eggs
Public Abstract Using human embryos produced from in vitro fertilization laboratories is always a major ethical concern. There is a great resource neglected in this field, which is the eggs containing two sperms. This type of egg, called tripronuclear zygotes, will be routinely discarded in the in vitro fertilization laboratory. Approximately 7% of fertilized eggs contain more than one sperm, with tripronuclear zygotes as a most common phenomenon. There is some evidence that tripronuclear zygotes could develop into normal embryos. Recently, a normal, live human birth was achieved by removing the extra male pronucleus in the zygote. Previously, 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. Therefore, in this proposal, the extra male pronucleus from the tripronuclear zygotes will be removed by a microsurgical procedure and the 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, the scientists will have a valuable resource in using the ESC lines for therapeutic applications and regenerative medicine. The most important aspect of this work is to further study the biology and differentiation of human embryonic stem cells. To date, none of the fertility clinics in the Northern California reserves or freezes these tripronuclear zygotes for research studies. Hence, there is an urgent need to bring attention to this unique resource in the field of human embryonic stem cells.
Statement of Benefit to California:
Summary of benefit to California Obtaining human embryonic stem cell lines is the prerequisite for developing therapeutic approaches. The proposed research is to use the eggs with two sperm inside that are routinely discarded in the in vitro fertilization laboratory. There is evidence that these eggs could develop normally after removing the extra sperm. It is a valuable resource to generate human embryonic stem cell lines without ethical concern of using human embryos.
This investigator proposes to establish new hESC lines, characterize them, and to optimize culture methods for maintenance of pluripotency. This proposal has three specific aims. The first is to establish new hESC lines using human polyspermic eggs produced by in vitro fertilization. The second is to optimize the culture methods for the purpose of maintaining their pluripotency. The third specific aim is to characterize the hESC lines by karyotype, activities of biochemical markers, expression of molecular markers, and analyses of in vivo/in vitro pluripotency. The NIH cannot fund this grant, as it proposed new derivation, albeit from abnormal embryos – polyspermic eggs discarded in the IVF laboratory. SIGNIFICANCE AND INNOVATION: This is a modestly innovative but potentially highly significant proposal to derive new human embryonic stem cell lines from embryos that were created by using eggs that are polyspermic, that is fertilized by two sperm such that they have three pronuclei. Effective use of these polyspermic eggs combined with derivation of ES cell lines separated from individual blastomeres have a highly significant impact in this field. The significance of this is based mostly on overcoming ethical concerns surrounding destruction of human embryos for the specific purpose of generating hESC lines. STRENGTHS: This proposal is relatively short but well laid out and the aims are straightforward, logical, consistent, and feasible. This group will have no problem having access to these polyspermic eggs and the methods described should be feasible. The characterization of the human ES cell lines with particular attention to karyotype analysis as well as evaluation of pluripotency in vitro and in vivo are nicely outlined. WEAKNESSES: 1. This group plans to derive ES cells only by separated blastomeres similar to what was recently reported by Lanza et al. It is unclear why they don’t also attempt to culture some embryos to blastocyst stage and extract the inner cell mass. Presumably this would be possible after the removal of the extra male pronucleus as these embryos are thought to effectively reach the 8-cell stage. It would be useful to compare this method directly to the separated blastomere method grid. 2. As the strategy to use separated blastomeres and place them into empty zona-pellucida growing to blastocysts and deriving ES cells remains largely unproven, alternative strategies would be useful. 3. There is no description of the number of embryos that are planned to be tested or needed for these studies and how many ES cells lines will be derived and thought to be sufficient. 4. Comparison of the methods described to more standard methods even using polyspermic eggs with one pronucleus removed would be useful. Perhaps additional studies to look at lineage-specific development of these new ES cell lines to well-defined lineages such as neurons, blood, or cardiomyocytes could be done as part of these studies. DISCUSSION: Generating new ES lines is important. Here, the PI proposes to separate blastomeres at the 8-cell stage as Advanced Cell Technologies (ACT) has done, which is work that has not been reproduced. The PI has relatively little expertise with the technology, and there are many technical issues with the grant. Another concern expressed in the discussion is that the approach of using polyspermic eggs is presumably taken to meet ethical objections, but is not compelling on those grounds.