Funding opportunities

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

Funding Type: 
New Cell Lines
Grant Number: 
RL1-00679
Funds requested: 
$909 619
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
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: 
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 Summary: 
Executive Summary 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. Reviewer One Comments Significance: 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. This is an under-explored source of potential embryos for hES cell derivation though by necessity the supernumerary nuclei will have to be removed which may prove technically demanding and not reproducible. Feasibility: The overall goal of this project is to develop new human embryonic stem cell lines from eggs that have been fertilized by multiple sperm that are routinely discarded due to their aberrant genetic makeup. To date there have not been reports of human embryonic stem cell lines derived from this source of eggs. One concern is the fact that these eggs would have abnormal numbers of nuclei. Therefore, one associated problem with generating stem cell lines is that it involves the removal of one of the male pronuclei, which in and of itself may be problematic as it will be difficult to determine with any certainty which nuclei are male. One additional feature is to isolate individual blastomeres from 8 cell embryos and to transfer each of them to an empty zona pellucida which will be obtained from eggs that fail to fertilize and to culture these to the blastocyst stage thus increasing the number of embryos for hES cell derivation. The applicants’ estimate that based on the number of IVF cycles in their local regional area per year, they could obtain 500 or so polyspermic eggs, suggesting this would give them a reasonable number of eggs from which to isolate cell lines. Although the research team has good reproductive medicine experience, their expertise with human embryonic stem cell technology would appear to be limited. Responsiveness to RFA: In terms of assessing pluripotency of resultant cell lines, the applicants propose to use standard methodology in terms of molecular, genetic and biochemical analysis of resultant cell lines, as well as teratoma assays. The applicants also say that once these cell lines are derived they will become available for clinical trials in the near future. This is naive in that these cells will not have been produced through GMP and will therefore not currently be acceptable for clinical applications. Reviewer Two Comments Significance: The investigator proposes to use discarded polyspermic eggs as a novel source of embryos from which to derive hES cells. Polyspermic eggs are readily available, yet their utility for generating hES cells remains to be determined. It is unlikely that they would serve any clinical utility as there are more feasible sources available. Though this is an innovative method, it is also cumbersome, in that it requires expertise in embryo manipulation. Feasibility: Dr. Ping and Vandevoort have extensive embryo culture / manipulation experience, but only Dr. Nolta has embryonic stem cell expertise and this appears to be more at an administrative level. The UC Davis stem cell program has adequate facilities, but I am surprised that the investigators did not identify specific personnel with more embryonic stem cell derivation and culture experience. This lack of hES cell derivation / culture experience is evidenced in the experimental methods, which are superficial and lacking in detail. Although Lanza’s 2006 paper on hES cell derivation from a single blastomere is referenced, the detailed Nature Protocols paper and a more recent report from Cell Stem Cells have not been referenced. The investigators who have yet to prove that polyspermic eggs / embryos can give rise to hES cells. Therefore, I see no scientific justification for adding a level of complexity by attempting to perform single blastomere hES cell derivation. Responsiveness to RFA: This proposal will likely generate pluripotent hES cell lines, but it is unclear how many lines. The utility of this method of derivation is limited (or at least unproven) and it is not clear how these hES cell lines will advance the field.
Conflicts: 

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