New Strategies to Understand Reprogramming Events in the Donor Nuclei Following Somatic Cell Nuclear Transfer
The possibility of making embryonic stem cells (NT-ESC) using a process called somatic cell nuclear transfer (SCNT), popularly known as “therapeutic cloning,” has captured the imagination of the scientific community because of the technology’s remarkable potential. Patient–specific embryonic stem cell lines have the potential to revolutionize regenerative medicine. When cells, tissue or even entire organs are damaged by disease, we may soon be able to make repairs using replacement cells created through the therapeutic cloning process. This technology involves removing the genetic material inside a donor egg (oocyte) and replacing it with a mature cell to generate embryos, and subsequently disease- or person-specific embryonic stem cell lines. For NT-ESC research to make any significant progress, a substantial number of eggs and embryos will be required. Because there is such a shortage, the eggs and embryos that are donated must be utilized in the most efficient way possible. Unfortunately past SCNT experience has taught us this technique can create embryos that appear normal, but because of improper cell development, are defective. A critical time in that development comes during the first few seconds after the replacement cell and empty egg come together. This project will focus on developing a new method of monitoring the molecular events during these important moments. This time frame is significant because genetic material in the donor cell must be rejuvenated (be returned to an immature state) for proper development of the embryo and the subsequent successful creation of an ESC line. Finding the source of defects will depend on observing how the replacement cells interact with the egg. Having the ability to observe this interaction without retarding further development would be an important scientific advance, one that could allow researchers to improve the success rate of SCNT, thus maximizing the limited genetic resources of eggs and embryos. Support for this project is intended to train an early career scientist who will collaborate with highly skilled individuals from a number of research disciplines, including human embryology, SCNT, non-toxic fluorescent imaging of cells and the assessment of developmentally important genes in cloned embryos. It presents a tremendous opportunity to bring a fresh mind into this research and train them under some of the foremost experts in the field. Although the proposed research is not primarily intended to lead to a specific therapeutic application (although it might), it is designed to provide a strong scientific basis for any such research in the future because the findings will be made available to all scientists through expected publication in scientific journals.
Statement of Benefit to California:
The projected annual economic impact of Alzheimer’s, Parkinson’s and other presently incurable degenerative diseases is staggering. Alzheimer’s disease alone is projected to cost California’s economy $10 Billion per year. These degenerative diseases, along with cystic fibrosis, spinal cord injury, macular degeneration, and others, are all potential targets for the development of treatments developed through the research of embryonic stem cells. Perhaps the most promising type of stem cell research is somatic cell nuclear transfer (SCNT), also referred to as “therapeutic cloning.” It involves removing the genetic material (DNA) from a donor egg (oocyte), and replacing it with an adult cell from a person with a degenerative disease who might benefit from stem cell therapy. After this new cell develops and multiplies, it may be used to create embryonic stem cells that would not be rejected by the person requiring treatment. These stem cells could then be used to replace brain cells destroyed by Alzheimer’s, nerve cells damaged by stroke or Parkinson’s, abnormal lung tissue from cystic fibrosis, or spinal cord nerves damaged in a traumatic injury, and, as has been shown recently in a study from the University of Washington, retinal cells damaged by macular degeneration. The efficient use of human eggs donated to the program is essential if the full potential of therapeutic cloning is to be realized. Unfortunately past research on SCNT has taught us that this technique can create embryos that appear normal, but because of improper cell development, are in actuality defective. A crucial time in development comes during the first few seconds after the replacement cell and donor egg come together. Critical to this process is an understanding of the events that transpire during this time. The newly created cell must resume the correct pattern needed for early embryonic development and the subsequent isolation of embryonic stem cell lines with therapeutic potential. This research is targeted at developing a new non-destructive technique that would allow these events to be monitored while permitting the embryo to continue its normal development. In this way, the usefulness of different cell types and procedures can be directly evaluated to improve the efficiency of SCNT and to reduce or eliminate those with abnormal development. The resulting cells will be carefully assessed using recognized procedures for assessing chromosome number and correct expression of developmentally important genes. An important additional benefit of this type of research is that it may assist in the development of more effective and efficient treatments for the many Californians with infertility, by providing a greater understanding of methods used to detect normal and abnormal early embryonic development. By identifying and utilizing only normal embryos for treatment, higher pregnancy rates with fewer embryos utilized may be possible.
SYNOPSIS: In this proposal the PI proposes the development of a new diagnostic tool, using in vivo imaging to dissect the events underlying donor nuclear remodeling and the formation of spindle chromosome complex (SCC) in cloned human embryos obtained by SCNT. The proposal is to fluorescently label a group of spindle associated proteins to permit live imaging of the assembly and formation of SCC followed by in vitro manipulations such intra-cytoplasmic sperm injection (ICSI), aparthenogenic activation and somatic/embryonic cell nuclear transfer. He attacks the problem at three different levels of specific aims. SIGNIFICANCE AND INNOVATION: The first specific aim is to observe the role of spindle-associated proteins in the formation of the spindle chromosome complex (SCC) following SCNT to that fertilized parthenogenetic human embryos using non-toxic fluorescent imaging technology. The second specific aim is to determine the consequence of adult versus embryonic donor cell type and modification to chromatin structure prior to nuclear transfer in their ability to form or restore normal spindle formation following SCNT. The third specific aim is to allow embryonic development to continue on that assessment of chromosome stability (mitotic defects) and reprogramming (correct expression of developmentally related genes) can be assessed. This is among the top innovative as well as significant proposals. It has become very clear that with human oocytes on short supply, it is imperative to develop consistent procedures that make the most effective use of them. This will lead ultimately to better-cloned embryos, which in turn will provide better ESC toward the realization of therapeutic cloning. This is a novel proposal with potentially important and significant results, though the proposal as outlined makes it unclear whether highly significant endpoints will be achieved. STRENGTHS: Previous studies have clearly demonstrated that cloning is very inefficient and this has been attributed to stochastic epigenetic phenomenon affecting nuclear reprogramming. Beginning with murine, bovine and porcine oocytes and ultimately directly in human oocytes, the simple and comparative observation that correlates the state of the donor nucleus, may it be in function of the cell cycle or timing of development, represents the strength of this application. Live imaging provides the ability to appreciate the dynamics of nuclear remodeling and would be a great substitute for the current steady-states type of measurement such as RT-PCR and biochemistry. This proposal does evaluate the basic biology of nuclear structures and spindle complexes that may be altered following SCNT. In particular, it is of interest to determine changes in differences in these cell processes following nuclear transfer from using nuclei of different cell sources such as fibroblasts or embryonic stem cells. The live imaging planned using fluorescently labeled spindle-associated proteins should provide a very interesting assessment of these spindle complexes and would likely achieve some important insight into these processes following nuclear transfer. WEAKNESSES: A weakness of the proposal remains the fact that there is no precedent for human cloning, and this study, while aesthetically beautiful and informative, might not help directly. On the other hand, this is the only way to figure it out. There are multiple problematic issues with this proposal: 1. There is no stated hypothesis, and this is a purely descriptive study of these changes post-SCNT. It would be helpful to more clearly state a hypothesis such as assessment of differences in spindle complexes using nuclei from different cell sources. 2. There is little information about how these spindle proteins will be fluorescently labeled. While they intend to use a commercially available labeling reagent, some reference or preliminary data showing feasibility of this method in other studies would be helpful. 3. There is no description of how these studies will be quantified. While they do state that six treatment groups will be studied, it is unknown how many nuclear transfer products or fertilized oocytes in each group will be evaluated and how it will be determined if there are distinct differences in these spindle complexes between these groups. 4. There is no description of potential pitfalls and possible solutions that could be expected from these studies. 5. There is no identified post-doctoral fellow to do these studies. While the timeline states they think a post-doc can be found and in one month’s time, in reality, it could take significantly more time than this. 6. The PI, Dr. French, has funding as PI on three other projects and his percent time on those projects are 80%, 50%, and 40%. Therefore, he already has 170% of his time accounted for and it is unclear how he will complete these planned studies. 7. The structure of the Stemagen Research Foundation that appears associated with the Reproductive Sciences Center in La Jolla, California, has an unclear administrative structure. Dr. Samuel Wood is listed as CEO and recipient of this award, and it would be helpful to know that there is appropriate oversight and administrative structure with this research foundation. This proposal needs to be more hypothesis-based, and a clearer plan for quantitative analyses of differences between different groups (oocyte treatments or spindle-associated proteins) needs to be outlined. There also needs to be better reference and feasibility of the methods for live fluorescent imaging of these various spindle-associated proteins. DISCUSSION: There was agreement that SCNT needs to be supported and that there is a limited number of places that can conduct this work. The science is interesting but the application lacks a hypothesis. Everything is descriptive, and the applicant neglected to mention many things: how results will be quantitated, the future impact of the work, the direction, as well as a description of anticipated problems and potential solutions. It was agreed that the work proposed is innovative but perhaps not ready for prime time. it was also noted that the applicant is funded at 170%.