Somatic cell nuclear transfer (NT) is a powerful research tool with the potential for creating unique cell and tissue sources for studies of disease pathogenesis and regenerative medicine. Creation of pluripotent mouse embryonic stem (ES) cells using NT has been achieved and the prospects for generating human ES cells by NT are promising. However, there are only a handful of researchers who have reported their experience with NT and development of this approach in California would benefit from increasing dedicated efforts toward this goal. We have assembled a team focused on NT that has achieved several experimental milestones that motivate these proposed studies of NT in human oocytes. These prior achievements include NT in mouse oocytes, efficient production of novel ES cells from mouse embryos, and controlled enucleation of recipient human oocytes. With CIRM SEED funds, we will use systematic approaches to identify conditions that generate multipotent human cells from NT into human oocytes, with the goal of eventually producing new patient-specific human ES cell (hESC) lines for studies of disease pathogenesis, transplantation and tissue regeneration.
Successful outcomes from this proposal would enable us and others to generate new ES cell lines to study the pathogenesis of human diseases. Discovery of molecular mechanisms underlying diseases inevitably will produce novel strategies for diagnosis, prognosis or therapeutics. For instance investigators here have provided evidence for dysregulated signaling through the NFAT/calcineurin pathway in Down syndrome (DS), which arises in patients with trisomy for chromosome 21. Development of diagnostics or therapies that exploit the tenets of this model would surely be accelerated by tests of the model with embryonic human tissue harboring the classic trisomy 21 karyotype. Currently, such embryonic tissues for experimental studies are not available we postulate that a human DS ES cell line generated by NT could be used to develop differentiating embryonic human tissues for study of dysregulated signaling in vitro. Similar logic would justify generating patient-derived ES cell lines to produce experimental systems for studies of other diseases, including childhood congenital malformations, sickle cell disease, or neurological disorders lacking models or a defined pathophysiologic basis, like amyotropic lateral sclerosis (ALS).
The generation of ES cell lines by nuclear transfer can be inefficient, and initial attempts to produce nuclear-transfer-derived blastocysts from human somatic donor cells have been unsuccessful. There are only a handful of researchers who have reported their experience with somatic cell nuclear transfer (SCNT) and development of this approach in California would benefit from increasing dedicated efforts toward this goal. Successful outcomes from the research proposed here would identify California as a center of nuclear reprogramming and SCNT. California institutions would benefit from the ability to create unique patient-specific embryonic stem cell lines and disease models from SCNT. In turn, this would accelerate progress in developing new therapeutic and diagnostic strategies for diverse human disorders. Progress in this area would attract researchers and others interested in using stem cells for disease study and treatment. Ultimately, these positive effects would likely improve human health in California and elsewhere.
SYNOPSIS: Armed with experience and success in somatic cell nuclear transfer (SCNT) in mouse, the investigator proposes to generate novel human embryonic stem cell lines from human embryos generated by SCNT. This proposal contains two specific aims. The first includes studies to optimize nuclear transfer in human oocytes. The second specific aim is the identification of methods to generate blastocyst-stage embryos with SCNT. Primary human fibroblasts or cultured fibroblast lines will serve as nuclear donors for specific aim 1. The oocyte donation will involve collaboration with Dr. Behr, who is the head of obstetrics and gynecology and the IVF laboratory at Stanford University Medical Center, and who has the appropriate protocols (no letter of collaboration was provided in the present application). This project can not be funded by the NIH and is the type of high risk, high reward project that CIRM is uniquely able to fund.
INNVOATION AND SIGNIFICANCE: This proposal is of the highest caliber in terms of innovation and significance. After the Korean debacle on SCNT, there is currently no evidence that SCNT approaches can be used in humans. SCNT, however, has been successfully performed in a variety of animals, clearly establishing the feasibility of the technology. There is no reason to believe that provided with the right combination of technology and talent it will not work in humans. This proposal has a very high significance, as it boldly addresses this issue, and the reviewers strongly believe that this technology will work, given the appropriate infrastructure. Finally, the NIH cannot fund this grant.
STRENGTHS: The lead investigator is part of a team assembled specifically to focus on nuclear transfer. In fact, the group assembled seems to be one of the most qualified to attempt this type of research. The team and the institutional environment are such that if success in SCNT is to be achieved, there is a high likelihood that it could be here.
Another strength of this proposal is that the investigator is proposing to take a systematic approach toward optimizing nuclear transfer and culture of embryos to blastocyst stage. Overall, the proposal is well written, well planned, and well substantiated by preliminary data.
WEAKNESSES: This is an extremely high-risk, but high-reward, project. As acknowledged by the author, the scientific risks of strategies to achieve nuclear reprogramming by SCNT with human cells are self-evident.
There is the lack of a good disease target for SCNT. The only disease mentioned is Downs Syndrome, which would be better modeled from available PGD embryos, not via SCNT. The best disease candidates for SCNT are complex genetic diseases not easily assessed by PGD. There is no such disease presented nor any description of how they might procure somatic donor cell lines from patients with a suitable disease.
An additional weakness is the proposed use of RNAi to modulate levels of DNMT1. Knock-down of DNMT1 in differentiated cells is incompatible with their growth and survival; while this could potentially be overcome in the donor somatic cells there is no strategy presented that would make the resulting hES cells usable in terms of differentiating them. In fact, there is no evidence that hES cells can tolerate a loss of DNMT1.
Many of the other proposed improvements in aim 2 have already been evaluated in normal human embryo culture with extremely limited success. Thus, there are no experiments presented that would perhaps ovecome the previous problem of cleavage arrest of SCNT embryos noted in the preliminary results. The PI should explore the use of cleavage stage embryos for the derivation of hESC lines as there is clear evidence that they can generate this type of embryo. They might also take a cue from Boiani and Scholer and consider aggregating cloned embryos to improve their development to the blastocyst stage.
Finally, a major issue here, as in any project of this nature, is the availability of enough human oocytes to perform all the experiments required to optimize SCNT in humans. What will the investigtors do if there are no oocytes donated/available for this research?
DISCUSSION: A discussant noted that SCNT has been done in UK with human cells, (University of Newcastle by Murdoch) where investigators made hESC blastocyst but did not derive any lines. The point was made that the investigators would be well advised to try to diversify the source of oocytes, so as not to have 'all of your eggs in one basket'.