Embryonic stem cells have tremendous potential value for the treatment of many diseases and injuries, but like other transplants their use is likely to be limited because the immune system of the recipient will probably reject the transplant unless there is a good genetic match between donor and recipient. The ideal solution to this problem would be to replace the genetic material of stem cells with genetic material from the prospective patient, and to use the genetically modified stem cells in therapy. Work on experimental animals has shown that it is possible to do this by implanting a nucleus from one of the patient’s body cells into an oocyte from which the nucleus is removed. The oocyte is then allowed to grow into an embryo, and stem cells are derived from the embryo when it reached the appropriate stage. This method suffers from several drawbacks, the most significant being the need for oocyte donors, the technical difficulty of performing the nuclear replacement, and ethical objections to the production of embryos which are later to be destroyed for the benefit of the patient. We have devised an alternative strategy in which the nuclei of existing stem cell lines will be replaced by nuclei from cells of the prospective patient, by physically fusing together the stem cell with a body cell from the patient under condition preventing the survival of the stem cell nucleus. The resulting cell lines, with the genetic material from the patient in stem cell cytoplasm, will then be grown up and should be suitable for use in cellular therapy. In the proposed research we will compare several methods for incapacitating or removing the stem cell nucleus from the fused cells, and we will rigorously test the resulting cell lines for the correct genetic makeup as well as the retention of developmental properties characteristic of stem cells.
Statement of Benefit to California:
California has taken the lead in the nation in providing support for stem cell research and especially in support for developing the use of human embryonic stem cells in cellular therapy for many diseases and disorders. Many laboratories are investigating methods to control the differentiation of these cells, so that they will no longer be likely to produce tumors in the patient, and they will be more suited to regenerative therapy in specific organs. But the full potential of these cells cannot be met until a solution is found to the problem of immune rejection by the patient. Claims made outside the U.S. for success in generation of stem cell lines that are genetically tailored to the patient turned out to be fraudulent, so the field still awaits a solution to this problem. The conventional approach to generating patient-specific stem cell lines would be to transplant a somatic cell nucleus into an oocyte obtained from a donor, then allow the oocyte to develop into an embryo and recover stem cells from that embryo. However, this has several drawbacks as stated elsewhere in this proposal. California could continue to lead the nation in embryonic stem cell research and in developing the use of these cells in cellular therapy if a solution cold be found to the problem of immune rejection. Here we propose a novel but feasible method of generating patient-specific embryonic stem cells from existing stem cell lines, avoiding the problems of obtaining egg donors, and of generating embryos for later destruction. If this method could be developed in California it would allow the state to continue the momentum that has built up in stem cell research, and to continue to build upon it by moving to the next stage where these cells are actually used in the clinic to treat some of the most devastating injuries and diseases affecting our people.
SYNOPSIS: The proposed studies will seek to develop alternative methods to re-program somatic cell nuclei to an ES cell state using cell fusion. The experiments will be performed in the human system using hES cells and human neural stem cells (hNSC). Briefly, the nucleus of the hES cells is incapacitated by treatment with Actinomycin D (to promote nuclear degeneration, either before or after fusion), while the hNSC cytoplasm will be incapacitated with a mitochondrial poison (e.g., Rhodamine 6G). Following fusion, the only cells that can survive in principle are hybrids with the nucleus from the somatic cell and the cytoplasm from the hES cells. An alternative method to label the hES cell nucleus with a vital dye, and destruction of this nucleus by irradiation following fusion will also be explored. Any surviving hybrid cells will be evaluated genotypically and phenotypically. The PI will utilize hNSCs and hES cells that are genotypically distinguishable using DNA microsatellite markers. These microsatellite markers will need to span the entire genome to insure the exclusion of ES cell-derived nuclear DNA. Haplotype specific mitochondrial DNA markers will be used to identify the origin of these organelles in any viable hybrids. Particular emphasis will be placed on the induction of pluripotency markers such as Oct4 and Nanog in the somatic nucleus. SIGNIFICANCE AND INNOVATION: If successful, the studies in this proposal may alleviate 2 major problems that will arise in re-programming of somatic nuclei to generate patient-specific hES cell lines. First, a need for oocytes (and oocyte donors) will be eliminated, and second there will be no need for sophisticated nuclear transfer technologies. The heart of the proposal lies in a complementation approach where an hES cell with an incapacitated nucleus, and a somatic cell with incapacitated cytoplasm are fused to obtain hybrids. These are then cultured in conditions that require both mitochondrial and nuclear functions for survival. It is hoped that any resultant viable hybrid will have the DNA content (genotype) of the somatic nuclear "donor" cell and the cytoplasm and mitochondrial component originating from the starting hES cells. It is further hoped that the hES cell cytoplasm will re-program the somatic nucleus to an ES cell fate. One reviewer felt that the proposed studies are innovative and significant, while another disagreed, saying that the work would seek to refine an already well established and widespread technique for reprogramming somatic cells via fusion with human embryonic stem cells. Nonetheless, reviewers clearly felt that the ability to re-program somatic nuclei without a need for oocytes would be a major advance. Whether this will be possible or not remains an open question; however, recent results suggest that a limited number of factors may be sufficient at least to approximate effective oocyte-mediated re-programming. The approaches in this proposal are novel, although with significant potential complications. Overall, these studies are of high risk, but potentially very high significance. The PI proposes to use non-federally approved hES cells. Thus, these studies are not eligible for NIH funding. There is no compelling reason why federally approved hES cells could not equally be utilized for the proposed studies. STRENGTHS: The strengths of the proposal include its collaborative nature and the attempt to establish a system for creating patient specific hES cells that would not involve new embryos or oocytes. In addition, the proposed back-up plan of specifically labeling the donor nucleus and ablating it after fusion using a newly engineered device for precise gamma-irradiation is interesting. While it has been shown that fusion of ES cells with somatic cells often re-reprograms the latter to an ES cell state (at least to a very good first approximation), the resultant heterokaryons have fused nuclei and are therefore tetraploid. Even if methods could be developed to reproducibly reduce ploidy to a diploid state, the preferential exclusion of the genetic complement originally derived from the ES cell is difficult to envision. Clearly, this is not a general problem when using oocyte donors. The key to this proposal is to develop ways to incapacitate the nucleus of the hES cells and the cytoplasm of the somatic cell, respectively, prior to (or simultaneously with) cell-cell fusion. The PI presents some preliminary data to indicate that both poisons to be used are effective in both hNSCs and hMSCs. The proposed studies are contingent on obtaining viable and genotypically verified hybrid cell lines; thereafter, the remaining studies to evaluate the extent of re-programming would be straightforward. WEAKNESSES: The proposed studies are very risky, given the lack of evidence that viable, growing hybrids can be obtained and that these can proliferate for prolonged periods using the proposed methodologies. This is a major drawback to the proposed studies. The PI's preliminary efforts with hNSCs and hMSCs have not been successful, and there is a question of how tight the selection procedure actually is. Growing cells were shown to be contaminant poison "escapees", and given the rate at which they observed “escape” from the nuclear and mitochondrial poisons it is possible the hybrid cells are tetraploid or even not the product of fusion. Thus, there is little confidence that the proposed hES/hNSC fusion studies will be successful. Even if permanently growing cell lines are obtained, it is not at all clear whether the hybrid cells generated via this method actually contain the correct nuclear composition. Are the genomic complements original-cell-type homogeneous or mixtures originating from the two donor genomes? There is also concern that the cytoplasm of ES cells may not be adequate to re-program somatic cell nuclei. There does not appear to be any direct proof of this, at least to the knowledge of reviewers, and there is no evidence that reprogramming can occur without nuclear factors. The most well established cell for reprogramming is the metaphase II oocyte, which has no nuclear envelope and free access of nuclear factors to the cytoplasm. In fact, recent studies have suggested that 4 nuclear proteins are sufficient for re-programming at least to an approximate ES-like state. These are very real considerations that substantially temper the enthusiasm for this proposal. DISCUSSION: One reviewer recommends that this proposal be re-written to emphasize a unique laser/gamma irradiation technique that is both highly innovative and avoids many of the reviewers’ concerns.