n therapeutic cloning, a patient’s cell is combined (fused) to an enucleated donated egg (oocyte) from an unrelated woman or from another animal. It is hoped that cellular factors in the egg cytoplasm will reprogram the patient’s cell nucleus making it capable of generating replacement cells for the patient’s body. Thus, if a patient is suffering from Parkinson Disease due to loss of brain cells, these cells could be replaced with differentiated, individualized, nuclear-transplantation, embryonic stem (ntES) cells. While this strategy should generate ntES cells with the patient’s nuclear DNA (nDNA), it overlooks the fact that another part of the cell, the mitochondrion, also has DNA, the mitochondrial DNA (mtDNA). While the nDNA contains the blueprints for assembling the structure of the cell and body, analogous to carpenter’s plans for a house; the mtDNA contains the blueprints for the cellular electrical system, the wiring diagram of the house. It is universally agreed that mixing the nDNAs from two different cells would be destructive, yet the potentially disastrous effects of mixing different mtDNAs has been overlooked. In electricity, randomly mixing the components of two different integrated electrical circuits will result in short circuits. The same appears to be true for the cell. In mice in which we artificially mixed two mtDNAs, the resulting mice aged and died prematurely, had a striking increased frequency of cancer, and an increased mtDNA mutation rate. Moreover, in human studies, the accumulation of mtDNA mutations has been associated with aging and the development of cancer. Therefore, to document what happens to the mtDNAs during the creation and growth of htES and hES cells, we propose to create ntES cells by fusion of human cells to enucleated rabbit eggs and then to monitor the fate of the human and rabbit mtDNAs. We will also determine if the mitochondria and mtDNAs of hES cells can influence the differentiated state of tissue cells, investigate the nature and extent of mtDNA mutations that accumulate in ES cells, and determine if mixing different mtDNAs in cells is deleterious. Then we will determine the effects of these various mtDNA genetic factors on the power output of the mitochondria and its effects on the ES cell’s capacity to differentiate and potentially to form tumors. Finally, we propose to develop a series of procedures to control the origin and nature of the mtDNAs in ntES and hES cells and to use changes in mitochondrial function to regulate ntES and hES cell growth and regulation. It is our hope that by utilizing mitochondrial biology and genetics it may be possible to develop strategies for creating individualized stem cells without using donated oocytes.
Prolonged cell culture of human Embryonic Stem Cells (hESCs) frequently results in the loss of the cell’s capacity to differentiate on command into well differentiated cells. This eliminates their utility for generating replacement cells for use in cell replacement therapy to repair damaged tissues and organs within the body. The reason for this loss of developmental capacity by the hESCs is currently unclear, but we believe that a major factor contributing to the decline in the therapeutic value of hESCs is the accumulation of deleterious mutations in the mitochondrial DNA (mtDNA) of the cultured hESCs. The mtDNAs are located in the mitochondria which are organelles in the cytoplasm of the human cell. The mitochondria are responsible for generating most of the energy used by the cell and as a toxic by-product, the mitochondrial generate most of the endogenous reactive oxygen species (ROS). The mtDNA encodes key elements of the mitochondrial energy generating apparatus, and since ROS is a mutagen, the mtDNA is highly prone to acquiring mutations in these energy genes. These mutations then inhibit mitochondrial energy production which also results in increased ROS production. Increased mitochondrial ROS production stimulates the cell growth, so the cells with the mutant mtDNAs out grow the normal cells. However, the more rapidly growing cells with the mutant mtDNAs also have reduced mitochondrial energy production, which together with the increased ROS production, inhibits the developmental capacity of the hESC. In this research, we propose to establish that deleterious mtDNA mutations do in fact accumulate in hESCs over time and that they play an important role in the loss of the developmental potential of hESC cells. If we can confirm that this is a fact, then we should be able to greatly increase the therapeutic potential of hESCs by developing procedures for protecting the mtDNA of the hESCs from oxidative damage. This might be accomplished by growth of hESCs in the presence of mitochondrially target antioxidants. Furthermore, cells that had lost their developmental potential might be revitalized by simply replacing the damaged mtDNAs with good mtDNAs using our trans-mitochondrial cybrid technique. Thus, the proposed research has the potential of greatly increasing the therapeutic potential of all hESCs that will be developed in the State of California.
SYNOPSIS: This proposal addresses the dangers of mixing mitochondrial DNAs (mtDNA) during therapeutic cloning. The PI points out that during routine culture hESC lines acquire deleterious heteroplasmic mtDNA mutations, which are associated with increased neonatal mortality, premature aging, and an increase in cancer rate. He proposes to create inter- and intra-specific ntESC lines and carry out five Specific Aims:
1) Determine the origins and fates of the mtDNA genotype in ntES and hES cells
2) Use intra-specific cybrids and hybrids to reprogram cell nuclei and examine the effects of mtDNA incompatibility and de novo mtDNA mutations on hES cell propagation
3) Determine the effects of mtDNA genotype on ntES and hES cell mitochondrial physiology
4) Determine the effects of mtDNA genotype on ntES and hES cell differentiation and tumorigenesis
5) Develop procedures for manipulating ntES and hES cell mitochondrial genotype and physiology to control cellular replication and differentiation.
IMPACT AND SIGNIFICANCE: When nuclear transplantation is performed, any resulting cloned animals have invariably shown heteroplasmy between the mitochondria of the donor cell and the mitochondria of the recipient cytoplast. This sort of heteroplasmy can have profound phenotypic implications, including abnormalities in respiration, aging, etc.. When somatic cell nuclear transplantation is performed, similar types of heteroplasmy would be expected to result. The implications of such heteroplasmy on ES cells function are the topic of this application. The major impact of the work described in this proposal is that it will address the issue of whether mixing of mtDNAs during therapeutic cloning, and accumulation of mutant mtDNAs during hESC propagation, cause serious adverse outcomes in the cell lines. The PI contends this will predispose recipients of stem cell therapy to increased risks of neonatal death, premature aging, and an increase in the incidence of cancer. The value of the proposed work is directly related to the extent to which he will identify mechanisms for, and can design experimental approaches to block, these adverse events. These experiments should illuminate these issues, which are important for the long term use of ES cells for cell replacement or disease modeling and for understanding the affects that SCNT will have any resulting ES cell lines.
This proposal is both innovative and original, and this highly experimental work doing therapeutic cloning via interspecies methodology should be supported. The PI begins by developing experiments to recreate human-rabbit ntES cells and then analyze their mtDNAs. It is of the utmost importance that this prominent US laboratory should first create rabbit embryonic stem cells published recently by Fang et al, 2006 and determine the efficiency of creating such ES lines before the lab proceeds to human-rabbit ntES cells lines. There will never be enough human oocytes to make ntES cells and it is still not clear whether this method will be successful. Prominent laboratories have been trying unsuccessfully to make monkey ntES cells using up to 300 oocytes (personal communication to reviewer). No alternative suggestions have been given for the use of other interspecies models to make human ntES cell lines. This may not be possible. According to the PI’s original report, these cells should contain both human and rabbit mtDNAs combinations and would be non-viable. This highlights our lack of a basic understanding of mitochondrial DNA replication. It is important to understand the reasons for these failures and determine if mitochondrial-nuclear incompatibilities may cause some of the problems.
QUALITY OF THE RESEARCH PLAN: This research plan covers a lot of ground but stays close to the topic of the affects of various types of mitochondrial heteroplasmy on ES cells. The plan itself is very clearly and concisely written with a broad spectrum of strategies proposed including: the creation of human-rabbit ntES cells for mtDNA analysis; the induction of heteroplasmy through prolonged culturing; the reprogramming of differentiated cells using reconstituted cells; the preparation of hESCs containing pathogenic mtDNA mutations; the characterization of mitochondrial respiration and oxidative phosphorylation enzyme status; the assessment of reactive oxygen species status, oxidative damage, and apoptosis; the influence of different mtDNA genotypes on tumorigenesis; and finally, the use of this new knowledge to produce safer hESCs that are easier to propagate yet retain maximum differentiation potential. Given the ambitious nature of this plan, it is questionable whether the PI will be able to accomplish all of these goals according to the proposed timetable. The proposal could be more focused on the experiments that would produce meaningful results since the topic of whether the mitochondrial genome will impact embryonic stem cells and differentiation is very important. In fact, the application could be subdivided into potentially four separate grant proposals. The major contribution to the field would be to answer whether heteroplasmic mtDNA haplogroups, due to cloning or reprogramming, have a negative impact on stem cell differentiation and therapeutics.
STRENGHTS: A major strength of this proposal is the PI and the consortium of investigators that specialize in the application of mitochondrial genetics and physiology to human stem cell biology. In particular, the PI is the Director for the Center for Molecular and Mitochondrial Medicine and Genetics at UC Irvine, and was elected to the National Academy of Sciences in 1995. He is an established expert in mitochondrial biochemistry and physiology, and is extremely well funded through peer-review mechanisms through 2010. The proposal itself is very authoritatively written, easy to follow, and logically moves from basic observations in stem cells through studies of tumorigenesis and the development of new procedures to control replication and differentiation. This group has substantial experience in performing the mitochondrial manipulations and analysis proposed. It is imperative that such collaborations are built in regenerative medicine since it is clear that mitochondrial biology and mitochondrial DNA (mtDNA) genetics are of central importance in human aging, cancer, and developmental biology.
WEAKNESSES: The major weakness of the proposal is its ambitious nature, perhaps due to the limited space for presentation, which may have caused the planned experiments to be presented rather superficially. In general, the PI lays out the goals and strategies to achieve them, and then simply states what he will do by giving extensive literature citations rather than providing experimental details. Moreover, it is not clear from the format used which are his own publications. In this regard, the proposal can be confusing to read. An exception to this is the Preliminary Data section where one can gain a better idea of experimental approaches. This is not to say the PI is not capable of performing the experiments, only that it is difficult to critique the experimental plan positively or negatively from the information as it is provided.
One reviewer feels that the human-rabbit ntES cells line experiments have a low probability of working, and suggests eliminating them from this grant. The ideas are compelling and important, but this proposal could be subdivided into at least two separate applications with the ntES cell ideas included as part of a separate grant. This research should be considered in a separate proposal since so little is known concerning mitochondrial replication in primate embryology.
While it is possible that accumulation of de novo mtDNA mutations during embryonic stem cell propagation may increase the probability that the cells will either die by apoptosis or transform into cancer cells, it is very important that the applicant is aware that culture conditions, passage number, and methods of passaging the cells (trypsin, collagenase, and manual passage) may also produce chromosomal abnormalities and/or mitochondrial mutations. Growth rate data could be mis-interpreted by picking the wrong or fast growing colonies in the human embryonic stem cells lines which are often chromosomally abnormal. Functional studies should be emphasized in these experiments since both embryonic and somatic cells have high copy numbers of mitochondrial genome in their cells. More physiological measurements are needed to determine whether the embryonic stem cell lines develop severe OXPHOS defects due to premature aging or cancer.
DISCUSSION: One reviewer commented that this one-of-a-kind proposal is the best s/he read. This beautifully written proposal represents a strong, very well-organized, excellent investigation. The main criticism is that it is over-ambitious, and there was perhaps not enough room to describe all procedures to be performed by this superb team.
Panel members recognize that this is an interesting and largely overlooked field, but one reviewer, who is largely positive about this application, feels that the grant needs to be reorganized into separate proposals. Aim 1 proposes to follow-up on a publication from China – this is the aim of most interest to CIRM. However, there does not appear to be supporting evidence for a link between mtDNA and cancer, and this part of the proposal could potentially be funded by NIH. In addition, there is a feasibility issue with the rabbit/human systems work, which is important, but the PI’s strength is not in nuclear transplantation or embryology, and there is no one on the team to provide this expertise. Perhaps this work should be in a separate grant where the PI would team up with additional experts to provide some advice. That being said, the potential difficulties with the rabbit/human experiments may in fact be due to mitochondrial heteroplasmy, and since some researchers have claimed success in cross-species work with primates, CIRM should support following this up. We don’t know whether haplotypes will be involved in incompatibility or the nature of mitochondrial transcription factors in rabbits/primates. Addressing these types of questions is certainly the strength of this excellent PI and group, and this type of work should be the focus of this grant.
Overall, reviewers feel that this is a terrifically important area of research where CIRM has the opportunity to recruit a world expert. The team is outstanding with each collaborator being a world authority in one field or another, and while they may not have a track record in stem cell research, it would be a tremendous benefit to bring them to the field. The group again suggests that the PI recruit an expert in stem cells/SCNT/embryology.
One reviewer commented on a number of additional conceptual points to consider. First, the PI stresses that mitochondrial mutations can cause aging, cancer and mitochondrial diseases; however, this reviewer wonders whether mitochondrial heteroplasmy is the underlying cause of the tumors and premature aging problems. It would have been very helpful to see more data on the heteroplasmic NZB mice. Recent work by the Prolla lab has shown that age-associated diseases arise more rapidly in mice where mtDNA mutations occur at higher rates than normal due to expression of an error-prone mtDNA polymerase. This mouse model may provide deeper insights into the relationship between mitochondria, aging, and susceptibility to age-associated diseases, such as cancer. Similarly, there is no evidence that mixing mtDNAs in the same cytoplasm causes premature aging or cancer in humans, and it is unclear if anything like this does really occur in normal aging in human. Nonetheless, this is a compelling hypothesis raised by the PI and it is extremely important to test whether two different mitochondrial genomes in human embryonic stem cell lines will cause such problems during differentiation into different tissues.
Second, the most intriguing aspect of this application is the proposed experiment to prepare a living library of mtDNA haplogroups for patient haplotype matching by transferring the mitochondria and mtDNAs for lymphoblastoid cell lines into his WAL2A ρo cell lines and fusing them with embryonic stem cells. This review feels that this topic should be expanded and that more specific experiments outlined to show this would be relevant to the embryonic stem cell area. It is not clear if different mtDNA haplotypes in human can cause nuclear-cytoplasmic incompatibilities. Thus, the most crucial question for this group to answer is whether different mtDNA haplogroups in humans will have a negative impact for tissue differentiation. Will there be problems with immunological rejection if these tissues are grafted into patients? Without these questions answered the other parts of the proposal are questionable. For example using intra-specific cybrids and hybrids to reprogram cell nuclei and to examine the effects of mtDNA incompatibility and de novo mtDNA mutations on hES cell propagation may not be relevant.
Third, regarding Specific Aim 2, it is still debatable whether there is an advantage to the prolonged exposure of the somatic nucleus to “reprogramming” factors from the ESC nucleus in hybrid cells. We cannot say that the somatic cell nucleus is reprogrammed as it coexists with the original chromosomes from the ESC in the hybrid cell. Thus, the ESC chromosomes need to be eliminated from the hybrid cell to see if the somatic cell nucleus continues to display its new identity. Takahashi and Yamanaka (2006) identified a set of ten cDNAs that when introduced using retroviral vectors induced formation of ES cell-like colonies. The resulting ES-like cells were termed “iPS-MEF10” cells, short for induced pluripotent stem cells from MEFs by 10 factors. While this is valuable experimentation it is still extremely experimental. If this group could show that some of their approaches could render fibroblasts “ES”- like it would be a significant breakthrough and certainly would be fundable. It is also not known if any of the nuclear encoded mitochondrial proteins are also important for epigenetic reprogramming as well as mtDNA replication.