Genetic factors are primarily responsible for multiple sclerosis (MS), although environmental factors also play a role. However, cell lines with a MS genome currently are not available for research. Recent studies suggest that human embryonic stem cell (hESC) lines with MS genome could be derived by fusing hESC to fibroblasts from MS patients. Neural and immune cell types could be differentiated from these hESC cells with MS genome. These hybrids would provide a platform to study the genetic factors of MS, and to study the interactions between environmental factors and MS susceptibility which is believed to be genetic. Most importantly, they could provide MS susceptible cells with identical genome to different research laboratories for conducting research and comparing results. These hybrid hESC lines could provide target MS cells to facilitate new drug and treatment developments, pre-clinical toxicity studies. We believe they will greatly accelerate MS research while avoiding the technological and societal difficulty of generating hESC by nuclear transfer. The overall goals of this proposal are: 1) Derive hESC lines with MS genomes for MS research and treatment development. 2) Identify genetic factors related to MS susceptibility. To achieve these goals, we propose to generate tetraploid hESC lines with MS genomes by fusing hESC lines with fibroblasts donated by MS patients and unrelated healthy individuals. Tetraploid hESCs containing normal genome could be used to factor out the tetraploid artifacts. Tetraploid hESC from a clone containing a MS genome and a clone with a normal genome would be terminally differentiated into astrocytes with our established protocols. Karyotyping and STR (short tandem repeat) genotyping would be performed to assess the stability of the tetraploid genome during neural differentiation (first year). Genetic factors related to MS would most likely cause difference in the maturation of neural progenitor. After the access of genome stability, tetraploid hESC containing MS or normal genomes along with diploid hESC would be differentiated to nestin positive neural progenitors. Gene expression profiling and network analysis would be used to investigate whether different gene regulation networks are involved in the neural differentiation of these cells. The unique gene networks in hESC with MS genome are the potential MS genetic factors. Gene expression profiling would be performed to access the difference of responding to inflammatory cytokines among these astrocytes (second year). Eventually, these cells would be used to study why remyelination fails in MS.
Statement of Benefit to California:
Approximately one of every thousand Californians suffered from multiple sclerosis (MS), which is the most common neural disorders without known cause, means of prevention, or cure. MS has a strong genetic component, but no definitive genetic factor has been identified despite intensive genetic screening at various populations. Here, we propose to derive hESC lines containing MS genomes, and study how a MS genome would alter the neural differentiation of hESC. This study could reveal biological pathways in the development of MS and MS susceptibility. These biological pathways could guide MS research into treatment, and perhaps even lead to the prevention of MS.
A recently report demonstrated that hybrid cells of hESC and fibroblasts maintained a stable tetraploid genome and displayed the developmental pluripotency and epigenetic state of hESCs. The hypothesis of this proposal is that introducing multiple sclerosis (MS) genomes into hESCs will affect neural maturation and reveal MS genetic factors. The cells could also be a valuable standard with identical genetic backgrounds for different laboratories to conduct MS research on the maturation and differentiation of MS neural and immune cells as well as the interaction of these MS cells in vitro and as targets for drug screens. In Aims I and II, hybrid hESC lines from MS patients or controls will be generated, reprogramming of the genomes tested. In Aim III, hybrid cells will be differentiated into astrocytes to assess stability of the fibroblast genomes and whether MS hybrid differentiation is similar to control. In Aim IV, differentially expressed genes in MS vs. normal hybrids will be sought. SIGNIFICANCE AND INNOVATION: MS is a not uncommon neurological disease of young adults. Genetic and environmental factors have been implicated in the disease; however, the cause and its pathogenesis remain unclear. The generation of "standard" MS cell lines with identical genetic backgrounds for research studies is a valuable goal. Proposals designed to provide clues as to the pathogenesis of MS or to generate reagents important in its study are significant ones. Even if this proposal fails to achieve its goals, it may answer important questions about the usefulness of hybrids in other diseases and difficulties that may arise with respect to their generation. This is a very creative proposal. The idea that MS cells might have abnormalities that will interfere with differentiation and development is novel. STRENGTHS: • The proposal is novel in its direction. • Even if this proposal fails to achieve its goals, it may answer questions about the usefulness of hybrids in other diseases and difficulties involved in their generation and differentiation. • The generation of a standard MS cell line that could be differentiated into different neural cell types or immune cells will be potentially valuable to the field • The inclusion of Dr. Leslie Weiner, an expert on MS, is a valuable one in this proposal WEAKNESSES: First reviewer: • This is a risky proposal. The hybrids may not grow well, they may be unstable, they may not differentiate, etc. • A hypothesis in this proposal is that MS genetic factors alter the maturation of normal hESCs towards neural lineages. The PI provides no data to support this hypothesis weakening some of the rationale for the planned experiments. • Many scientists think that MS is really “many diseases” and that many genes contribute slightly to the susceptibility to an environmental factor(s). If this is true, it would seem that the present proposal is unlikely to clarify this pathogenesis of the disease, and other genetic approaches will be far more fruitful • The PI notes that one of his long- term goals is to provide hESC lines with MS genetic factors for MS research and drug development and that “most importantly” these MS cells would be a standard, easily accessible, and with identical genetic backgrounds. The fact that the hybrid cells may be unstable leads to some concern about the identical genetic background. Also, it would have been valuable for the PI to have prvoided more detailed about what his plans are for the hybrid lines and future experiments. How could these cells "help us understand how MS susceptible immune cells ... process infections" more that an MS patient's peripheral blood mononuclear cells (before, during and after an attack), cerebrospinal fluid cells, or a T cell clone? • The PI states that the “difference [in microarray gene expression profiling] between hybrids containing MS and normal genomes is most likely related to MS genetic factors.” I am not convinced by this statement. There are many possible reasons for the differences that could be genetic but unrelated to MS susceptibility genes. • It is not clear to this reviewer why the PI plans in the third specific aim to differentiate the hybrids into astrocytes and in the fourth specific aim to differentiate the hybrids into nestin+ cells, leaving the differentiation into OPCs as a bit of a brief comment – even thought they “recognize that the most relevant cell type in MS is [the] oligodendrocyte". One would have thought that defects that affect oligodendrocyte growth or differentiation are more likely than those affecting other neural cell types. This needs some further explanation. • The proposal is difficult to follow at times. • The PI obtained a PhD in Biochemistry and Molecular Biology at USC in 2003, and has been an Assistant Professor in the Department of Neurology at USC since 2005. He has had modest productivity and lists 5 publications. • The inclusion of additional collaborators would have been valuable. Second reviewer: The rationale behind the hypothesis that genetic factors related to MS will cause differences in neural progenitor maturation is weak. There is little evidence for a developmental aberration in the brains of MS patients. “The genes that fail to return to embryonic levels would be further investigated.” I can understand wanting to compare hybrids with similar reprogramming levels, but what might be the connection to MS and how will the PI sort out differences due to variable reprogramming and differences due to MS? Why would differences between the hybrids containing MS and normal genomes is most likely related to MS genetic factors? Presumably the MS patients and controls have many (unknown) genetic differences that are unrelated to MS. DISCUSSION: This is a creative and novel proposal, but highly risky and poorly conceived. The PI has lofty and perhaps over-ambitious goals for the new cells, e.g. differentiating into astrocytes, performing whole genome arrays versus control hybrids, developing nestin-positive progenitors, without much detail on how the PI would use the cells. The rational behind the hypothesis is unclear, and the proposal is poorly-written. Although a standard MS line would be very valuable, this approach may not be the best. MS may not be the right disease to use as a cell source for tetraploid hybrids given the believed genetic heterogeneity and complexity of the disease. Hybrids might be better used in a clear-cut autosomal dominant disease. Alternatively, hybrid formation with cells from a genetically and phenotypically well characterized MS family might be more useful for this type of analysis. Another issue is how would differentiation be teased apart from other factors. Why not try to differentiate cells into oligodendrocytes?