Embryonic stem cells (ESC), originating from preimplantation embryos, are able to differentiate into any type of cells in our body. The generation of ESC lines deriving from human embryos (hESC) has attracted a lot of dispute among researchers, but raised the hope that one day hESCs can be use as a source of cells for cell replacement therapy in the treatment of degenerative diseases and cancer. Substantial efforts are currently focused on unveiling the full potential of hESCs by developing culture systems aimed at the selective differentiation in the cell type of interest. We set up specific culture conditions that allow hESC differentiation in the originator cells (mesenchymal precursors) that form the bones, cartilage and muscles in our body. Muscular dystrophies (MD) are a group of diseases affecting the muscles in our body characterized by progressive muscle weakness and atrophy. There is no cure or treatment available for MD. In our preliminary studies, we further defined conditions for the specific generation of skeletal muscle cells from the hESC-derived mesenchymal precursors. We then converged our attention on their potential therapeutic applications. The specific aims of our proposed studies are: 1) optimization of this culture system to enhance skeletal muscle development during hESC differentiation. 2) Transplantation of these cells into animal models of MD (dystrophic mice and dystrophic dogs), to evaluate their in vivo functionality and their potential to repair or replace dystrophic muscle fibers.
Statement of Benefit to California:
The establishment of pluripotent stem cell lines derived from the human blastocyst (hESC) opened a new era in biomedical research. Because of their embryonic origin, hESCs can be virtually differentiated in all the cells of all tissues in our body. There is a great hope that one day hESC-derived specialized progeny will be used for cell-based therapy in the treatment of a variety of degenerative diseases and cancer. The California stem cell initiative with the institution of the CIRM will boost hESC research through the funding of innovative projects. Muscular dystrophies (MD) are a group of > 20 genetic diseases characterized by progressive weakness and degeneration of the skeletal muscles that control movement. There are many forms of muscular dystrophy, some noticeable at birth (congenital muscular dystrophy) and others in adolescence (Becker and Duchenne MD). Duchenne MD is perhaps the most common of them, with a worldwide incidence of 1 in 3,500 male births. Duchenne MD is the result of mutations in the gene that regulates dystrophin - a protein involved in maintaining the integrity of muscle fibers. Despite the substantial advances made in identifying the genetic defects causing these diseases, there is no treatment or cure available and affected children usually die in their teens. We propose to investigate the potential clinical applications of ESC-derived skeletal muscle cells upon transplantation in animal models of muscular dystrophy. We also investigate the molecular basis of skeletal muscle development during hESC differentiation. This research proposal will benefit the State of California and its citizens in the following ways. First of all, Californians are not immune to any form of MD and the overall incidence of this group of diseases is the same as elsewhere, with devastating consequences for the affected individuals and their families. If our hypothesis proves to be correct and skeletal muscle cells derived from hESCs can efficiently repair dystrophic muscle fibers in animal models, the likelihood of the use of these cells for transplantations in MD patients is going to be high. Therefore, Californians will be the first to benefit from the outcome of the proposed research. In addition, successful ESC therapy of MD will certainly encourage and stimulate research for other ESC-based therapy for related diseases. In conclusion, the CIRM initiative will undoubtedly lead to the discovery of a therapy and/or the developmental mechanisms leading to some disease and that in itself will put California at the top of ESC research with enormous benefits for all Californians.
SYNOPSIS: The goals of this proposal are to optimize strategies for directed differentiation of hES into skeletal muscle myocytes and to investigate their ability to engraft in vivo in mouse and canine models of muscular dystrophy. The applicant will use a 2-step in vitro differentiation strategy to generate myogenically committed cells that will be isolated by FACS. The process of myogenic committment will be analyzed by genomic and proteomic approaches, and the in vivo engraftment potential measured by transplant of GFP or luciferase-marked cells into immunocompromised injured or dystrophic mice or immunosuppressed dystrophic dogs. SIGNIFICANCE AND INNOVATION: Muscular dystrophies are a group of related muscle degenerative diseases for which there currently are no effective treatments or cures. This research would investigate the possible use of differentiated hES cells for replacement of diseased muscle cells in muscular dystrophy. Thus, if successful, it would provide novel preclinical data that could lead to the development of new treatments for muscle disease. The application is innovative in using a novel 2-step differentiation strategy that the applicant developed during his postdoc at Sloan-Kettering to generate myogenically committed cells. QUALITY OF THE RESEARCH PLAN: While the overall rationale of the proposed experiments is straightforward, the description of the research plan in several cases lacks sufficient detail to allow appropriate evaluation of its likely success. For example, the applicant proposes genomic and proteomic approaches to identify soluble factors that may influence myogenic specification in this model; however, the description does not clearly indicate which cells will be profiled at what timepoints in the differentiation protocol, how promising candidates will be selected, or how they will be assayed for activity (particularly if combinatorial action of multiple factors is required). This aim seems highly ambitious given the 2-year timeframe proposed for identification of the factor. The plan also needs better support from relevant preliminary data. This is particularly true for the dog studies, in which they will transplant differentiated cells into cyclosporine-treated dystrophic animals. It is not clear that the immunosuppressive regimen will be sufficient to overcome rejection of the xenograft. Also, this strategy necessitates shipping the differentiated cells from California to North Carolina, but no data is given to indicate how well the cells will survive and engraft after shipment. There is also some concern about whether the small number of dogs to be included in the study will provide sufficient statistical power for comparison. Likewise, the strategy for transducing cells for subsequent detection of GFP-labeled or luciferase-labeled progeny is not well described. At what stage will the cells be transduced? How efficient is this? Is there variability in expression of the markers in vivo in different cell types? The BLI data presented in Fig. 7 does not show negative control data, and does not correlate photons/sec. with # engrafted fibers, and so it is unclear whether this provides a quantitative measure of muscle regeneration. It seems that for measuring specifically myogenic engraftment, the stragegy would benefit from the use of a tissue-specific promoter (which would express only in myogenically committed cells) to drive marker gene expression. STRENGTHS: The proposed experiments address an important medical problem. The applicant has already isolated muscle stem cells from hESCs, which is an important discovery since myoblasts and even adult muscle satellite cells, though potentially available for transplantation into DMD patients, have not been very successful as transplant reagents. A series of clinical trials using human myoblasts in these patients proved unsuccessful for two major reasons: the cells were highly immunogenic (which can certainly be overcome) and they also failed to migrate from sites of injection. Thus a highly proliferative muscle progenitor that expresses genes that mediate muscle stem cell self-renewal is highly significant. Given that there is also enormous experience in the muscle biology community with transplant models, the experimental focus using this potential new source of cells should be very translational. The expertise of the collaborators in dystrophic dog models is also a strength. WEAKNESSES: There is lack of detail in the experimental design especially in reference to genomic/proteomic approaches, the strategy for systemic delivery of cells, the timing of cell isolation and transduction with marker proteins. There is also a lack of preliminary data to support some key aspects of the approach, especially in the dog studies. Perhaps the greatest weakness of the proposal is the functional measures of the engraftment. The investigators have developed protocols to differentiate the cells and have begun engraftment studies. However they provide no data on how they will determine whether or not this engraftment is truly functional. The proposal is limited to characterizing the engraftment, but does not truly show that these cells have any real functional value. Optimally such a characterization would include: 1) strength measurements in the animals; 2) electrophysiological evidence of appropriate myocyte function; and 3) perhaps most important -evidence of innervation of the new myocytes by neuromuscular junctions with appropriate histological and electrophysiological measures. The protocal as described could lead to the engraftment of cells which really do not add to the overall muscular function of the skeletal muscle. They do suggest in their methods that the collaborator may have some of these skills including a muscle strength test, at least in dogs, and neuromuscular reinnervation-- but it is not clear that this investigator is capable of carrying out and interpreting these studies, nor that such studies have been previously done in all of the mouse and canine models. Lastly, as a more general statement, this approach has been studied quite extensively and is yet has failed to show a benefit other then focal tiny effects, thus the therapeutic overall approach of focally injecting cells into muscle may not necessarily be a promising approach. The widespread delivery of skeletal myocytes via intra-arterial injection is interesting but there is great difficulties regarding pharmacokinetics and distribution of cells that would have to be worked out and were not addressed in this proposal. Although intr-arterial injections are proposed, but no experiments are described to determine the distribution of the injected cells. If the cells indeed have great migratory capacity, and can home to sites of injury, this would be important information in designing ideal translation strategies. But the experiments are almost mentioned as an afterthought in the optimization of culture conditions. In Aim 1a optimization consists of changing the timing of sorting for CD73 (one time-point only different than they have studied), and use of Noggin (one dose only? A single timing plan?) In 1b, the microarray studies will be done on a custom array of known muscle developmental factors, so it is unlikely to lead to any novel insights. The PI presumes (probably correctly) that secreted factors from CD73-negative cells are important for hES myogenesis, and so the microarray might also miss the important factors on many levels. Also a ‘targeted’ proteomics approach forms the centerpiece of Aim 1b. But there is absolutely no explanation of how the studies will be performed (or at least a letter from the person who will perform them), so the Aim is fairly incomprehensible. There is no plan presented for analysis at all, only an acknowledgement that analysis is not straightforward. In Aim 2a, SCID/Beige mice are used to determine the regenerative capacity of the hES-derived muscle progenitors after cardiotoxin injection into the TA muscle. The PI mentions that some pre-treatments of the cells can be examined using this model, pre-treatments to enhance regenerative capacity, but the treatments are not defined. It is unclear why these same experiments in the mdx mice do not give the same information. And, since the injection site is localized, and the ultimate assay for regenerative capacity is the pathology and immunohistochemical stains, the use of the optical imaging for these experiments seems optional and not likely to give substantial information. In sum, the major weakness of the proposal is that the experiments are simply not well-thought out. Given that translational research should be a priority for these cells, the most important parts of this application are the issues of (1) optimizing production of these muscle stem cells in vitro, and perhaps even more importantly, (2) scale-up to a large animal model, with details like the optimal site of injection and number of cells needed, the ability of the cells to migrate, and of course effectiveness in rebuilding functional muscle in an animal model of the disease. Unfortunately, these important issues are lost in the unfocused nature of the proposal. More detail is needed to determine feasability of the proposal. DISCUSSION: The discussion centered around the fact that the transplantation and functional studies would be done in a distant lab and that there were too few details and too many potential problems in them. The rationale for using all of the different disease models was also unclear.