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RN1-00525-1: Skeletal muscle development from hESC and its in vivo applications in animal models of muscular dystrophy
Recommendation: Recommended for funding
Scientific Score: 80
First Year Funds Requested: $309,794
Total Funds Requested: $1,623,064
Public Abstract (provided by applicant)
Embryonic stem cells (ESC) originating from early stage embryos are able to differentiate into any type of cells in the body. The generation of ESC lines from human embryos (hESC) has attracted a lot of dispute among researchers, but raised the hope that one day hESCs can be used in cell replacement therapy for the treatment of degenerative diseases and cancer. Substantial efforts are currently focused on unveiling the full potential of hESCs by developing culture systems supporting the selective differentiation into the cell types of interest. We have reported the specific culture conditions that allow hESC differentiation in the originator cells (mesenchymal precursors) that form the bones, cartilage and muscles in our body. Furthermore, we then defined the conditions for selective generation of skeletal muscle cells from the hESC-derived mesenchymal precursors. Transplantation of these muscle cells into the limb muscle of immunodeficient mice showed their ability to survive and integrate in the host’s tissue. Muscular dystrophies (MD) are a group of diseases affecting the muscles in our body. MD is characterized by progressive muscle weakness and atrophy for which there is no cure or treatment available. Based on our previous studies, we are proposing to optimize the culture conditions for the in vitro generation of skeletal muscle cells from hESCs and study the developmental mechanisms involved in this process. Furthermore, we will transplant these cells into dystrophic dogs, an animal model of MD, to evaluate their in vivo functionality and potential to repair or replace dystrophic muscle fibers. The accomplishment of our aims will contribute to the understanding of human skeletal muscle development and will provide the basis for the clinical application of hESC-derived cells in muscle diseases.
Statement of Benefit to California (provided by applicant)
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 great hope that in the near future hESC-derived specialized progeny will be used in cell-based therapy for a variety of degenerative diseases and cancer. The California stem-cell initiative, through CIRM, gives a significant boost to hESC research by funding pioneering projects in the field.
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 form, with a worldwide incidence of 1 in 3,500 male births. This dystrophy occurs as 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 hESC-derived skeletal muscle cells upon transplantation in animal models of muscular dystrophy. In parallel, we propose to study 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. We already showed that hESC-derived skeletal muscle cells can integrate and survive in a host muscle when transplanted in immunodeficient mice. Therefore, we expect that these cells will efficiently repair dystrophic muscle fibers in animal models of MD, such as dystrophic dogs. If our hypothesis proves to be correct, it is very likely that these cells will be used for transplantations in MD patients. Californians will then be the first to benefit from the outcome of the proposed research. In addition, a successful ESC-based therapy of MD will certainly encourage and stimulate research for other ESC-based therapies 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. That in itself will put California at the top of ESC research with enormous benefits for all Californians.
Review
SYNOPSIS: This proposal will explore the differentiation of human embryonic stem cells (hESCs) to skeletal muscle cells. These cells will be used to explore the developmental differences between wild type cells and those with the Duchenne muscular dystrophy (MD) gene. Finally, the applicant will explore the potential of hESC-derived skeletal muscle cells in the therapy of Duchenne MD using a canine model of this disease.
STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: This is an extremely interesting, well-written proposal from a well qualified young investigator. One of the major strengths is the candidate who has had excellent training in ES cell biology from a leading expert in stem cell biology. The applicant has published significant embryonic stem (ES) cell papers that are the springboard for this application. The applicant has chosen an important area for a translational application of ES cells; Duchenne muscular dystrophy is an important medical target and has long been pursued as potentially being amenable to cell therapy. The proposal is well organized, logical and flows well.
The applicant has experience in the generation of hESC derived sketetal myoblasts. Having recently described the isolation of mesenchymal precursors from differentiating hESCs that were able to further differentiate into skeletal myoblasts, the Principal Investigator (PI) now proposes to characterize a specific subpopulation of cells and evaluate their ability to differentiate into bone, dermis and skeletal muscle. Thereafter, the PI proposes to standardize culture conditions which promote their selective differentiation into skeletal myoblasts from a variety of human ES cell lines.
Overall, the aims are wide reaching but do-able, particularly with the support of core facilities at the applicant’s institution. Preliminary data demonstrates the ability to culture and differentiate hESCs into skeletal myoblasts and muscle, and to transplant the cells into immunodeficient mice. The PI also demonstrates the ability to identify and sort a specific subpopulation of cells, and to guide their differentiation in culture to skeletal myoblasts. One goal is more translational and complex, since it will introduce these hESC derived myoblasts into a canine model of Duchenne muscular dystrophy.
Major strengths of the proposal include clearly described, well thought out, and well presented experiments. Another major strength is the comparison of wild type cells and dystrophin mutation carrying hESCs, which could reveal very interesting information. The applicant is perhaps disarmingly honest in stating that accomplishing the work detailed in the first aim will be required for achieving the remaining aims. Considerable technical success is required in the isolation of the cells needed for subsequent aims. Nonetheless, the applicant would appear to have the technical skill and experience to succeed.
A number of points of concern/criticism were raised. For example, to confirm the somitic origin of precursor cells in the first aim, the PI proposes to select and sort cells based on the expression of a marker gene driven by a specific transcriptional promoter. However, as the PI admits, the promoter may not be specific for somitic mesoderm but may also be expressed by neural crest cells, and the stability of transgene expression in hESCs may be an issue. Also, some of the reagents needed to perform expression studies are not yet produced. These considerations and concerns about the absence of absolute specificity of the promoter lower the enthusiasm for this approach.
Following this, the PI intends to test their differentiation protocol with several hESC lines. What major biological insights are to be learned from these experiments? The PI also proposes to perform high throughput screening using the core facility to identify one or several small molecules that promote differentiation of their specific precursor subpopulation into skeletal myoblasts. The rationale for conducting these experiments, since a known protein appears to be quite effective, is not precisely clear. In addition, these methods of high throughput screening depend on a marker cell line that is not in place, and the high content screening capability that also is not currently in place.
The second aim proposes to take advantage of a novel hESC line which carries a mutation in the coding sequence of the gene of interest such that muscles derived from this line will lack the dystrophin specific protein. By conducting experiments involving in vitro differentiation of sorted cells with both wild type and the mutant cell line, the PI may be able to detect differences in cell function. However, the experimental design could be developed more insightfully. The PI proposes to compare cells harvested at different stages during muscle differentiation from both wild type and mutant hESC lines and compare their gene expression profiles. This might be viewed as a fishing trip since, as acknowledged by the applicant, gene expression analysis may lead to an unwieldy number of candidate genes. The PI will undertake this analysis based on the idea that there are developmental differences between wild type and dystrophin negative muscle fibers. However, and perhaps equally possible, changes in gene expression might only be found prior to and during muscle fiber degeneration. In addition, if different cells exist during this differentiation process then different gene expression profiles will be observed that are based on different cell populations, not changes in gene expression within cells, so what will this experiment tell us? It is expected that skeletal muscle differentiation programs from Duchenne MD hESCs will be different from those of wild type hESCs given that patients with Duchenne muscular dystrophy are born with essentially normal muscle phenotype and function. Unfortunately, there does not appear to be a plan to test chemical or traumatic insults and their effects on myotubes or myoblasts during skeletal muscle differentiation from these different cell lines (i.e., test possible differential response to injury). In general, this aim is not hypothesis-driven and could be developed in a more structured manner.
Major pitfalls which have been identified include that this an immunocompetent xenotransplant model with potential sequelae of either required exogenous immunosuppression or rejection of the potential regenerative cells.
For the third aim, the applicant has initiated a collaboration for transplantation of the cells into a model of Duchenne muscular dystrophy. While moving to a large animal model has attractions, it is perhaps surprising that the PI is jumping from studies in mice to the canine without having attempted to treat the xmd mouse. The collaboration to achieve the canine model experiments is essential. However, the collaborator appears to have some reservations as failures have occurred with a myoblast transplant approach in the canine and in the xmd mouse, potentially due to poor immunosuppression (although one reviewer noted that immunosuppressive therapy in the canine model ought to be relatively straightforward). The collaborator has just moved his/her lab, and notes that the supply of affected dogs may be limited. Since this aim will be carried out at the end of the project, this concern may be allayed. A second reviewer echoed concerns about transport of cells which requires cryopreservation with uncertain recovery and function. The proposal would benefit from preliminary data either in this model or in a murine immunodeficient model.
QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: The applicant qualified with undergraduate work in 1997 and a PhD in 2005. The applicant did post doctoral work from 1998 until 2006, in the latter years working with a stem cell expert on embryonic stem cells. In 2006 the applicant moved to the current institute as an Assistant Professor. The applicant also directs an hESC core.
The applicant has a strong publication record with many publications in high impact journals, and the PI has active research support through two modest grants. The PI’s training and publication underscore his/her expertise in the ES cell field. The applicant has a strong background in stem cell research and appears to be in an institution that is highly committed to recruiting new faculty. Indeed, many new faculty studying aspects of stem cell biology have been recruited over the last five years to join a number of already established laboratories in stem cell research. The applicant’s career development plan is well thought through; informal and formal mentoring is in place.
INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: A significant strength of the proposal is the institutional commitment that the applicant institution is making toward the candidate. They will provide the applicant with salary, sufficient space, equipment, core facilities and mentoring. The applicant will not be working on stem cells alone, as the applicant institution has hired several new faculty in stem cell research. There are numerous core facilities available at the applicant institution, including those in biostatistics, biomedical informatics, analytical cytometry, mass spectrometry, proteomics, functional genomics, high throughput screening, small animal imaging and synthetic biopolymer chemistry, among others. Thus the PI and institutional commitment both appear to be quite strong.
DISCUSSION: Two reviewers called this a “very promising” and “very strong” proposal from a well qualified investigator. The third reviewer was in general agreement, but did comment that gaps in the field were not delineated in the proposal.
Overall, the enthusiasm from the panel for this translational-oriented application was strong. There was significant discussion about the canine animal model experiments. One panelist who is an expert in the field noted a large clinical experiment using human myoblasts failed because of: 1) an underestimated immune response to the cells, and 2) a failure of the cells to migrate. In response to immunosuppression concerns, one reviewer commented that the proposed immunosuppressive regimen is well-worked out in the canine model. A panelist commented that cyclosporine itself ameliorates the dystrophic phenotype (citing a recent paper), and therefore, having the correct controls will be key. In response to the cell migration concern, one reviewer commented that the PI has designed experiments to select for more primitive cells. A recommendation from the panel was for the PI to pay attention to stage of differentiation versus migration. There was also discussion among the reviewers about the method of delivery of the cells. Despite the fact that no consensus was achieved on this issue, the panel remained enthusiastic for this well-written translational application from a strong, talented candidate, addressing a clinically important target.
The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:
- None