Funding opportunities

Funding Type: 
New Faculty I
Grant Number: 
Principle Investigator: 
Funds requested: 
$1 623 064
Funding Recommendations: 
Grant approved: 
Public Abstract: 

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: 

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 Summary: 

SYNOPSIS: This proposal will explore the differentiation of myoblasts from human embryonic stem cells (hESCs), the use of such cells to explore the developmental differences between wild type cells and those with the Duchenne muscular dystrophy gene, and the potential of hESCs in the therapy of this disease using a canine model of Duchenne MD.

The applicant describes three specific aims to achieve his objectives. In Specific Aim 1 he has three goals. In the first he will characterize the in vitro differentiation of hESCs into skeletal myocytes. He will modify his earlier technique of sorting using CD73+ mesenchymal precursors isolated from differentiating hESCs, only a small percentage of which are committed to muscle differentiation. In the second goal of this aim, he will standardize the culture conditions required to selectively differentiate hESCs into myoblasts. Thirdly, he will use high throughput screening to identify molecules involved in the differentiation of sorted precursors toward myoblasts. Here, sorted cells from the Pax3/GFP line will be used.

In Specific Aim 2, he will utilize a wide-genome analysis to explore genes involved in myogenesis in wild-type cells and those from Duchenne muscular dystrophy.

In Specific Aim 3 he will explore the therapeutic potential of hESC-derived myoblasts by assessing survival and integration of myoblasts in dogs with induced muscle injury. Cells will also be injected intra-arterially into a canine model of Duchenne muscular dystrophy.

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 himself who comes from an excellent training in ES cell biology at the Sloan Kettering with Lorenz Studer. There he published two significant ES cell papers that are the springboard for this application. He has chosen an important area for the translational application of ES cells. Duchenne muscular dystrophy is an important medical target and has long been sought after as potentially being amenable to cell therapy. The proposal is well organized, logical and flows well.

The applicant has published on the generation of hESC derived sketetal myoblasts. Having recently described the isolation of CD73+ mesenchymal precursors from differentiating hESCs that were able to further differentiate into skeletal myoblasts, the PI now propose to characterize a population of P75+/EpCAM+ cells and evaluate their ability to differentiate into bone, dermis and skeletal muscle, thereby defining their somitic origin and thereafter propose to standardize culture conditions which promote their selective differentiation into skeletal myoblasts from a variety of human ES cells lines.

Their preliminary data demonstrates the ability to culture and differentiate hESCs into skeletal myoblasts and muscle and transplant into immunodeficient mice. They also demonstrate the ability to identify and sort P75+/EpCAM+ cells and to guide their differentiation in culture to skeletal myoblasts defined by myogenin/MF20 expression under specific culture conditions including Wnt agonists.

Overall, the three aims are wide reaching but do-able, particularly with the support of core facilities at City of Hope. Aim 3 is more translational and more complex since it will introduce these hESC derived myoblasts into a dog model of Duchenne muscular dystrophy.

Major strengths of the proposal include: clearly described, well thought out, and well presented experiments; and, the comparison of the wild type 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 Specific Aim 1 will be required for achieving the remaining aims. Clearly the success of this aim will require considerable technical success in the isolation of the cells required for Aims 2 and 3, nonetheless he would appear to have the technical skill and experience to succeed.

A number of points of concern/criticism were raised:

Regarding Aim 1: To confirm the somitic origin of P75+/EpCAM+ precursors in Aim 1, they propose to select and sort cells based on Pax3 gene expression driving a green fluorescence protein, based on the premise that Pax3 is a transcriptional regulator essential for trunk and limb muscle cell development. However, they admit that the Pax3 promoter may not be specific as a single marker for somitic mesoderm but may also be expressed by neural crest cells. They will also correlate their GFP+ cells with CD73 expression and therefore be able to interrelate expression patterns between cell surface markers CD73 and Pax3 as a nuclear transcription factor. Although this is an important aim, the important caveat is that the gene trap vector and targeted hESCs are not produced yet, and therefore the reagents to perform these studies are not available. These considerations and concerns about the absence of absolute specificity of Pax3 expression lower the enthusiasm for this approach. Following this, they intend to test their differentiation protocol with several hESC lines. What major biological insights are to be learned from these experiments? What advantage is there to test the Millipore lines? Then, they propose to perform high throughput screening using the core facility directed by Dr. Yip. By testing a chemical library of 70,000 compounds, they hope to identify one or several small molecules that promote differentiation of their P75+/EpCAM+ precursor population into skeletal myoblasts. The rationale for conducting these experiments, since Wnt3A appears to be quite effective, is not precisely clear. In addition, these methods of high throughput screening depend on the Pax3 GFP cell line which is not in place, and the high content screening capability that also is not currently in place.

A second reviewer echoed the concerns about Aim 1: There are some issues that arise such as the stability of Pax3:GFP transgene expression in the hESCs; whether the high throughput screen using Pax3:GFP as the read-out on p75dim/EpCAM + cells from the transgenic cell line will be specific enough for muscle cells (would a muscle promoter be better?).

Regarding Aim 2: Overall, this Aim might be viewed as a fishing trip and, as acknowledged by the applicant, microarray 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.
It was noted as a strength that the comparison of the wild type and dystrophin mutation carrying hESCs which could reveal very interesting information; however, the experimental design could be developed more insightfully. Aim 2 proposes to take advantage of a novel hESC line from Stemride International #SI283 which carries a mutation in the coding sequence of the dystrophin gene such that muscles derived from this line will lack the dystrophin protein as this is a male line. By conducting experiments involving in vitro differentiation of sorted cells with both wild type and the mutant cell line, they may be able to detect different abilities to form myotubes. Then they propose to compare cells harvested at different stages during muscle differentiation from both wild type and mutant hESC lines and compare them by Affymetrix whole genome microarrays for their gene expression profiles. However, 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? In addition, is it 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.

Regarding Aim 3: Specific Aim 3 has a number of concerns. While moving to a large animal model has attractions, it is perhaps surprising that they are jumping from their studies on the SCID/Beige mice to the canine without having attempted to treat the xmd mouse. Major pitfalls which have been identified include that this an immunocompetent xenotransplant model with potential sequelae of eitherrequired exogenous immunosuppression or rejection of the potential regenerative cells.The collaboration with Dr. Kornegay is essential. However, Kornegay appears to have some reservations. He identifies past failures with a myoblast transplant approach both by himself in the canine and in the xmd mouse. Dr. Kornegay suggests that poor immunosuppression might be at fault, but one reviewer is surprised by this, as cyclosporine therapy in the dog ought to be relatively straightforward. Dr. Kornegay also notes that, as he has just moved, the supply of affected dogs may be limited. But, since this aim will be carried out at the end of the project, this concern may be allayed. Dr. Kornegay is also located in North Carolina, and the transport of cells to North Carolina for transplantation is cumbersome and may introduce problems. A second reviewer echoed the concerns about transport of cells over long distance, 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: Dr. Tiziano Barberi qualified with a DSc from the University of Rome in 1997 and a PhD from the University of Tubingen in Germany in 2005. He did post doctoral work at Memorial Sloan-Kettering from 1998 until 2006, in the latter years working with Dr. Lorenz Studer on embryonic stem cells in the Laboratory of Stem Cell and Tumor Biology. In 2006 he moved to the Beckman Research Institute of the City of Hope where he is an Assistant Professor and Director of the Human Embryonic Stem Cell Core facility.

Dr. Barberi has a strong publication record with many publications in high impact journals such as Nature Biotechnology, PNAS, and Nature Medicine, with the exciting reports of inducing mesenchymal precursors from hESCs to form myoblasts that survived and intergrated in vivo. The PI has active research support through two modest grants from the NIH and Stop Cancer Foundation. His training and publication underscore his expertise in the ES cell field.

Dr. Barberi 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 embryonic, neural, and mesenchymal stem cell biology have been recruited over the last five years which joined eight already established laboratories using human and mouse stem cells in their research. His career development plan is well thought through; informal and formal mentoring is in place. He has initiated a collaboration with Dr. Kornegay in North Carolina for transplantation of his cells into a dog model of Duchenne muscular dystrophy.

INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: A significant strength of the proposal is the institutional commitment that the City of Hope is making toward the candidate. They will provide him with salary, sufficient space, equipment, core facilities and mentoring. They are obviously making an investment to ES cell research by establishing a high throughput screening core facility, along with a functional genomics core. He will not be working on stem cells alone, as City of Hope has five Assistant Professors over the last five years who are also working on stem cells.

There are numerous core facilities available at City of Hope, 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. City of Hope is part of a consortium of six Southern California institutions with research programs in embryonic stem cells that plans to submit a proposal to CIRM for production of large facilities for stem cell research. 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 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 immunosupression concerns, one reviewer commented that cyclosporine and ketoconazole was identified as the experimental regimen, and having been used for years, is well-worked out in the canine model. A panelist commented that cyclosporine itself ameliorates the dystrophic phenotype (citing a Nature 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 by using early markers. A recommendation from the panel was for the PI to pay attention to stage of differentiation versus migration. A follow-on question from the panel was why has the PI chosen an intra-arterial delivery strategy, versus directly injecting the cells into the muscle? A panelist commented that the precedent for intra-arterial injection was reported from an Italian group in Nature approximately one year ago. A second panelist commented that extravasation may be cell-type specific. 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.