Muscular Dystrophy

Coding Dimension ID: 
302
Coding Dimension path name: 
Muscular Dystrophy

Combination therapy to Enhance Antisense Mediated Exon Skipping for Duchenne Muscular Dystrophy

Funding Type: 
Early Translational from Disease Team Conversion
Grant Number: 
TRX-05426
ICOC Funds Committed: 
$0
Disease Focus: 
Muscular Dystrophy
Pediatrics
Stem Cell Use: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
Duchenne muscular dystrophy (DMD) affects 1 in every 3,500 boys worldwide. DMD is caused by mutations in the gene encoding dystrophin, a protein key to muscle health. DMD patients are typically weaker than normal by age 3, and with progressive muscle weakness most loose the ability to walk by age 11. DMD progresses to complete paralysis, respiratory insufficiency heart failure, and death, usually before the age of 25. No therapies exist that address the primary defect or dramatically alter the debilitating disease. Exon-skipping is an emerging therapy in which anti-sense oligonucleotide (AO) guided-RNA splicing rescues expression of a partially functional dystrophin; but it is unclear if efficacy will be optimal for clinical gain. We identified a combination therapy that improves the efficacy of exon-skipping in mouse muscle and human DMD patient-stem-cell-derived muscle cells. The DMD mouse model will be used to establish dosing and efficacy. To determine if combination therapy promotes exon skipping in human DMD patient cells with different DMD mutations, DMD patient derived stem cells converted into muscle-like cells in culture and screened for efficacy of combination drug relative to AO alone. The proposed research program will complete studies to identify a single drug/AO combination as a developmental candidate anticipated to treat up to 13% of DMD patients; although the strategy is likely generalizable to enable treatment of 70% of DMD patients.
Statement of Benefit to California: 
Duchenne muscular dystrophy (DMD) is a fatal genetic disorder, caused by a defect in the gene that produces dystrophin, a protein critical for normal skeletal muscle function. DMD affects more than 1,000 boys in California. Muscle weakness first appears in boys in the hips and legs and progressively extends to every muscle in the body such that most affected individuals require a wheelchair by age 11, have trouble feeding themselves by their late teens and ultimately loose most muscle function. Patients usually die by age 25 from respiratory or cardiac insufficiency. In addition to the human suffering, DMD places a large economic burden on patients, their families and society. Patients require intensive medical care because they cannot perform the simplest activities of daily living. Eventually, each individual requires ventilation and 24/7 care. The proposed combination therapy is predicted to cause skeletal muscle cells to skip DMD exon 51 and express a partially functional dystrophin protein, lessening the severity of DMD. A therapy that effectively slows or reverses disease will allow patients to lead longer, more productive lives and reduce costly supportive services—progress that will benefit patients, their families and society. Our proposal stands to specifically benefit Californians in another way: Because the University of California owns the intellectual property to the combined therapy, our success could ultimately lead to revenue for a state institution.
Progress Report: 
  • Duchenne muscular dystrophy (DMD) is the most common muscular dystrophies and the most common fatal genetic disorder of childhood. Approximately one in every 5,000 boys worldwide is affected with DMD often caused by spontaneous mutations. Extrapolating from population based studies, there are over 15,000 people currently living with DMD in the US alone. DMD is a devastating and incurable muscle-wasting disease caused by genetic mutations in the gene that codes for dystrophin, a protein that plays a key role in muscle cell health. Children with DMD are typically weaker than normal by age three, and progressive muscle weakness of the legs, pelvis, arms, neck and other areas result in most patients requiring full-time use of a wheelchair by age 11. Eventually, the disease progresses to complete paralysis and increasing difficulty in breathing due to respiratory muscle dysfunction and heart failure, with death usually occuring before the age of 25. While corticosteroids can slow disease progression and supportive care can extend lifespan and improve quality of life, no therapies exist that address the primary defect or dramatically alter the debilitating disease course.
  • Exon-skipping is a promising therapy that aims to repair the expression of the dystrophin protein by repairing the RNA. We have identified a combination therapy that improves the effectiveness of exon-skipping therapy in mouse muscle and in human DMD patient stem cell derived muscle cells in culture. In exon skipping the genetic defect is directly repaired inside of each muscle cell. Thus, this therapy is predicted to lessen the disease severity.
  • Early research on this combination therapy for Duchenne used human DMD patient stem cells including: reprogrammed patient fibroblasts converted into muscle-like cells in culture or when transplanted in mice. We have made a panel of these cells with different mutations to assess efficacy in a range of DMD mutations. These cells are necessary because each patient’s mutation in the dystrophin gene is different. In order to know who will or will not benefit from the exon-skipping therapy, individualized cell culture and mouse transplant models from a number of DMD patients must be created to effectively characterize the combination therapy. At 12 months of the CIRM-funded research program, we have established optimal oral dosing of dantrolene that is compatible with 6 month long-term testing in dystrophic mice and optimal dosing of morpholino antisense oligo. The combination therapy is well tolerated by mice, and dystrophin rescue is increased in short term experiments. 6 month treatment experiments are being initiated that will test if the induction of dystrophin can reduce the severity of the disease in the dystrophic mice. Since exon-skipping therapy relies on knowing individual patients exact DNA mutation, this is a form of personalized genetic medicine. While the specific combination therapy being developed here will treat up to 13% of DMD patients, the strategy is likely to be generalized to be able to treat up to 70% of DMD patients.

Phenotypic Analysis of Human ES Cell-Derived Muscle Stem Cells

Funding Type: 
Basic Biology III
Grant Number: 
RB3-05041
ICOC Funds Committed: 
$1 381 296
Disease Focus: 
Muscular Dystrophy
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
We study human muscle development, and are actively investigating potential cell-based therapies for the treatment of degenerative muscle diseases, such as muscle dystrophy. This project will define the pathway that muscle stem cells follow as they form new muscle, and identify which muscle stem cells are most useful for therapy. Our approach will be to examine human embryonic stem cells as they become muscle stem cells and mature muscle in culture, to define the stages of normal muscle development. We will then transplant these stem cells at various stages of development into the leg muscles of mice with muscular dystrophy, and study how these cells become new muscle tissue, how this impacts the animals’ ability to exercise, and the strength of the treated muscles. Our goal for this research is to fully understand the normal process of human muscle stem cell development, and to identify specific stem cells that provide therapeutic benefit when transplanted into dystrophic muscle.
Statement of Benefit to California: 
Muscular dystrophies are profoundly debilitating disorders that affect more than 1 in 3,500 male births. They comprise a group of genetic diseases that cause progressive weakness and damage to skeletal muscle resulting from abnormal proteins critical to muscle health. These abnormal proteins are thought to predispose muscle to damage from normal activity, leading to premature depletion of normal muscle stem cells that maintain muscle health during normal use. This research will identify human embryonic stem cells that are able to repair damaged muscle, thereby providing a new approach to therapy for patients with muscle disease. The medical treatments developed as a result of these studies will not only benefit the health of Californians with muscular dystrophy and other degenerative muscle diseases, but also should result in significant savings in health care costs. This research will push the field of muscle regenerative medicine forward despite the paucity of federal funds for embryonic stem cell research, and better prepare us to utilize these funds when they become available in the future.
Progress Report: 
  • The overall goal of this project was to use hESCs to define the cellular and functional phenotypes of human muscle stem cells as they differentiate along the muscle lineage, and specifically evaluate their ability to augment tissue and function of dystrophic muscle. Toward this goal, we have evaluated one muscle-specific reporter hESC line, generated a second line using another muscle-specific promoter, developed three alternative methods for directing myoblast differentiation from hESCs in culture, and piloted injections of hESC-derived myogenic precursor cells into hind limb muscle of immunodeficient mice.
  • Muscle cells derived from human embryonic stem cell (hESC) can be potential source for cell therapy to regenerate muscular diseases. The focus of this grant has been to develop efficient methods for isolating muscle stem cells from hESCs that avoid animal products so that we can use these to both understand how muscle cells form, and determine which of these may be best for treating muscular dystrophy.
  • Our progress over the past year has significantly advanced the aims of this work: 1) Determine the cellular phenotypes of human muscle stem cells as they differentiate into myoblasts, and 2) Determine the ability of human muscle stem cells at different stages of development to engraft, proliferate and differentiate into muscle in a mouse model of muscular dystrophy, and determine their functional and myo-mechanical effects on dystrophic muscle. We now have a working system to derive early progenitor muscle cells from human embryonic stem cells. The differentiation protocol has been developed sufficiently such that skeletal muscle cells can be generated from human ES cells. We have identified points along the differentiation process at which muscle cells that are less mature and possibly more stem-like are prevalent. The data suggests that based on the genes the cells express at early stages, isolation and transplantation of cells at that stage but not further along will be most beneficial for transplantation and clinical application. This brings us a step closer to obtaining useful muscle cells that can be transplanted to treat muscle disorders. The current plan is to test these cells in muscle injury preclinical models to evaluate their capacity to regenerate injured muscle.

Identification of hESC-mediated molecular mechanism that positively regulates the regenerative capacity of post-natal tissues

Funding Type: 
New Faculty I
Grant Number: 
RN1-00532
ICOC Funds Committed: 
$2 246 020
Disease Focus: 
Aging
Muscular Dystrophy
Pediatrics
Trauma
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
The tissue regenerative capacity deteriorates with age in animals and in humans, leading to the loss of organ function, which is well exemplified in skeletal muscle, but is poorly understood in molecular terms. Our recent work uncovered that factors produced by human embryonic stem cells have a unique ability to enhance the regenerative responses of organ stem cells, dedicated for tissue maintenance and repair, be they young or old and located in young or old organism. This proposal seeks to understand the molecular mechanism of this novel phenomenon, which is two-fold important: in expanding our knowledge of the stem cell biology and in developing entirely novel embryonic stem cell-based therapeutic applications that do not have the side-effects associated with immune rejection. Importantly, this uncovered enhancement of tissue repair is conserved between mice and humans , which allows use of an animal model for identifying these therapeutically-relevant human factors and greatly facilitates the pre-clinical data collection, interpretation and translation to clinic. The main goals of this Proposal are to identify the embryonic pro-regenerative factors, to understand their mode of action and to validate their efficiency for enhancing and rejuvenating repair of injured and pathological tissues in an animal model. Notably, using the infrastructure of [REDACTED] the data generated by this work will be quickly disseminated to [REDACTED] clinicians and will be applied through Clinical Affiliates Program for clinical studies and human trials. Identifying these embryonic stem cell-produced pro-regenerative factors will help counter the loss of tissue maintenance and repair in the old, generally and not just in skeletal muscle, and will be of immediate therapeutic value without a need for “humanization” and without the risk of immune rejection. Additionally, for muscle wasting caused by diabetes and immobility, and in Duchenne/Becker and Limb-Girdle myopathies, these factors will boost the performance of satellite cells struggling to repair continuous myofiber deterioration, thus countering degeneration and improving organ function.
Statement of Benefit to California: 
Degenerative diseases in which the bodies capacity to regenerate new tissue can no longer keep up with tissue death is a major problem for society in general and for State of California in particular. The lack of tissue repair that eventually leads to the loss of organ function is undeniable and devastating trait of aging that causes many degenerative disorders, exemplified by Parkinson’s, Alzheimer’s and muscle atrophy. Therefore, Californians with life-long skills, expertise and invaluable knowledge can no longer contribute to society and do not enjoy life fully. In recent years biologists and clinicians realized that practical therapies would only emerge when the balance between the regenerative and the degenerative processes were properly understood in biomedical terms. Comprehensively, the proposed research seeks to uncover novel evolutionary conserved molecular regulation that is mediated by human embryonic stem cells and promotes regenerative capacity of postnatal stem cells (likely, generally and not just in skeletal muscle). Qualified scientists from underrepresented minorities will be involved with this academic and translational stem cell project, hence allowing expand the representation of all Californians in the cutting-edge biomedical research. This proposal describes steps to rejuvenate stem cell responses in the old and to rescue tissue repair in people suffering from debilitating degenerative diseases. The outcomes of this work will insure that the health prognosis is significantly improved for older Californians, especially those afflicted with degenerative disorders, and that the results of these studies are translated as rapidly as possible to the clinical setting where their practical benefit can be fully utilized. Thus this work seeks not only to improve the quality of life for our older citizens, but also to reduce the health-cost associated with treating currently incurable degenerative diseases. The developing therapies will be immediately applicable for all Californians irregardless of their ethnic background, gender or age.
Progress Report: 
  • In 2009 beginning of 2010 we have focused on investigating what factors human embryonic stem cells (hESCs) may produce that enhance regeneration and if those factors have any effects by themselves on regeneration. We have published three papers and four book chapters funded at least in part by this award. One patent application has been filed with our University. We have used a proteomic antibody array to examine over 500 common signaling proteins at once to see if any are produced in much higher or lower levels by hESCs. We found that hESCs produce both positive growth factors and negative regulators of the TGF-beta family. We confirmed that typical growth factor signaling was in fact occurring in muscle cells exposed to hESC produced factors, and that hESCs produce a TGF-beta antagonist. This fits with our recently published work showing that young muscle regenerates well from strong growth factor signaling and low TGF-beta signals while old muscle regenerated poorly due to weak growth factor signaling and high TGF-beta signaling. Our current running hypothesis is that the positive growth factors produced by hESCs trigger injured muscle to initiate and maintain regeneration, the TGF-beta inhibitors produced by hESCs reduce the TGF-beta signaling, and the combination assures the robust regeneration of muscle. We also found a surprising increase in insulin production by hESCs and are integrating that result with ongoing regeneration experiments. In the next reporting period we will re-confirm that the levels of candidate proteins from the 500 antibody array actually are very highly produced by hESCs and that the signals from these proteins are perceived by regenerating muscle cells. For Aim 4 we have examined the effects on live regenerating muscle of administering the TGF-beta inhibitors that we found in Aim 2. Preliminary data indicates the effects on regeneration of old muscle look very promising. What was surprising is that administering these inhibitors to the whole animal appears to reduce TGF-beta levels in the whole animal, suggesting some kind of feed-back and perhaps effects on other tissues as well as muscle. For the next reporting period we will confirm these results. In addition we will analyze the effect on regeneration of administering the growth factors that we found in Aim 2, both alone and in combination with the inhibitors of TGF-beta.
  • In 2010 beginning of 2011, we have approached the identification and characterization of the proteins that are produced by hESCs and have the rejuvenating and pro-regenerative activity on adult muscle. Specifically, our data suggest that several other ligands of MAPK pathway secreted by hESCs are likely to enhance and rejuvenate the regeneration of old muscle tissue. Our work is at the stage of understanding the molecular mechanisms by which the aging of the regenerative potential of organ stem cells can be reversed by particular human embryonic factors that are capable of neutralizing the affects of aged niches on tissue regenerative capacity. We have submitted the several manuscripts on topics of enhanced tissue regeneration and we are preparing the manuscript that identifies hESC-based novel strategies for restoring high regenerative capacity to old muscle. Additionally, our data in progress suggest that muscle and brain age by similar molecular mechanisms and thus, therapeutic strategies for rejuvenating muscle repair might be applicable to the restoration of neurogenesis in aged brain. Finally, our data suggest that muscle stem cells either do not accumulate DNA damage with age or can efficiently repair such damage, when activated for tissue regeneration. Thus, the use of hESC-produced pro-regenerative factors for boosting the regenerative capacity of organ stem cells is likely to yield healthy, young tissue. Our plan is to develop further these projects that cross-fertilize each other and have a main theme of enhancing and rejuvenating tissue regeneration. In the next funding period we also plan to accomplish transition from mouse model to human cells and studies.
  • Although functional organ stem cells persist in the old, tissue damage invariably overwhelms tissue repair, ultimately causing the demise of an organism. The poor performance of stem cells in an aged organ, such as skeletal muscle, is caused by the changes in regulatory pathways such as Notch, MAPK and TGF‐β, where old differentiated tissues and blood circulation inhibit the regenerative performance of organ stem cells. While responses of adult stem cells are regulated extrinsically and age‐specifically,
  • our work recently published puts forward experimental evidence suggesting that embryonic cells have an intrinsic youthful barrier to aging and produce soluble pro‐regenerative proteins that signal the MAPK pathway for rejuvenating myogenesis. Future identification of this activity will improve our understanding of embryonic versus adult regulation of tissue regeneration suggesting novel strategies for organ rejuvenation. Comprehensively, our progress of the last year indicates that if the age‐imposed decline in the regenerative capacity of stem cells was understood, the debilitating lack of organ maintenance in the old could be ameliorated and perhaps, even reversed.The same understanding is also required for successful transplantation of stem and progenitor cells into older individuals and for combatting many tissue degenerative disorders: namely, productive performance of transplanted cells is dependent on the niche into which they are placed and the inhibitory factors of the aged and pathological niches need to be identified and neutralized. Additional recently published work was focused on developing new strategies for providing new source of regenerative cells to people who suffer from genetic myopaties (where their own muscle stem cells become exhausted due to the progression of the disease). Muscle regeneration declines with aging and myopathies, and reprogramming of differentiated muscle cells to their progenitors can serve as a robust source of therapeutic cells. We utilized small molecule inhibitors to dedifferentiate muscle into dividing myogenic cells, without gene over expression, which is clinically adaptable. The reprogrammed muscle precursor cells contributed to muscle regeneration in vitro and in vivo and were unequivocally distinguished because of the lineage marking method. These findings enhance understanding of cell-fate determination and create novel therapeutic approaches for improved muscle repair. Moreover, one more of our recently published papers has identified new ways of making muscle progenitor cells to fuse more robustly into muscle fibers, hence enabling deliberate control of muscle tissue formation. We are at the latest stages in our work on the design of novel biomimetic stem cell niches, which based on our current results make easy to expand in culture progenitor cells (e.g. derived from paints) akin to muscle stem cells and enhancing the efficiency of cell transplantation to such an extent that progressive muscle loss in genetic myopathies is predicted to be averted. We have also deciphered some of the fundamental properties of embryonic stem cells, which would enable deliberate control of their self-renewal and tissue specific differentiation and the manuscript describing these findings has been submitted to Cell.
  • Since the last progress report we have confirmed and extrapolated our studies and, as proposed last year, we have identified specific proteins that are secreted by human embryonic stem cells and that enhance muscle regeneration. We have extrapolated the mouse findings and see that these therapeutic embryonic proteins have the pro-regenerative activity on human muscle cells, and excitingly, show that these factors also enhance proliferation of neuronal stem cells and even combat the Alzheimer’s disease (modeled in human cortical neurons derived from embryonic stem cells). We are starting to understand how these pro-regenerative proteins act (which will help to optimally harness their therapeutic potential). We are also in the process of attempting rejuvenation of tissue repair in live aged animals and the preliminary results are encouraging. Notably, the manuscripts, which were listed in the last progress report as in preparation or submitted have been published.
  • The work on the hESC-secreted pro-regenerative factors has been published in two papers and the third manuscript is under review. Additionally, an invention disclosure has been filed with UC Berkeley on the enrichment of the pro-regenerative activity in proteins with heparin-containing domains and thus, our ability to enrich these therapeutic factors by heparin-coated beads.

Skeletal muscle development from hESC and its in vivo applications in animal models of muscular dystrophy

Funding Type: 
New Faculty I
Grant Number: 
RN1-00525
ICOC Funds Committed: 
$1 623 064
Disease Focus: 
Muscular Dystrophy
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
Embryonic Stem Cell
oldStatus: 
Closed
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.

A Novel Microenvironment-Mediated Functional Skeletal Muscle from Human Embryonic Stem Cells and their In Vivo Engraftment

Funding Type: 
New Faculty II
Grant Number: 
RN2-00945
ICOC Funds Committed: 
$2 300 569
Disease Focus: 
Muscular Dystrophy
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Muscle wasting is a serious clinical problem associated with a number of diseases and health conditions, affecting individuals of all ages. Muscular dystrophy (MD) is a form of muscle wasting disease resulting from genetic mutations. Duchenne muscular dystrophy (DMD) is the most common form of MD that limits motility and life expectancy of children. It is characterized by progressive skeletal muscle degeneration, and occurs in 1 out of every 3,500 male births. Currently, there is no effective treatment to stop or reverse DMD. One of the potential clinical solutions for treating DMD is cell transplantation where the implanted cells contribute to the functional muscle regeneration. Adult stem cells such as mesoangioblasts and AC133 cells have already been shown to ameliorate dystrophic muscle pathology in animals. Although embryonic stem cells (ESCs) can provide unlimited numbers of progenitor cells with the ability to contribute to the formations of functional skeletal muscle, their ability to reverse/repair wasting muscle has not been explored in detail. To this end, we seek to develop an ESC-based cell transplantation therapy for treating muscle-wasting pathology, focusing on DMD. Three specific aims are proposed to achieve this overall goal. (i) Optimize the microenvironment factors comprising of cell-matrix interaction, cytokines, and cell-secreted morphogens on differentiation of ESC into muscle cells using a combinatorial array technology. (ii) Derive and characterize a population of clinically viable ESC-derived muscle progenitor cells using a dynamic, three-dimensional (3D), bioengineered niche. The tailored niche would be comprised of a multifunctional hydrogel that exhibits “excitation-contraction” dynamics. We will then functionalize this hydrogel with optimized ECM components and use it to investigate myogenic differentiation of ESCs in a 3D environment, and to derive engraftable muscle progenitor cells from ESCs. (iii) Investigate the therapeutic efficacy of ESC-derived progenitors for treating DMD pathology using animal models. The molecular pathways involved in myogenic differentiation of ESCs will be analyzed using proteomics and transcriptomics tools. Successful completion of this study could offer broad applicability of stem-cell-based therapies for treating all types of muscle wasting diseases and other pathologies associated with dysfunctional muscles. Unraveling the microenvironment factors and molecular events determining the commitment of ESCs into specific lineages will significantly increase our understanding of stem cell biology and their application in regenerative medicine. In addition, the multifunctional hydrogel (mechanical actuator) developed here could find many applications in the field of artificial functional implants where a muscle-like response is desirable, cardiac tissue repair, drug delivery, and biomimetic devices.
Statement of Benefit to California: 
The proposed study seeks to develop a clinically viable cell transplantation therapy based on pluripotent embryonic stem cells (ESCs) for treating muscular dystrophies (MDs), especially Duchenne muscular dystrophy (DMD). DMD is the most common form of MD that limits motility and shortens life expectancy of children, and is estimated to occur 1 in 3500 male births. DMD is characterized by progressive muscle wasting, limited ambulation, compromised lung or cardiac function, paralysis, and ultimately premature death. DMDfund (http://www.dmdfund.org), a California-based forum dedicated to families of children affected by DMD, emphasizes the devastating nature of this disease “... in contrast to cancer, DMD currently is uniformly fatal. As a result there is only a 3-fold difference in the number of childhood deaths due to cancer vs. DMD, but DMD research is relatively scarce. The paucity of research may be the reason that there is no cure for this devastating neuromuscular disease. Without research, there is no hope. Shouldn't all people at least been given the chance to hope?” Clearly, there is a need to develop new paradigms for treating DMD. The PI has proposed one possible approach to address this need and it will contribute to the State of California and its citizens in the many ways: Clinical benefits: Successful completion of the project will help develop new stem-cell-based therapies for treating DMD and other muscle wasting diseases. Unraveling the microenvironment factors and molecular events that determine the commitment of ESCs will significantly increase their treatment potential. Concepts and knowledge gained from the study can translate into other areas of regenerative medicine. Economic benefits: There are two ways the proposed work could contribute to the economy of California. First, improvements in DMD treatment could bring down the healthcare costs. Second, tools and technologies developed during the course of this study (soft mechanical actuators, smart biomimetic hydrogels, etc.) can find various other applications in biomedical fields such as artificial functional implants, cardiac tissue repair, drug delivery, and biomimetic devices. Such devices could be of great interest to California-based biotechnology companies. The PI’s past work involving musculoskeletal tissues has contributed to a start-up biotech company located in California, and the PI hopes to contribute similarly through the proposed studies. Education, training, and awareness: One of the most important components of the PI’s laboratory is training future leaders in the biomedical/stem cell field. We expect to train 10-12 researchers within the course of this project (clinical fellows, postdoctoral fellows, graduate students, undergraduate students, and high school students). These very students and trainees will, in the future, lead to the development of newer and more advanced strategies for regenerative medicine.
Progress Report: 
  • The overarching goal of this proposal is to develop robust clinically viable stem cell based therapeutics for muscle wasting focusing on Duchenne Muscular Dystrophy. Three specific aims were proposed to achieve this goal, and during the past year we have made significant progress towards this goal. The focus of aim one is to optimize the microenvironment factors comprising of cell-matrix interaction, cytokines, and cell-secreted morphogens on differentiation of stem cells into muscle cells. Stem cells require complex signals for differentiation and many of which are unknown, making it a challenge to direct tissue specific differentiation. We have adopted a stepwise differentiation strategy to promote myogenic differentiation of pluripotent stem cells (embryonic stem cells, ESCs and induced pluripotent stem cells, iPSCs). This involves differentiating the pluripotent stem cells into mesoderm progenitor cells followed by differentiating them into muscle cells. Our experimental findings show that our approach promotes efficient mesoderm differentiation of pluripotent stem cells. These extensively expandable ESC/iPSC-derived mesoderm progenitor cells are now being subjected to myogenic differentiation. We have also identified and optimized the combination of soluble factors that promote myogenic differentiation of muscle progenitor cells and stem cells. These soluble factors were found to promote myotube formation of muscle progenitor cells, in addition to promoting myogenic differentiation of stem cells. In an effort to understand the role of cell-matrix interactions, we have developed a novel method to decellularize the native skeletal muscle while maintaining their structural, biochemical composition, and mechanical properties unaltered. This approach has much more implications than supporting myogenic differentiation because it can be used as a model system to examine the changes ECM undergoes with pathological changes and how these changes affect stem cell myogenic differentiation and engraftment.
  • The second aim of the proposed study is to test the hypothesis that a bioengineered niche exhibiting “excitation-contraction” dynamics encoded with biochemical cues can be used as a 3D microenvironment to promote myogenic differentiation of stem cells. The first goal of this aim is to develop a bioengineered synthetic niche recapitulating the biophysical cues of native skeletal muscle. To this end, we have developed synthetic hydrogel based biocompatible electro-mechanical matrices, which not only provide three-dimensional structural support to the embedded cells but also can simultaneously provide dynamic mechanical and electrical cues to the cells. A unique aspect of these matrices is that they can undergo reversible, anisotropic bending dynamics in an electric field and functions like multi-functional mechanical actuators. The direction and magnitude of this bending can be tuned through the hydrogel crosslink density while maintaining the same electric potential gradient, allowing precise control over the mechanical strain imparted to the cells in a three-dimensional environment. Our results showed that these bioengineered electro-mechanical niches not only support stem cell culture but also promotes their proliferation and differentiation. The third aim of the proposed study is on evaluating the engraftment and differentiation of in vitro conditioned stem cells using animal models. Based on the progress made in aims 1 and 2, we are certain that we will be able to initiate the animal studies soon.
  • In sum, we have made significant progress in aims 1 and 2 of the proposed study. We have submitted one manuscript and three more manuscripts are under preparation. Another important achievement is training the researchers in stem cells. In addition to the postdoctoral and graduate fellows, we also trained undergraduate and high school students; Jomkuan Theprungsirikul from United World College, Montezuma, NM, had spent the summer in our lab to learn more about regenerative medicine and its potential in irradiating the public health problems. The PI visited Francis Parker School and gave a talk on stem cells on September 23 (Stem Cell Awareness Day). The PI also gave an invited talk on stem cell and regenerative medicine at the 3rd International Congress of NanoBiotechnology & Nanomedicine NanoBio 2009 held in San Francisco, CA.
  • N/A
  • During the reporting period, we successfully developed a protocol for deriving progenitor cells from human embryonic stem cells, which is being written up for a publication. The ESC-derived progenitor cells were found to undergo both myogenesis in vitro and in vivo. We were able to significantly expand these cells in vitro and the in vitro cultured cells expressed a number of muscle markers Pax3, Myf5, desmin, and MyoG. A muscle injury model was then used to investigate the in vivo viability and engraftment efficiency of these cells. A significant fraction of the transplanted cells were found to be engrafted. Additionally, we observed localization of a large number of transplanted cells in the centers of muscle fibers indicating the contribution of the transplanted cells towards the muscle regeneration. We have also developed a muscle-like synthetic material (termed as electromechanical material) that simultaneously provides mechanical and electrical cues to the embedded cells. A manuscript based on these results is published in Advanced Functional Materials, a highly regarded journal in interdisciplinary materials science. This work also received significant press coverage. The above-developed system will allow us determine the effect of various physicochemical cues of the matrix on myogenic commitment and maturation of progenitor cells.
  • During the reporting period, we successfully developed a protocol for deriving progenitor cells from human embryonic stem cells, which is being written up for a publication. The ESC-derived progenitor cells were found to undergo both myogenesis in vitro and in vivo. We were able to significantly expand these cells in vitro and the in vitro cultured cells expressed a number of muscle markers Pax3, Myf5, desmin, and MyoG. A muscle injury model was then used to investigate the in vivo viability and engraftment efficiency of these cells. A significant fraction of the transplanted cells were found to be engrafted. Additionally, we observed localization of a large number of transplanted cells in the centers of muscle fibers indicating the contribution of the transplanted cells towards the muscle regeneration. We have also developed a muscle-like synthetic material (termed as electromechanical material) that simultaneously provides mechanical and electrical cues to the embedded cells. A manuscript based on these results is published in Advanced Functional Materials, a highly regarded journal in interdisciplinary materials science. This work also received significant press coverage. The above-developed system will allow us determine the effect of various physicochemical cues of the matrix on myogenic commitment and maturation of progenitor cells.
  • We have developed a protocol devoid of genetic manipulations to derived progenitor cells with myogenic differentiation potential from human embryonic stem cells (hESCs). These hESC-derived cells underwent myogenic differentiation in vitro both in the presence and absence of serum in culture medium. When transplanted in vivo into an injured muscle, these pre-myogenically committed cells were viable in tibialis anterior muscles 14 days post-implantation. A manuscript describing these results have been published (Hwang Y, Suk S,Lin S, Tierney M, Du B, Seo T, Mitchell A, Sacco A, Varghese S. Directed in vitro myogenesis of human embryonic stem cells and their in vivo engraftment. PLoS One, 8, e72023 (2013). We have further modified the culture conditions to improve myogenic differentiation. When transplanted into injured muscles of immune-deficient NOD/SCID mice, a significant portion of fraction of the transplanted cells were found to be engrafted. Additionally, we observed localization of a large number of transplanted cells in the centers of muscle fibers indicating the contribution of the transplanted cells towards the muscle regeneration. Additionally, we also observed contribution of the transplanted donor cells towards the satellite compartment. The high proliferative capacity of hESCs along with their ability to differentiate into functional skeletal myogenic progenitor cells and engraft in vivo without teratoma formation highlights their potential therapeutic applications to ameliorate skeletal muscle defects and injuries.

Functional Genomic Analysis of Chemically Defined Human Embryonic Stem Cell

Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00100
ICOC Funds Committed: 
$2 628 635
Disease Focus: 
Genetic Disorder
Muscular Dystrophy
Pediatrics
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Regenerative medicine holds the promise that tissues can be engineered in vitro and then transplanted into patients to treat debilitating diseases. Human Embryonic Stem Cells differentiate into a wide array of adult tissue types and are thought to be the best hope for future regenerative therapies. This grant has three main goals: 1. The creation of new human embryonic stem cells in animal free conditions which will allow for future therapeutic uses. 2. The creation of human embryonic stem cell that contain mutations in their genomes that cause diseases, including cystic fibrosis, muscular dystrophy, Downs Syndrome and many others. These lines can be used to study these diseases and to test potential therapies 3. A close biological assessment of one of the first tissues to arise during differentiation of human embryonic stem cells – the endoderm. Since the endoderm eventually, during many days of development, becomes the pancreas, liver, and gut. It is critically important that we know everything about this very specialized tissue if we are to attempt to engineer these organs in the laboratory. Our overwhelming goal is to provide tools that clinician can use to treat disease whether it is to establish new and improved human embryonic stem cell lines or to find new ways of creating endodermal tissues within the laboratory for future therapeutic uses.
Statement of Benefit to California: 
This grant will provide to the state of California new human embryonic stem cell lines that could be used in future therapeutic uses. It will also provide disease specific human embryonic stem cell lines that can be used to study disease and as models to test pharmacological compounds to treat disease. We will also provide a characterization of tissues generated from the new human embryonic stem cells which we hope will someday aid in the formation of liver, lung and pancreas.
Progress Report: 
  • We have made significant progress during the previous granting period which has resulted in a publication in Genome Research detailing genomic DNA methylation changes in a variety of human embryonic stem cells and their derivatives. We have also been successful in identifying regions of the genome bound by of histone modifications and transcription factors in hESCs and derived endoderm. These targets have led to a greater understanding of how Nodal signaling and chromatin configuration maintain pluripotent state and subsequently trigger differentation. We expect 3 publications to result from this work during the next granting period.
  • Toward a goal of developing endodermal lineages from hESCs, including liver, pancreas, lung and intestine, we have developed new tools and approaches to identify these subtypes as well as a molecular understanding of how these subtypes emerge. These advances are highlighted in three papers which are currently under review for publication and one in preparation. Two of these papers redefine endodermal subtypes derived from hESCs, including new methods to isolate lineage restricted endodermal populations and a means to distinguish between single endodermal cells. The third paper provides an unprecedented view of the Nodal signaling pathway and its intersection with bivalent domains in both hESCs and derived endoderm. This chromatin signature which consists of Smad transcription factors and both histone repressive and active marks is the most conducive for mediating downstream targets of Nodal, providing inroads into how signaling pathways and chromatin cooperate during fate specification in hESCs. This analysis has led to the elucidation of new proteins that mediate Nodal signaling; ones that play a key role in endoderm specification.
  • During the past year, we have completed three research projects as a result of this grant. Two of these have recently been published in Genes and Development and Developmental Biology. The third is currently in review at Development. The discoveries reported in these papers are highlighted below:
  • HEB and E2A function as SMAD/FOXH1 cofactors
  • Nodal signaling, mediated through SMAD transcription factors, is necessary for pluripotency maintenance and endoderm commitment. We have identified a new motif, termed SMAD Complex Associated (SCA) that is bound by SMAD2/3/4 and FOXH1 in human embryonic stem cells (hESCs) and derived endoderm. We demonstrate that two bHLH proteins - HEB and E2A - bind the SCA motif at regions overlapping SMAD2/3 and FOXH1. Further, we show that HEB and E2A associate with SMAD2/3 and FOXH1, suggesting they form a complex at critical target regions. This association is biologically important, as E2A is critical for mesendoderm specification, gastrulation, and Nodal signal transduction in Xenopus tropicalis embryos. Taken together, E proteins are novel Nodal signaling cofactors that associate with SMAD2/3 and FOXH1 and are necessary for mesendoderm differentiation.
  • Chromatin and Transcriptional Signatures for Nodal Signaling During Endoderm Formation in hESCs
  • The first stages of embryonic differentiation are driven by signaling pathways hardwired to induce particular fates. Endoderm commitment is controlled by the TGF-β superfamily member, Nodal, which utilizes the transcription factors, SMAD2/3, SMAD4 and FOXH1, to drive target gene expression. While the role of Nodal is well defined within the context of endoderm commitment, mechanistically it is unknown how this signal is manifested at binding regions within the genome and how this signal interacts with chromatin state to trigger downstream responses. To elucidate the Nodal transcriptional network that governs endoderm formation, we used ChIP-seq to identify genomic targets for SMAD2/3, SMAD3, SMAD4, FOXH1 and the active and repressive chromatin marks, H3K4me3 and H3K27me3, in human embryonic stem cells (hESCs) and derived endoderm. We demonstrate that while SMAD2/3, SMAD4 and FOXH1 binding is highly dynamic, there is an optimal signature for driving endoderm commitment. Initially, this signature is marked by both H3K4me3 and H3K27me3 as a very broad bivalent domain in hESCs. Within the first 24 hours, at a few select promoters, SMAD2/3 accumulation coincides with H3K27me3 reduction so that these loci become relatively monovalent marked by H3K4me3. JMJD3, a histone demethylase, is simultaneously recruited to the promoters of the endodermal gene GSC and EOMES, suggesting a conservation of mechanism at multiple promoters genome-wide. The correlation between SMAD2/3 binding, monovalent formation and transcriptional activation suggests a mechanism by which SMAD proteins coordinate with chromatin at critical promoters to drive endoderm specification.
  • Distinguishing Cells with Housekeeping Transcripts
  • Distinguishing between cell types is central to a broad array of biological fields and is at the heart of advancing regenerative medicine and cancer diagnostics. In this report, we use single cell gene expression to identify transcriptional patterns emerging during the differentiation of human embryonic stem cells (hESCs) into the endodermal lineage. Endoderm specific transcripts are highly variable between individual CXCR4+ endodermal cells, suggesting that either the cells generated from in vitro differentiation are distinct or that these embryonic cells tolerate a high degree of transcript variability. Housekeeping transcripts, on the other hand, are far more consistently expressed within the same cellular population. However, when we compare the levels of housekeeping transcripts between hESCs and derived endoderm, patterns emerge that can be used to clearly separate the two embryonic cell types. We further compared 4 additional human cell types, including 293T, iPSC, HepG2 and endoderm derived iPSC. In each case the relative levels of housekeeping transcripts defined a particular cell fate. Interestingly, we find that three transcripts, LDHA, NONO and ACTB, contribute the most to this diversity and together serve to segregate all 6 cell types. Overall, this suggests that levels of housekeeping transcripts, which are expressed within all cells, can be leveraged to distinguish between human cell types and thus may serve as important biomarkers for stem cell biology and other disciplines.

Derivation and characterization of human ES cells from FSHD embryos

Funding Type: 
SEED Grant
Grant Number: 
RS1-00455
ICOC Funds Committed: 
$632 500
Disease Focus: 
Genetic Disorder
Muscular Dystrophy
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Facioscapulohumeral muscular dystrophy (FSHD) is the third most common hereditary muscular dystrophy. It is autosomal dominant, meaning that if one of the parents has the disease, their children have a 50:50 chance of getting it, too. FSHD is characterized by progressive weakness and atrophy of facial, shoulder and upper arm musculature, which can spread to other parts of the body. In some cases, it is accompanied by hearing loss and, in severe cases, mental retardation. There is no cure or treatment of this disease since the gene(s) responsible for this disease has not been identified. One thing that is clear is that the majority of FSHD is linked to a decrease in the number of repeats of a DNA sequence called D4Z4 located at the end of chromosome 4. When shortening of this repeat region occurs in either chromosome 4, the person gets FSHD. However, it is unclear how shortening of this repeat leads to the disease. We found that this D4Z4 repeat cluster contains “heterochromatin” structure, which is associated with gene silencing. This heterochromatin structure includes specific methylation of histone H3 and the recruitment of heterochromatin binding proteins HP1 and cohesin. HP1 is known to be required for gene silencing. Importantly, we found that this heterochromatin structure is uniquely lost in FSHD patient cells. Surprisingly, the minor population of FSHD patients who do not exhibit any repeat shortening also lack this heterochromatin structure in the D4Z4 repeat on chromosome 4. Therefore, FSHD is a “heterochromatin abnormality” disease, in which loss of heterochromatin at D4Z4 repeats leads to disease manifestation. We hypothesize that the normal heterochromatin structure spreads silencing effects on to other genes, but in FSHD this effect is lost and these genes that are normally silent may be abnormally expressed. Since we found that this heterochromatin structure is already established in embryonic stem (ES) cells under normal circumstances, it is of vital importance to examine this process in FSHD ES cells. This would be important to understand how heterochromatin establishment is compromised during development and, as a result, which genes are affected. However, no FSHD ES cells are currently available. Generous and courageous families with FSHD in their history donated in vitro fertilized embryos for research use in the hope of improving the life of FSHD patients in the future. Therefore, our major goal is to establish FSHD ES cell lines not only for our research, but also for use in the FSHD research community. We hope to optimize a protocol to differentiate these cells into skeletal muscle cells for a comparative analysis between normal and FSHD ES cells during development. I believe that the proposed project will make significant contributions to understanding the etiology and pathogenesis of FSHD as well as to possibly develop therapeutic strategies to improve the physical functioning of FSHD patients.
Statement of Benefit to California: 
Facioscapulohumeral dystrophy (FSHD) is the third most common hereditary muscular dystrophy in the United States. California is the most represented state in the U.S. in terms of membership in the FSH Society. Almost 250 families in California with an average of 3-4 affected members per family belong to the FSH Society, which would translate to 750 to 1,000 total registered patients. FSHD is reported to have a 1 in 20,000 incidence. However, the Society estimates that the actual incidence is probably considerably higher, with a likely incidence of 1/7,000. This higher estimate is based on clinician expert opinion that FSHD is at least three times more prevalent due to misdiagnosis, which reflects the difficulty associated with recognizing those patients with a mild clinical disease presentation. This is consistent with the opinions of some people who track FSHD cases that improved molecular diagnostic techniques will give a more accurate assessment of the full range of this disease in the population. Since this disease is dominantly inherited, a large family can have a significant number of affected individuals. In one documented case a family of 2,500 people traced to a settler who had FSHD had1600-1700 affected members, many of whom ultimately moved to California. Many families affected by this disease are reluctant to come forward to seek help, and therefore are not included in the membership rolls of the FSH Society. FSHD is not necessarily lethal and many patients must live and cope with progressive disability during their lives without any effective treatment. There is no effective treatment because the disease mechanism is unclear. The fact that the upper limbs are predominantly affected suggests that the abnormality may be initiated during embryonic development. Thus, the proposed project to obtain FSHD ES cells is critical for studying this crucial time point in manifestation of the disease. We plan to make these cells available to scientists in the FSHD field to further facilitate investigation of many different aspects of FSHD pathogenesis. We also hope that these cells become valuable reagents for development of better molecular diagnostics as well as fostering new ideas concerning possible treatment strategies. The primary goal is to offer new insight into improving the lives of the many FSHD patients in California and across the international community.
Progress Report: 
  • Facioscapulohumeral muscular dystrophy (FSHD) is the third most common hereditary muscular dystrophy. There is no cure or treatment for this disease since the disease mechanism is not understood. We found evidence that FSHD is a “chromatin abnormality” disease, in which specific histone methylation and factor binding affecting gene silencing is lost at specific genomic regions. We hypothesize that the chromatin structure important for gene regulation is compromised in FSHD. Since we found that this chromatin structure is established in normal human embryonic stem (hES) cells, it is critical to examine how this process goes awry in FSHD during differentiation of hES cells into skeletal muscle cells. Therefore, our major goal is to establish hES cell lines from affected and normal embryos and to optimize a protocol to differentiate hES cells into skeletal muscle cells for comparative analyses. This is not only for our research, but also for use in the FSHD research community, which should help to understand the etiology and pathogenesis of FSHD as well as to possibly develop therapeutic strategies. During the past one year, we continued to optimize skeletal myoblast differentiation from hES cells by using several different protocols. While we were able to detect expression of muscle lineage-specific marker genes (i.e., Pax7 and MyoD) during ES cell differentiation, the expression level remained low and we continue to investigate different protocols for better gene induction and enrichment of the proper cell population by cell sorting. It is essential to attain a sufficiently high density of derived myoblasts in order to allow them to form myotubes, which is the basic fundamental element of skeletal muscle. The very recent publication (i.e., June, 2009) of a detailed protocol for derivation of skeletal muscle cells from hES cells should be of great assistance to us, as it highlights several key factors that are likely to be crucial for success. In addition, we have acquired with proper hSCRO approval the first batch of embryos of seven donated by an FSHD patient donor. Out of seven embryos, we have thawed five embryos initially and followed their growth in culture dishes. However, these embryos were frozen five years ago under suboptimal conditions, and failed to further develop in culture for us to derive any ES cells. We are in the process of thawing the remaining two embryos, although similar problems may persist. Presently, we have identified two additional sources of FSHD embryos. The acquisition process has been considerably prolonged due to recent changes in NIH regulations that necessitated the modification of the consenting form and process. However, with the help of our collaborator from Scripps, Dr. Jeanne Loring, we are in the process of obtaining five additional embryos and we have requested a “no cost” extension of this project in order to complete the derivation of hES cells from these embryos. During the past year, we published our initial characterization of the chromatin structure in FSHD patient cells by comparison with normal cells of different cell types, including hES cells. We detected differences in how one of the histone proteins is chemically modified in certain regions of the genomes in normal versus FSHD cells. This is important since histone proteins not only help to package DNA to form the basic element of chromatin, they also play a huge role in controlling gene expression. Since the effects of FSHD likely result from abnormal regulation of expression of certain genes, our work identified a relevant chromatin “marker” which is potentially useful for diagnostic purposes. In addition, our findings define a way to follow any changes in target gene regulation during the differentiation process (i.e., hES cells to myoblasts and myotubes) in normal and FSHD hES cells. The published manuscript was selected for “Faculty of 1000”, and we have already been asked to write a review on this subject. Furthermore, we have obtained NIH R01 funding for further high-throughput analysis of chromatin structure in FSHD. Taken together, with the support of the CIRM SEED grant, we are moving forward to understand the chromatin abnormality during cellular differentiation linked to FSHD pathogenesis.

Generation of clinical grade human iPS cells

Funding Type: 
New Cell Lines
Grant Number: 
RL1-00681
ICOC Funds Committed: 
$1 382 400
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Melanoma
Cancer
Muscular Dystrophy
Neurological Disorders
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 
The therapeutic use of stem cells depends on the availability of pluripotent cells that are not limited by technical, ethical or immunological considerations. The goal of this proposal is to develop and bank safe and well-characterized patient-specific pluripotent stem cell lines that can be used to study and potentially ameliorate human diseases. Several groups, including ours have recently shown that adult skin cells can be reprogrammed in the laboratory to create new cells that behave like embryonic stem cells. These new cells, known as induced pluripotent stem (iPS) cells should have the potential to develop into any cell type or tissue type in the body. Importantly, the generation of these cells does not require human embryos or human eggs. Since these cells can be derived directly from patients, they will be genetically identical to the patient, and cannot be rejected by the immune system. This concept opens the door to the generation of patient-specific stem cell lines with unlimited differentiation potential. While the current iPS cell technology enables us now to generate patient-specific stem cells, this technology has not yet been applied to derive disease-specific human stem cell lines for laboratory study. Importantly, these new cells are also not yet suitable for use in transplantation medicine. For example, the current method to make these cells uses retroviruses and genes that could generate tumors or other undesirable mutations in cells derived from iPS cells. Thus, in this proposal, we aim to improve the iPS cell reprogramming method, to make these cells safer for future use in transplant medicine. We will also generate a large number of iPS lines of different genetic or disease backgrounds, to allow us to characterize these cells for function and as targets to study new therapeutic approaches for various diseases. Lastly, we will establish protocols that would allow the preparation of these types of cells for clinical use by physicians investigating new stem cell-based therapies in a wide variety of diseases.
Statement of Benefit to California: 
Several groups, including ours have recently shown that adult skin cells can be reprogrammed in the laboratory to create new cells that behave like embryonic stem cells. These new cells, known as induced pluripotent stem (iPS) cells should have, similar to embryonic stem cells, the potential to develop into any cell type or tissue type in the body. This new technology holds great promise for patient-specific stem-cell based therapies, the production of in vitro models for human disease, and is thought to provide the opportunity to perform experiments in human cells that were not previously possible, such as screening for compounds that inhibit or reverse disease progression. The advantage of using iPS cells for transplantation medicine would be that the patient’s own cells would be reprogrammed to an embryonic stem cell state and therefore, when transplanted back into the patient, the cells would not be attacked and destroyed by the body's immune system. Importantly, these new cells are not yet suitable for use in transplantation medicine or studies of human diseases, as their derivation results in permanent genetic changes, and their differentiation potential has not been fully studied. The goal of this proposal is to develop and bank genetically unmodified and well-characterized iPS cell lines of different genetic or disease backgrounds that can be used to characterize these cells for function and as targets to study new therapeutic approaches for various human diseases. We will establish protocols that would allow the preparation of these types of cells for clinical use by physicians investigating new stem cell-based therapies in a wide variety of diseases. Taken together, this would be beneficial to the people of California as tens of millions of Americans suffer from diseases and injuries that could benefit from such research. Californians will also benefit greatly as these studies should speed the transition of iPS cells to clinical use, allowing faster development of stem cell-based therapies.
Progress Report: 
  • The goal of this project is to develop and bank safe, well-characterized pluripotent stem cell lines that can be used to study and potentially ameliorate human diseases, and that are not limited by technical, ethical or immunological considerations. To that end, we proposed to establish protocols for generation of human induced pluripotent stem cells (hiPSC) that would not involve viral vector integration, and that would be compatible with Good Manufacturing Processes (GMP) standards. To establish baseline characteristics of hiPSCs, we performed a complete molecular characterization of all existing hiPSCs in comparison to human embryonic stem cells (hESCs). We found that all hiPSC lines created to date, regardless of the method by which they were reprogrammed, shared a common gene expression signature, distinct from that of hESCs. The functional role of this gene expression signature is still unclear, but any lines that are generated under the guise of this grant will be subjected to a similar analysis to set the framework by which these new lines are functionally characterized. Our efforts to develop new strategies for the production of safe iPS cells have yielded many new cell lines generated by various techniques, all of which are safer than the standard retroviral protocol. We are currently expanding many of the hiPSCs lines generated and will soon demonstrate whether their gene expression profile, differentiation capability, and genomic stability make them suitable for banking in our iPSC core facility. Once fully characterized, these cells will be available from our bank for other investigators.
  • For hiPSC technology to be useful clinically, the procedures to derive these cells must be robust enough that iPSC can be obtained from the majority of donors. To determine the versatility of generation of iPS cells, we have now derived hiPSCs from commercially obtained fibroblasts derived from people of different ages (newborn through 66 years old) as well as from different races (Caucasian and mixed race). We are currently evaluating medium preparations that will be suitable for GMP-level use. Future work will ascertain the best current system for obtaining hiPSC, and establish GMP-compliant methodologies.
  • The goal of this project is to develop and bank safe, well-characterized pluripotent stem cell lines that can be used to study and potentially ameliorate human diseases. To speed this process, we are taking approaches that are not limited by technical, ethical or immunological considerations. We are establishing protocols for generation of human induced pluripotent stem cells (hiPSCs) that would not involve viral vector integration, and that are compatible with Good Manufacturing Practices (GMP) standards. Our efforts to develop new strategies for the production of safe hiPSC have yielded many new cell lines generated by various techniques. We are characterizing these lines molecularly, and have found hiPSCs can be made that are nearly indistinguishable from human embryonic stem cells (hESC). We have also recently found in all the hiPSCs generated from female fibroblasts, none reactivated the X chromosome. This finding has opened a new frontier in the study and potential treatment of X-linked diseases. We are currently optimizing protocols to generate hiPSC lines that are derived, reprogrammed and differentiated in the absence of animal cell products, and preparing detailed standard operating procedures that will ready this technology for clinical utility.
  • This project was designed to generate protocols whereby human induced pluripotent stem cells could be generated in a manner consistent with use in clinical trials. This required optimization of protocols and generation of standard operating procedures such that animal products were not involved in generation and growth of the cells. We have successfully identified such a protocol as a resource to facilitate widespread adoption of these practices.

Engineered iPSC for therapy of limb girdle muscular dystrophy type 2B

Funding Type: 
Early Translational IV
Grant Number: 
TR4-06711
ICOC Funds Committed: 
$1 876 253
Disease Focus: 
Muscular Dystrophy
Pediatrics
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
Limb girdle muscular dystrophy type 2B (LGMD 2B) is a form of muscular dystrophy that leads to muscle degeneration and disability. In LGMD 2B, a vital muscle protein is mutated, and its absence leads to progressive degeneration of muscles in the body that are needed for mobility. To create a therapy, we will provide a new supply of stem cells that carry the missing protein that is lacking. These cells will be delivered to the body in such a way that they will engraft into the muscles and produce new, healthy muscle tissue on an ongoing basis. We now possess methods to create stem cells that can become muscle cells out of adult skin cells by a process known as “reprogramming”. By reprogramming adult cells, together with addition to them of a correct copy of the gene that is mutated in LGMD 2B, we will create stem cells that have the ability to create new, healthy muscle cells in the body of a patient. This is the type of strategy that we are developing in this proposal. The corrected muscle stem cells will be transplanted into mice with LGMD 2B, and the ability of the cells to generate healthy new muscle tissue and increased muscle strength will be evaluated. This project could lead to a new stem cell therapy that could improve the clinical condition of LGMD 2B patients. If we are successful with this disease, similar methods could be used to treat other degenerative disorders, and perhaps even some of the degeneration that occurs during muscle injury and normal aging.
Statement of Benefit to California: 
The proposed research could lead to a stem cell therapy for limb girdle muscular dystrophy type 2B (LGMD 2B). This outcome would deliver a variety of benefits to the state of California. There would be a profound personal benefit to the Californians affected directly or indirectly by LGMD 2B. Progress toward a cure for LGMD 2B is also likely to accelerate the development of treatments for other degenerative disorders. The most obvious targets would be other forms of muscular dystrophy and neuromuscular disorders. Muscle injury, and even some of the normal processes of muscle aging, may be treatable by a similar strategy. An effective stem cell therapy for LGMD 2B would also bring economic benefits to the state by reducing the huge burden of costs associated with the care of patients with long-term degenerative disorders. Many of these patients would be more able to contribute to the workforce and pay taxes. Another benefit is the effect of novel, cutting-edge technologies developed in California on the business economy of the state. Such technologies can have a profound effect on the competitiveness of California through the formation of new manufacturing and health care delivery facilities that would employ California citizens and bring new sources of revenue to the state. Therefore, this project has the potential to bring health and economic benefits to California that are highly desirable for the state.

Combination therapy to Enhance Antisense Mediated Exon Skipping for Duchenne Muscular Dystrophy

Funding Type: 
Disease Team Therapy Planning I
Grant Number: 
DR2-05426
ICOC Funds Committed: 
$86 414
Disease Focus: 
Muscular Dystrophy
oldStatus: 
Closed
Public Abstract: 
A drug was identified through the use of muscle stem cells that can enhance the effectiveness of exon skipping by antisense oligonucleotides to the DMD gene to restore dystrophin expression and at least partially correct the defect responsible for loss of muscle function in Duchenne. We propose to test the effectiveness of this drug in combination with antisense oligonucleotides as a novel therapeutic strategy for Duchenne muscular dystrophy (DMD). DMD is the most common muscular dystrophy and leads to progressive muscle loss in boys resulting in severe weakness, and is caused by mutations in the DMD gene. DMD generally leads to death in the teens or early 20’s, making Duchenne one of the most severe disorders in humans. Further, Duchenne occurs in 1/3500 boys, making it one of the most common genetic disorders. There are no highly effective therapies. Thus, there is an urgent need to develop new and highly effective therapies. We propose to perform the necessary studies using DMD patient-derived iPS and animal models to perform safety studies that will permit regulatory approval to test the safety and efficacy of the combination therapy in Duchenne muscular dystrophy. The goal of the treatment is to make a functional dystrophin protein the patient’s body by altering the RNA in each muscle cell. Preliminary results indicate that the process is relevant to about 70% of those afflicted by Duchenne.
Statement of Benefit to California: 
Since Duchenne muscular dystrophy is the most common lethal genetic disorder, there are over 1,000 patients affected in the state of California alone, 15,000 nationwide, and 300,000 worldwide. Duchenne muscular dystrophy has a large direct economic impact with intensive medical care with substantial disability. There is an obvious huge impact on the family as well. More effective therapies will directly benefit these families, lead to increased productivity. Further, a California based company will have developed a key therapy for an otherwise lethal genetic disorder further demonstrating California’s leadership in medical science, and generating novel business opportunities within the Biotechnology industry in California.
Progress Report: 
  • Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy and one of the most common fatal genetic disorders. Approximately one in every 3,500 boys worldwide is affected with DMD. Extrapolating from population based studies, there are over 15,000 people currently living with DMD in the US. DMD is a devastating and incurable muscle-wasting disease caused by genetic mutations in the gene that codes for dystrophin, a protein that plays a key role in muscle cell health. Children are typically weaker than typical by age three, and progressive muscle weakness of the legs, pelvis, arms, neck and other areas result in most patients requiring full-time use of a wheelchair by age 11. Eventually, the disease progresses to complete paralysis and increasing difficulty in breathing due to respiratory muscle dysfunction and heart failure. The condition is terminal, and death usually occurs before the age of 25. While corticosteroids can slow disease progression and supportive care can extend lifespan and improve quality of life, no therapies exist that address the primary defect or dramatically alter the debilitating disease course.
  • The Planning Award funded the organization of the scientific and clinical team to apply for a well-defined CIRM Disease Team Therapy Development Award. The PI and project manager travelled to San Francisco to a CIRM sponsored workshop to gain training and insight in developing a Target Product Profile, to meet with other CIRM disease team grantees and aid in developing the most effective grant application. During that trip, we also met with investigators at the California based CRO, SRI, toured the SRI facilities, and planned for the assembly of the team that would us develop appropriate toxicology package for the proposed drug development. Several face-to-face meetings and conference calls with leaders in Duchenne advocacy, DMD clinic directors and industry partners were orchestrated to assess enthusiasm for the proposed project, which was across the board very high. A day long planning grant meeting was held on Dec 9th, 2011 in Santa Monica with participation of over 30 national and local academic, CRO, and industry experts in exon skipping, Duchenne Muscular Dystrophy, clinical trials planning, FDA regulatory practices and drug development. We identified and secured a leading industry partner necessary for streamlining the proposed work. Following the meeting, a series of weekly/daily conference calls between team members from UCLA, SRI and the industry partner enabled us to develop details of the proposal. During the planning grant period we were awarded a provisional patent for the combination therapeutic that is being moved forward in this proposal. Through discussions with leaders in the clinical care of Duchenne Muscular Dystrophy, leaders in antisense mediated exon skipping, and leaders in pre-IND drug development, we built a strong team to propose all IND enabling work to bring a proposed combination therapy for exon skipping as a novel Duchenne muscular dystrophy therapy.
  • Exon-skipping is a promising therapy that aims to repair the expression of the dystrophin protein by altering the RNA, but it is unclear whether it will be effective enough to lead to clinical improvements. We have identified a combination therapy that improves the effectiveness of exon-skipping therapy in mouse muscle and in human DMD patient stem cell derived muscle cells in culture. Because the genetic defect is being directly repaired inside of each muscle cell, this therapy is predicted to lessen the disease severity. The early research and further development of the proposed combination therapy require screening for drug efficacy and toxicity using human DMD patient stem cells including: reprogrammed patient fibroblasts converted into muscle-like cells in culture or when transplanted in mice. These cells are necessary because each patient’s mutation in the dystrophin gene is different. In order to know who will or will not benefit from the exon-skipping therapy, individualized cell culture and mouse transplant models from a number of DMD patients must be created to effectively characterize the combination therapy. The proposed research program will complete necessary efficacy and toxicity studies to allow submission of appropriate material to the FDA to allow testing of this novel combined therapeutic in children with DMD. It will also involve a team of clinical trialists who will incorporate findings in planning optimal trial design and ensure clinical trial readiness by the grants end. Since exon-skipping therapy relies on knowing individual patients exact DNA mutation, this is a form of personalized genetic medicine. While the specific combination therapy being developed here will treat up to 13% of DMD patients, the strategy is likely to be generalized to be able to treat up to 70% of DMD patients.

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