Muscular Dystrophy

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

MicroRNAs in Human Embryonic Stem Cell Self-Renewal and Differentiation

Funding Type: 
SEED Grant
Grant Number: 
RS1-00455
ICOC Funds Committed: 
$0
Disease Focus: 
Genetic Disorder
Muscular Dystrophy
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
Embryonic Stem Cell
Public Abstract: 
Drs. Andrew Fire and Craig Mello won this year’s Nobel Prize in Physiology or Medicine for their discovery of RNA interference. Their discoveries revealed fundamental new paradigm in gene regulation and demonstrated that animal genomes consist of not only the transcriptional programs controlled by transcription factors but also the post-transcriptional programs controlled by RNAs. MiRNAs are ~22-nt small regulatory RNAs that are thought to control gene expression at the post-transcriptional level by targeting cognate target mRNAs for either degradation or translational repression. MiRNAs are individually encoded by their own set of genes and represent an integral component of animal genetic programs. MiRNA genes constitute about 1-5% of the predicted genes in worms, mice, and humans, and many miRNAs are conserved from worm to human. Moreover, each miRNA has the potential to regulate as many as 200 target genes, which implies that miRNA-mediated gene regulation may have a broad impact on gene expression and likely represents a fundamental layer of the genetic programs at the post-transcriptional level. Not surprisingly, miRNAs have been shown to play important roles in regulating various cellular, developmental, and disease processes in worm, fly, mouse, and human and several lines of evidence have implicated miRNAs in the developmental regulatory decisions of stem-cell maintenance and differentiation in flies and mice. However, the roles of miRNAs in human embryonic stem cell maintenance and differentiation are still unknown. The proposed research plan will address the fundamental questions regarding the roles of miRNAs and miRNA-mediated posttranscriptional genetic programs in human embryonic stem cells.
Statement of Benefit to California: 
To realize the clinical potential human embryonic stem cells, one has to understand the fundamental molecular mechanisms that govern stem cells self-renewal and differentiation. Human embryonic stem cells have a number of important properties: they can propagate under the right culture conditions and they can differentiate into cell types of all human tissues if properly guided. However, until today the molecular processes that regulate these properties are still quite elusive. The proposed research plan will address the fundamental molecular mechanisms that govern stem cells self-renewal and differentiation from a new angel –– the recently discovered post-transcriptional genetic programs controlled by small regulatory RNAs. Thus, these studies are likely to reveal important missing puzzle pieces that may be required for solving the ultimate question and yield clues to harness the power of human embryonic stem cells.
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.

Role of PK2 on the differentiation of human neural stem cells and ischemia-induced neurogenesis

Funding Type: 
SEED Grant
Grant Number: 
RS1-00455
ICOC Funds Committed: 
$0
Disease Focus: 
Genetic Disorder
Muscular Dystrophy
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
Embryonic Stem Cell
Public Abstract: 
Neurodegenerative disorders, including Alzheimer's Disease and Parkinson's Disease, have become an increasingly important concern due to the expanding elderly people. The cost to society is immense, and successful new drug or cell therapies would result in an enormous savings to society, in addition to alleviating patient suffering. Neural damage as a result of stroke or trauma to the brain or spinal cord is also a leading cause of death and disability, and neuronal death is an important cause of some of the most intractable forms of epilepsy. Few therapies are available for these neurological disorders. Providing a supply of new, functional neural cells at the site of the lesion, including new neurons, astrocytes and glial cells, have been proposed as means to ameliorate neural degeneration or damage. One proposed strategy for achieving this goal is to transplant neural stem and progenitor cells into the brain or spinal cord, such that they develop into specialized neural cells. An alternative strategy is to boost or initiate endogenous neurogenesis by delivering biologically active molecules to the brain or spinal cord, so as to stimulate the proliferation, differentiation and migration of endogenous neural stem or progenitor cells. Ideally, with either approach, the new cells will correctly reconstruct neuronal circuits, produce neurochemically active substances, and integrate into existing functional neural circuity. However, the development of effective therapies for treating neural repair is currently limited by the lack of understanding of the mechanisms that control neurogenesis. To develop improved therapies will thus require the identification of signal transduction pathways involved in neurogenesis and the regulators of these pathways. By identifying such pathways and regulators, new drugs can be developed that modulate neurogenesis in either ex vivo or in vivo applications. Thus, there exists a need to identify signal transduction pathways for modulating neurogenesis. Identification of extrinsic factors coordinating the proliferation and maturation of neural stem cells and progenitors in normal brain as well as after brain injury may lead to novel drug candidates and/or therapeutic targets for neural repair.
Statement of Benefit to California: 
Neurodegenerative disorders, including Alzheimer's Disease, Parkinson's Disease, have become an increasingly health concern due to the expanding elderly population. Neural damage as a result of stroke or trauma to the brain or spinal cord is also a leading cause of death and disability. No adequate therapy is available for these delabiliting neurological disorders. California, with one of highest ratio of retired population, has increasing number of people suffer from these debilitating neurological disorders, sometimes for many years. The cost is immense, and progress at prevention and new drug or cell treatment would result in an enormous savings to our state, in addition to alleviating patient suffering. Here we propose to study the effect of a newly discovered molecule on the differentiation of cultured human neural stem cell. We will also study whether the delivery of this molecule into brain can ameliorate the neural damage caused by stroke. The successful completion of these aims may lead to validation of a potential novel drug therapy or new drug target for treating stroke and other neurological disorders. This grant application will also benefit the State of California and its citizens by creating intellectual properties, and help the training of next generation scientists of neural repair and neural regeneration.
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.

Role of RXR in Nurr1-induced Differentiation of Human ES Cells and in Parkinson's Disease

Funding Type: 
SEED Grant
Grant Number: 
RS1-00455
ICOC Funds Committed: 
$0
Disease Focus: 
Genetic Disorder
Muscular Dystrophy
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
Embryonic Stem Cell
Public Abstract: 
Background. Human embryonic stem (hES) cells hold promise for replacement therapies. One of the promising targets for such therapy is Parkinson’s disease (PD). However, successful application of hES cell-based therapy depends on our ability to produce a large number of dopaminergic (DA) neurons and to control their survival once these neurons are transplanted into patients. This largely depends on our understanding of cellular components and pathways governing the proliferation, differentiation and survival of hES cells. Recent studies show that a protein called Nurr1 and its closely related protein Nur77 play a critical role in mouse ES cell differentiation, maturation, and survival. Introduction of Nurr1 into mouse ES cells induces their differentiation. However, the efficiency of generating DA neurons by Nurr1 is low and Nurr1-induced DA neurons are functionally immature, suggesting the necessity of additional factor(s). Nurr1 and Nur77 bind to RXR, a receptor protein for vitamin A-derived compounds and certain fatty acids. Interestingly, docosahexaenoic acid (DHA), which is enriched in brain and promotes the survival of newborn DA neurons, acts as a RXR ligand. Furthermore, synthetic RXR ligands enhance the differentiation of Nurr1-expressing cells and increase the survival of DA neuron. Recently, we discovered a novel Nur77-mediated survival/death pathway, in which Nur77 acts in the nucleus to confer cell survival, whereas its migration to mitochondria results in cell death. In addition, we showed that the survival and death activities of Nur77 are controlled by RXR and its ligands (such as DHA). Hypothesis: We hypothesize that RXR and its ligands are required for efficient hES cell differentiation, maturation and survival through their interaction with Nurr1 and Nur77 proteins. Objective: The objectives of this proposal are to establish an effective and reliable protocol for generating DA neurons from hES cells and to identify novel agents for the prevention and treatment of PD. We propose three specific aims: 1). To analyze the role of RXR and ligands in Nurr1-induced hES cell differentiation. Nurr1 and RXR proteins will be introduced into hES cells, which will be differentiated in the presence or absence of RXR ligands. 2). We will employ computer-based approaches to design and identify DHA analogs, which will be evaluated for their ability to modulate RXR activities, to induce hES cell differentiation, and to promote the survival of hES-derived neurons. 3). To evaluate hES cells in animals. The hES-derived DA neurons will be evaluated in a mouse PD model by transplantation. Two most effective DHA analogs will be also evaluated for their PD preventive effects. Results from these studies will not only enhance our understanding of the molecular pathways governing the differentiation of hES cells but may also lead to the identification of effective RXR-based agents for prevention and treatment of PD.
Statement of Benefit to California: 
Parkinson’s disease (PD) is a progressive disorder of central nervous system, which occurs when a group of brain cells called dopamine (DA) neurons begin to malfunction and die. According to the National Institutes of Health, PD affects at least 500,000 people in the United States and some 50,000 new cases are diagnosed each year, a number that is expected to rise as the population ages. Parkinson's disease affects both men and women almost equally. People of every race, economic class, and ethnicity can get PD. Although age is a clear risk factor, the commonly used herbicide, paraquat, which is structurally similar to the neurotoxic chemical MPTP, is known to be able to induce parkinsonism. Thus, there is an increased PD mortality in California, as California uses approximately a quarter of all pesticides in the US. According to the National Parkinson Foundation, PD patients spend an average of $2,500 a year for medications. Thus, there is of great interest to Californian residents for the development of new agents for the prevention and treatment of human PD. Current therapies, including the administration of L-dopa and dopamine receptor agonists and on deep-brain stimulation in the subthalamic nucleus, are effective only for some symptoms. They are associated with side effects and do not stop the progression of the disease. Recent progress shows that PD is a prime candidate for treatment by stem cell transplantation. If cells with properties of DA neurons can be generated in vitro from human embryonic stem (hES) cells, they can be used to replace damaged brain cells in PD patients. Thus, hES cells hold promise for generating an unlimited supply of brain cells for PD therapy. In fact, the potential to use ES cells to replace damaged cells has already been demonstrated in animal. However, little is known whether human ES cells can be successfully instructed to brain cells with therapeutic value. Our proposed studies aim at the development of new approach for facilitating the generation of DA neurons from hES cells by genetic manipulation consisting of the specific activation of Nurr1, Nur77, and RXR proteins, which are known to regulate the development, maturation and survival of DA neurons. Results from these studies may lead to improved generation of DA neurons for PD therapy. Our proposed studies of identifying RXR-binding DHA analogs may also lead to the identification of new DHA-based agents for the prevention and treatment of PD. Thus, California residents who suffering PD will benefit from this research. Development of novel preventive and therapeutic strategies and agents will certainly stimulate economic growth in California.
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.

Stem Cell Therapy for Duchenne Muscular Dystrophy

Funding Type: 
Early Translational II
Grant Number: 
TR2-01756
ICOC Funds Committed: 
$2 325 933
Disease Focus: 
Muscular Dystrophy
Pediatrics
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
Duchenne muscular dystrophy (DMD) is the most common and serious form of muscular dystrophy. One out of every 3500 boys is born with the disorder, and it is invariably fatal. Until recently, there was little hope that the widespread muscle degeneration that accompanies this disease could be combated. However, stem cell therapy now offers that hope. Like other degenerative disorders, DMD is the result of loss of cells that are needed for correct functioning of the body. In the case of DMD, a vital muscle protein is mutated, and its absence leads to progressive degeneration of essentially all the muscles in the body. To begin to approach a therapy for this condition, we must provide a new supply of stem cells that carry the missing protein that is lacking in DMD. These cells must be delivered to the body in such a way that they will engraft in the muscles and produce new, healthy muscle tissue on an ongoing basis. We now possess methods whereby we can generate stem cells that can become muscle cells out of adult cells from skin or fat by a process known as “reprogramming”. Reprogramming is the addition of genes to a cell that can dial the cell back to becoming a stem cell. By reprogramming adult cells, together with addition to them of a correct copy of the gene that is missing in DMD, we can potentially create stem cells that have the ability to create new, healthy muscle cells in the body of a DMD patient. This is essentially the strategy that we are developing in this proposal. We start with mice that have a mutation in the same gene that is affected in DMD, so they have a disease similar to DMD. We reprogram some of their adult cells, add the correct gene, and grow the cells in incubators in a manner that will produce muscle stem cells. The muscle stem cells can be identified and purified by using an instrument that detects characteristic proteins that muscles make. The corrected muscle stem cells are transplanted into mice with DMD, and the ability of the cells to generate healthy new muscle tissue is evaluated. Using the mouse results as a guide, a similar strategy will then be pursued with human cells, utilizing cells from patients with DMD. The cells will be reprogrammed, the correct gene added, and the cells grown into muscle stem cells. The ability of these cells to make healthy muscle will be tested by injection into mice with DMD that are immune-deficient, so they will accept a graft of human cells. In order to make this process into something that could be used in the clinic, we will develop standard procedures for making and testing the cells, to ensure that they are effective and safe. In this way, this project could lead to a new stem cell therapy that could improve the clinical condition of DMD patients. If we have success with DMD, similar methods could be used to treat other degenerative disorders, and perhaps even some of the degeneration that occurs during normal aging
Statement of Benefit to California: 
The proposed research could lead to a stem cell therapy for Duchenne muscular dystrophy (DMD). This outcome would deliver a variety of benefits to the state of California. First, there would be a profound personal impact on patients and their families if the current inevitable decline of DMD patients could be halted or reversed. This would bring great happiness and satisfaction to the thousands of Californians affected directly or indirectly by DMD. Progress toward a cure for DMD 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. However, the impact would likely also stimulate medical progress on a variety of conditions in which a stem cell therapy could be beneficial. These conditions may even extend to some of the normal processes of aging, which can be traced to depletion of stem cells. An effective stem cell therapy for DMD would also bring economic benefits to the state. Currently, there is a huge burden of costs associated with the care of patients with long-term degenerative disorders like DMD, which afflict thousands of patients statewide. If the clinical condition of these patients could be improved, the cost of maintenance would be reduced, saving billions in medical costs. 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.
Progress Report: 
  • The goal of this project is to develop a new approach for therapy of Duchenne muscular dystrophy (DMD). In our strategy, we use skin cells from patients as the starting material and convert these cells into stem cells by adding “reprogramming” genes. We then add the therapeutic dystrophin gene to the stem cells to correct the mutation that causes DMD. The corrected stem cells are grown in a manner so that they will become muscle precursor cells. This process is called differentiation. The differentiated muscle precursor cells are injected into diseased muscles to restore healthy muscle fibers. The overall goal of the project is to demonstrate the entire strategy in a mouse model of DMD, using both mouse and human cells.
  • The milestones for the first year of the project were 1A) to demonstrate the complete strategy for making stem cells from the mouse model and adding a correct dystrophin gene at a precise location in the chromosomes, and 1B) to demonstrate differentiation of mouse and human stem cells into muscle precursor cells that will later be used for engraftment. We achieved both of these milestones.
  • Milestone 1A. Our project takes advantage of the recent discovery that ordinary skin cells can be “reprogrammed” into stem cells that are similar in their properties to embryonic cells. The reprogramming process is carried out by introducing four genes into skin cells that can change the pattern of expression of genes in the cells to that of embryonic cells. The reprogramming genes are usually introduced into cells by putting them into viruses that can incorporate, or integrate, themselves into the chromosomes. This process is effective, but leaves behind viruses embedded in the chromosomes, which can activate genes that cause cancer. My laboratory has developed a safer method for reprogramming, in which no viruses are used. Instead, we utilize an enzyme that can place a single copy of the reprogramming genes into a safe place in the chromosomes.
  • In our method, the reprogramming genes are present on small circles of DNA that are easily made from bacteria grown in the laboratory. The circles of DNA, along with DNA that codes for the integration enzyme, are introduced into patient skin cells. The enzyme causes the reprogramming genes to incorporate into a chromosome at a single, safe location. After the cells are reprogrammed, the reprogramming genes, which are no longer needed, are precisely removed from the chromosomes by using another enzyme. These cells appear to be safe to use in the clinic.
  • In addition, we developed a method to add the therapeutic dystrophin gene to the reprogrammed cells at a precise location. By adding a correct copy of the dystrophin gene, the stem cells now have the potential to make healthy muscle. These corrected stem cells were used to create muscle precursor cells in Milestone 1B.
  • Milestone 1B. In these experiments, we demonstrated that cells reprogrammed and corrected by our methods can be grown in such a way that they differentiate into muscle precursor cells that have the capacity to become healthy muscle fibers. This process of differentiation takes place over a period of about two to three weeks, while the stem cells are grown in plastic dishes in an incubator. The cells are grown in culture medium that contains substances that allow the cells to differentiate from generalized stem cells into cells that are committed to produce muscle.
  • We followed two procedures published in the literature for differentiation of the cells. We analyzed the cells at different time points to see if they had the characteristics of muscle precursor cells. First, we observed the cells under the microscope and saw that they fused together into long fibers, which is characteristic of muscle cells. Moreover, the fibers began to contract and twitch, which is typical of muscle fibers.
  • To analyze the cells at the molecular level, we stained them with antibodies that recognize proteins that are made in muscle precursor cells. We were able to detect staining in some of the cells in the culture, indicating that they were becoming muscle precursor cells. This was demonstrated for both human and mouse stem cells.
  • To measure what fraction of the cells had become muscle precursors, we mixed the culture containing differentiated mouse stem cells with an antibody that binds to the surface of muscle precursor cells. The cells were analyzed with an instrument that can measure how many cells in the culture bind the antibody. We found that 5 – 10% of the cells stained with the antibody. This result indicated that a significant fraction of the cells had become muscle precursors cells, with the potential to be engrafted.
  • In the coming year, these corrected and differentiated mouse stem cells will be introduced into DMD mice to repair muscle damage. We will also apply our reprogramming and correction methods to human cells from DMD patients.
  • The goal of this project is to develop a new approach for therapy of Duchenne muscular dystrophy (DMD). In our strategy, we use skin cells from patients as the starting material and convert these cells into stem cells by adding “reprogramming” genes. We then add the therapeutic dystrophin gene to the stem cells to correct the mutation that causes DMD. The corrected stem cells are grown in a manner so that they will develop into muscle precursor cells. This process is called differentiation. The differentiated muscle precursor cells are injected into diseased muscles to restore healthy muscle fibers. The overall goal of the project is to demonstrate the entire strategy in a mouse model of DMD, using both mouse and human cells as starting material.
  • The first milestones for the project were 1A) to demonstrate the complete strategy for making stem cells from the mouse model and adding a correct dystrophin gene at a precise location in the chromosomes, and to do the same for human cells (2B).
  • Our project took advantage of the recent discovery that ordinary skin cells can be “reprogrammed” into stem cells that are similar in their properties to embryonic cells. The reprogramming process is carried out by introducing four genes into skin cells that can change the pattern of expression of genes in the cells to that of embryonic cells. The reprogramming genes are usually introduced into cells by putting them into viruses that can incorporate, or integrate, themselves into the chromosomes. This process is effective, but leaves behind viruses embedded in the chromosomes, which can activate genes that cause cancer. Our laboratory developed a safer method for reprogramming, in which no viruses are used. Instead, we utilize an enzyme that can place a single copy of the reprogramming genes into a safe place in the chromosomes.
  • In our method, the reprogramming genes are present on small circles of DNA that are easily made from bacteria grown in the laboratory. The circles of DNA, along with DNA that codes for the integration enzyme, are introduced into skin cells. The enzyme causes the reprogramming genes to incorporate into a chromosome at a single, safe location. After the cells are reprogrammed, the reprogramming genes, which are no longer needed, are precisely removed from the chromosomes by using another enzyme. In addition, we developed a method to add the therapeutic dystrophin gene to the reprogrammed cells at a precise location. By adding a correct copy of the dystrophin gene, the stem cells now have the potential to make healthy muscle. These corrected stem cells were used to create muscle precursor cells in Milestone 1B.
  • In the Milestone 1B experiments, we demonstrated that cells reprogrammed and corrected by our methods can be grown in such a way that they differentiate into muscle precursor cells that have the capacity to become healthy muscle fibers. This process of differentiation takes place over a period of several weeks, while the stem cells are grown in plastic dishes in an incubator. The cells are grown in culture fluids that contain substances that allow the cells to differentiate from generalized stem cells into cells that are committed to produce muscle. We analyzed the cells at different time points to see if they had the characteristics of muscle precursor cells. We observed the cells under the microscope and saw that they fused together into long fibers, which is characteristic of muscle cells. Moreover, the fibers began to contract and twitch, which is typical of muscle fibers.
  • To analyze the cells at the molecular level, we stained them with antibodies that recognize proteins that are made in muscle precursor cells and also demonstrated that they contained messenger RNA that encoded muscle proteins. We also verified that the cells expressed the dystrophin gene that we inserted into them and produced normal dystrophin protein. To measure what fraction of the cells had become muscle precursors, we mixed the culture containing differentiated mouse stem cells with an antibody that binds to the surface of muscle precursor cells. The cells were analyzed with an instrument that can measure how many cells in the culture bind the antibody. We found that 20 - 50% of the cells stained with the antibody. This result indicated that a significant fraction of the cells had become muscle precursors cells, with the potential to be engrafted.
  • In Milestone 2A, we introduced these corrected and differentiated mouse stem cells into DMD-model mice to repair muscle damage. We injected the cells into a leg muscle, and three weeks later, we detected engrafted cells by staining for dystrophin. In the coming year, we will carry out the final experiments, Milestone 3, in which human cells that have been reprogrammed and corrected are engrafted into disease model mice.
  • This project has led to great progress in the development of a stem cell therapy for Duchenne muscular dystrophy. During the project period, we went from a conceptual strategy to making all parts of the strategy work, while at the same time discovering improvements in all aspects. The studies began with developing a new and potentially safer way to reprogram mouse cells. We started with skin cells from mdx disease model mice and introduced a plasmid, or circle of DNA, that encoded four genes that could reprogram the skin cells back into embryonic-like cells. We used an enzyme from bacteria called a “recombinase” to paste the reprogramming genes into a safe place in the mouse chromosomes. The next step was to use a second recombinase enzyme to place a correct copy of the dystrophin gene, the gene that is mutated in this form of muscular dystrophy, into a precise position next to the reprogramming genes. Once this was accomplished, we used a third recombinase to delete the portions of inserted DNA that were no longer needed, including the reprogramming genes. These steps left us with “induced pluripotent stem cells”, or iPSC, that were corrected for the disease-causing mutation. In the next step, we used methods to grow the iPSC that induced them to become muscle precursor cells. We measured these changes by monitoring several proteins that are typical of muscle cells. These muscle proteins began to appear in the iPSC as they were undergoing the differentiation process. Once the cells were differentiated, we injected them into the leg muscles of living mice that had muscular dystrophy. We showed that the cells we injected were able to engraft into the muscle, where they could repair and replace damaged muscle fibers. Having successfully carried out the complete stem cell strategy using mouse cells, we published our findings in a scientific journal and sought to develop a similar strategy using human cells. We found that the reprogramming strategy that we had used in mouse cells did not work well in human cells. Therefore, we turned to a reprogramming method that was recently reported by two labs, in which plasmids based on Epstein-Barr virus are used to carry the reprogramming genes into human cells. The long-lasting plasmids provided a sufficient dose of the reprogramming genes, such that the human cells became iPSC. In order to supply a correct copy of the mutated gene, we developed a new method of genome engineering called DICE, for dual integrase cassette exchange. In this method, a short DNA sequence called a “landing pad” was positioned in a special place in the chromosomes called H11. This location has features that make it favorable as a spot to place introduced genes. The landing pad contains recognition sequences for two different recombinase enzymes. When a piece of DNA carrying the genes we want to insert is flanked by recognition sequences for the two enzymes, the landing pad is replaced by the gene we would like to insert. By using this method, we generated iPSC that had a new gene inserted precisely at the H11 location. The next step is to differentiate the cells into muscle precursor cells. The procedure that had worked in mouse cells was not effective for the human cells. We tried two new methods, and both generated human muscle precursor cells at good efficiency. We transplanted the differentiated muscle precursor cells into leg muscles of immune-deficient mice. The mice needed to be immune-deficient in order to accept grafts of human cells without rejecting the cells. We obtained evidence that the human cells successfully engrafted into the muscle. Until now, we had been introducing the stem cells by injecting them directly into a muscle with a needle. This procedure works well in the small muscles of a mouse, but would not work well in the much larger muscles of a human. Therefore, we also began developing a new stem cell delivery method in which the stem cells are introduced into an artery, where they can access muscle tissue by passing through the blood vessel wall and into the muscle tissue. We generated preliminary results suggesting that this arterial delivery system might be a successful means to distribute healthy stem cells to diseased muscles throughout the body. We intend to continue developing this stem cell strategy so that it can be used to help repair the muscles in patients with muscular dystrophy.

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.

Purified allogeneic hematopoietic stem cells as a platform for tolerance induction

Funding Type: 
Transplantation Immunology
Grant Number: 
RM1-01733
ICOC Funds Committed: 
$1 403 557
Disease Focus: 
Blood Disorders
Immune Disease
Muscular Dystrophy
Stem Cell Use: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
Blood and immune cells originate and mature in the bone marrow. Bone marrow cells are mixtures of blood cells at different stages of development, and include rare populations of blood-forming stem cells. These stem cells are the only cells capable of generating the blood system for the life of an individual. Bone marrow transplants (BMT) have been performed > 50 years, to replace a diseased patient’s blood system with that of a donor. Unfortunately, BMT have associated dangers which make the procedure high risk. Major risks include a syndrome called graft-versus-host disease (GvHD) which results when the donor’s mature blood cells attack the organs of the host, and toxicity from the treatments (radiation and chemotherapy) required to permit the donor cells to take in the recipient. These risk factor limit the use of BMT to only immediate life-threatening diseases. If made safer, BMT could cure many other debilitating diseases. In addition to being curative of blood cancers and non-malignant blood diseases (such as sickle-cell anemia), these transplants can cure autoimmune diseases, such as juvenile (type I) diabetes and multiple sclerosis. In addition, simultaneous BMT with organ transplants induces “tolerance” to the new organ, meaning the recipient will not reject the graft because the new blood system provides continuous proteins to re-train the recipient immune system not to attack it. This establishment of tolerance eliminates the need for drugs that suppress the immune system. In efforts to make BMT safer, our research has focused on isolating the blood stem cells away from the other bone marrow cells because transplants of pure stem cells do not cause GvHD. We developed the methods to purify the blood stem cells from mouse and human blood forming sources and showed in mice that transplants of blood stem cells can cure autoimmune disease and induce tolerance to solid organ transplants. However, this technology has not been tested in human clinical trials because safer methods must be developed that permit the stem cells to engraft in recipients. Our studies in mice show that we can replace the toxic drugs and radiation used to prepare recipients for BMT with non-toxic proteins that target the cells responsible for rejection of blood stem cells. The goal of this study is to translate this technology from mice to patient clinical trials. If successful, the studies will open the door to the use of blood stem cell transplants to the many thousands of patients who could benefit from this approach. The science behind achieving blood stem cell engraftment by the methods we propose look toward the future when blood stem cells and other tissues will be developed from pluripotent stem cells (ES, NT and iPS). We envision that the blood stem cells will induce tolerance to tissues derived from the same pluripotent stem cell line, in the same way that adult blood stem cells induce tolerance to organs from the same living donor.
Statement of Benefit to California: 
The science and the preclinical pathway to induce human immune tolerance in patients with degenerative diseases so that new blood and tissue stem cells can regenerate their lost tissues: For stem cell biology to launch the era of regenerative medicine, stem cells capable of robust and specific regeneration upon transplantation must be found, and methods for safe patient administration must be developed. In the cases where cell donation cannot come from the host, immune responses will reject the donor stem cells. Successful transplants of blood-forming stem cells (HSC) leads to elimination immune cells that reject organ grafts from donors. While bone marrow or cord blood transplants contain immune cells called T cells that will attack the host in a potentially lethal graft against host immune reaction, purified HSC do not do this. Pluripotent stem cells (ES, NT, iPS) can make all cell types in the body and provide a shortcut to find tissue and organ stem cells. Just as co-transplants of adult HSC prevent rejection of organs from the same donor, co-transplants of HSC derived from pluripotent cells should protect tissues derived from the same pluripotent line. Attack by a patient's blood system against one’s own organs cause the syndromes of autoimmune disease including juvenile diabetes, multiple sclerosis, and lupus. Transplanted HSC from donor mice genetically resistant to these diseases end the autoimmune attack permanently. We have in mice, substituted minimally toxic antibodies for toxic chemoradiotherapy to prepare the host for HSC transplants. Now it is time to take these advances to humans, with human immune cell and HSC-targeting antibodies. Long-term potential benefits to the state of California and its residents: The justification for Proposition 71 was to establish in California centers of research not funded adequately in the areas of stem cell biology and regenerative medicine. This research, if successful, is the platform for the application of stem cell biology to regenerative medicine. The costs for long-term immune suppression to patients who receive organ transplants are enormous, both in terms of quality of life, even survival, and healthcare resources. Add to that the lifetime costs of insulin to treat juvenile diabetes, with the inevitable premature diseases of compromised blood vessels and organs, and the shortened lifespan of patients. Add to that the costs to lives and the healthcare system of lupus, of multiple sclerosis, of other autoimmune diseases like juvenile and adult rheumatoid arthritis and scleroderma, and of muscular dystrophy, to mention a few, and the value to Californians and people everywhere is obvious. If our studies are successful, and if the clinical trials were first done in California, our citizens will have the first chance at successful treatment. Further, if these studies are successful - new antibodies, if produced by CIRM funds, will generate royalties which eventually will return to the state.
Progress Report: 
  • The successful transplantation of blood forming stem cells from one person to another can alter the recipient immune system in profound ways. The transplanted blood forming cells can condition the recipient to accept organs from the original stem cell donor without the need for drugs to suppress their immune system; and such transplantations can be curative of autoimmune diseases such as childhood diabetes and multiple sclerosis. Modification of the immune system in these ways is called immune tolerance induction.
  • Unfortunately, the current practice of blood stem cell transplantation is associated with serious risks, including risk of death in 10-20% of recipients. It has been a long-standing goal of investigators in this field to make transplantations safer so that patients that must undergo this procedure have better outcomes, and so that patients who need an organ graft or that suffer from an autoimmune disorder can be effectively treated by this powerful form of cellular therapy. The major objectives of our proposal are to achieve this goal by developing methods to prepare patients to accept blood forming stem cell grafts with reagents that specifically target cell populations in recipients that constitute the barriers to engraftment, and to transplant only purified blood forming stem cells thereby avoiding the potentially lethal complication call graft-vs-host disease.
  • The proposal has four Specific Aims. Aims 1 and 2 focus on development of biologic agents that specifically target recipient barrier cells. Aims 3 and 4 propose to test the reagents and approaches developed in the first two aims in mouse models to induce tolerance to co-transplanted tissues and to cure animals with Type 1 diabetes mellitus or multiple sclerosis. These aims have not changed in this reporting period.
  • One parameter of success in this project is the development of one or more biologic reagents that can replace toxic radiation and chemotherapy that can be used in human clinical trials by the end of the third year of funding (Aim 2). In this regard, significant progress has been made in the last year. A reagent critical to the success of donor blood forming stem cell engraftment is one that targets and eliminates the stem cells that already reside in the recipients. Recipient blood stem cells block the ability of donor stem cells to take. In our prior mouse studies we determined that a protein (antibody) that specifically targets a molecule on the surface of blood forming stem cells called CD117 is capable of eliminating recipient blood stem cells thus opening up special niches and allowing donor stem cells to engraft. This antibody was highly effective in permitting engraftment of purified donor blood stem cells in mice that lack a functional immune system. In this application we proposed to develop and test reagents that could target and eliminate human blood forming stem cells by targeting human CD117. This year we have identified and tested such an antibody which is manufactured by a third party. This anti-CD117 antibody has been evaluated in early clinical trials for an indication separate from our proposed use and appears to be non-toxic. In mice that we generated to house a human blood system, the antibody was capable eliminating the human blood forming stem cells. We plan to pursue the use of this reagent in a clinical trial as a non-toxic way to prepare children with a disease called severe combined immunodeficiency (SCID) for transplantation. Without a transplant children with SCID will die. The use of the anti-CD117 antibody and transplantation of purified blood forming stem cells has the potential to significantly reduce the complications of such transplants and improve the outcomes for these patients. The trial will be the first step to using this form of targeted therapy and serve as a pioneering study for all indications for which a blood forming stem cell transplant is needed, including the induction of immune tolerance.
  • The transplantation of blood forming stem cells from one individual to another can alter the recipient immune system in profound ways. Transplanted blood forming cells can condition the recipient to accept organs from the original stem cell donor without the need for drugs to suppress their immune system. Such transplantations can also be curative of autoimmune diseases such as childhood diabetes and multiple sclerosis. Modification of the immune system in these ways is called immune tolerance induction.
  • The major goal of this project is to enable the use of blood forming stem cell transplantation for the purpose of immune tolerance induction without unwanted side effects. The current practice of blood stem cell transplantation is associated with serious risks, including risk of death in 10-20% of recipients due to complications of transplant conditioning and graft-versus-host disease. We aim to abolish or reduce the risks of these transplantations so that this curative form of stem cell therapy can safely treat patients who need an organ graft or who suffer from an autoimmune disorder. To achieve our goals, we proposed the development of methods to prepare patients to accept blood forming stem cell grafts with reagents that specifically target recipient cell populations that constitute the barriers to engraftment, and to transplant only purified blood forming stem cells, thereby avoiding graft-versus-host disease.
  • The proposal has four Specific Aims. Aims 1 and 2 focus on development of biologic agents that specifically target recipient barrier cells. Aims 3 and 4 propose testing the reagents and approaches developed in the first two aims in mouse models to induce tolerance to co-transplanted tissues and to cure animals with muscular dystrophy, Type 1 diabetes mellitus and multiple sclerosis. These aims have not changed in this reporting period.
  • In this reporting period, significant progress has been made in the first three aims. In prior years we identified a biologic reagent that has the potential to replace toxic radiation and chemotherapy. Radiation and chemotherapy are used in transplantation to eliminate the blood forming stem cells of recipients because recipient stem cells block the ability of donor cells to take. The novel reagent we have studied is a protein, called a monoclonal antibody, which differs from radiation and chemotherapy because it specifically targets and eliminates recipient blood stem cells. This antibody reagent recognizes a molecule on the surface of blood stem cells called CD117. In years 1 and 2 we began testing of an anti-human CD117 (anti-hCD117) antibody in mice. Mice were engrafted with human blood cells and we showed that this antibody safely and specifically eliminated the human blood forming cells. These studies were proof-of-concept that the antibody is appropriate for use in human clinical trials.
  • This last year we were awarded a CIRM Disease Team grant to move the testing of this anti-hCD117 from the experimental phase in mice to a clinical trial for the treatment of children with a disease call severe combined immunodeficiency (SCID), also known as the “bubble boy” disease. Children with SCID are missing certain types of white blood cells (lymphocytes) so they cannot defend themselves from infections. Without a transplant, children with SCID will die. The use of the anti-CD117 antibody and transplantation of purified blood forming stem cells has the potential to significantly reduce the complications of such transplants and improve the outcomes for these patients. The use of the anti-CD117 antibody and transplantation of purified blood forming stem cells has the potential to significantly reduce the complications of such transplants and improve the outcomes for these patients. The trial will be the first step to using this form of targeted therapy and serve as a pioneering study for all indications for which a blood forming stem cell transplant is needed, including the induction of immune tolerance.
  • In the last year we have moved forward with the purification of skeletal muscle stem cells based upon labeling and sorting of primitive muscle cells that express an array of molecules on the cell surface. We have also transplanted a special strain of mice (mdx) that are a model for muscular dystrophy with blood forming stem cells from normal mouse donors. In the coming year we will perform simultaneous transplants of blood forming stem cells and skeletal muscle stem cells from normal donor mice into the mdx mice. We will determine if the blood stem cells permit the long-term survival of the muscle stem cells in recipients transplanted across histocompatibility barriers. Our ultimate goal is to achieve long-term recovery of muscle cell function in the recipients of these co-transplantations.
  • The transplantation of blood forming stem cells from one individual to another is widely used to treat patients with otherwise incurable cancers. Because such transplantations alter the recipient immune system in profound ways there are many other applications for this powerful form of therapy. The studies proposed in this grant focused on the use of blood stem cell transplantation for the purpose of immune tolerance induction. Tolerance induction in this setting means that transplantation of blood stem cells trains the body of a recipient to accept organs from same stem cell donor without the need for drugs to suppress their immune system. Blood stem transplantations can also reverse aberrant immune responses in individuals with autoimmune diseases such as childhood diabetes and multiple sclerosis.
  • In this project we sought to develop new ways to perform blood stem cell transplants to make the procedure safer and therefore more widely useable for a broad spectrum of patients. Transplants can be dangerous and sometimes fatal. Serious complications are caused by the toxic chemotherapy or radiation which are used to permit stem cells to engraft, and by a syndrome called graft-versus-host disease. Our research has aimed to replace the toxic treatments by testing novel reagents that more specifically target and eliminate the cells in recipients that constitute the barriers to stem cell engraftment. Furthermore, we perform transplantations of purified blood forming stem cells, and thus are able to avoid the problem of graft-versus-host disease which is caused by non-stem cell “passenger” immune cells in the donor grafts.
  • The proposal has four Specific Aims. Aims 1 and 2 focus on development of biologic agents that specifically target recipient barrier cells. Aims 3 and 4 propose testing the reagents and approaches developed in the first two aims in mouse models to induce tolerance to co-transplanted tissues and to cure animals with muscular dystrophy, Type 1 diabetes mellitus and multiple sclerosis. These aims have not changed in this reporting period.
  • Our prior reports highlighted our progress in Aim 2, which is now complete. Aim 2 focused on the identification and testing of an antibody directed against a molecule called CD117 present on surface of human blood stem cells. We demonstrated that this antibody can safely target and eliminate human blood stem cells in mice that had been previously engrafted with human cells. Based upon these studies we were awarded a CIRM Disease Team Grant, which will test this anti-human CD117 antibody in a clinical trial for the treatment of children with severe combined immune deficiency (SCID), also known as the “bubble boy” disease. Children with SCID are missing certain types of white blood cells (lymphocytes) so they cannot defend themselves from infections. Without a transplant, SCID patients usually die before the age of two. Our proposed clinical study has the potential to significantly improve the success of transplants for these patients. This clinical trial will be a first to test a reagent that specifically targets recipient stem cells to clear niche space and allow replacement therapy by healthy donor stem cells.
  • In the last year we have continued to make significant progress on Aims 1, 3 and 4. Aim 1 proposed to study how to improve blood stem cell engraftment using novel agents in mice that have intact immune systems. The anti-CD117 antibody discussed above works well in recipients that lack lymphocytes but not recipients with normal immune function. We have tested the anti-CD117 antibody in mice that lack more defined lymphocyte subsets to narrow down which lymphocyte type must be neutralized or eliminated. We have also tested novel reagents that inhibit the activity of specific immune cells and observed a stronger effect of the anti-CD117 antibody when co-administered with these reagents. For Aims 3 and 4, we have successfully achieved our goal of performing blood stem cell transplants that result in the stable mixing of blood cells between donor and recipients (called partial chimerism). For Aim 3, recipients are from a specialized mouse strain that models muscular dystrophy (MDX mice). We have transplanted purified skeletal muscle stem cells (SMSC) and observed engraftment of SMSC in MDX mice injected with genetically-matched SMSC. The next step is to test if co-transplants of blood stem cells plus SMSC from genetically mismatched donors will permanently engraft and expand in MDX recipients. For Aim 4, two mouse models are studied: (1) NOD mice which model childhood diabetes, and (2) mice that develop multiple sclerosis. We can successfully block the progression of disease in these animals with blood stem cell transplants. Our next steps are to apply the therapies developed in Aim 1 to these disease models. In the post-award period we will continue to carry out studies testing the novel approaches developed here in models of tolerance induction.

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.

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.

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.

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