Muscular Dystrophy

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

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.

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