Statement of Benefit to California:
Year 1Facioscapulohumeral 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.