MODELING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY USING PRIMARY AND PATIENT-DERIVED INDUCED PLURIPOTENT STEM CELLS

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MODELING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY USING PRIMARY AND PATIENT-DERIVED INDUCED PLURIPOTENT STEM CELLS

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2021-01

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Abstract

Facioscapulohumeral Muscular Dystrophy (FSHD) is an autosomal dominant degenerative muscle disease with no cure or treatment. The genetic cause of FSHD is the reduction of the copy number of subtelomeric D4Z4 repeats at 4q35 encoding Double Homeobox 4 (DUX4) protein, which is a potent transcription factor that is toxic to the cell. Contractions lead to loss of repeat-induced silencing, allowing for transcription of DUX4. A common diagnostic method for FSHD shows demethylation of D4Z4 using gDNA of blood cells, and Kyba lab has shown that D4Z4 is demethylated in FSHD iPS cells, meaning that any cell type in an FSHD patient could be subject to deregulation by DUX4. DUX4 recruits p300 to induce global changes in histone acetylation, massively disrupting gene expression, thus pathology is not obviously exclusive to myogenic cells. Why the disease is muscle-specific and which cells in muscle may be deregulated by DUX4 are unknowns. Here are examined non-myogenic, muscle-associated cells derived from primary and induced pluripotent stem cells (iPSC) sources for evidence of DUX4 and target gene activation. To better visualize DUX4 expressing cells in vitro and in situ, these studies outline a visualization tool which uses signal amplification combined with a newly developed commercial antibody in both mouse and human tissue. Using this method, DUX4 positive cells have been identified in the human biopsy for the first time, providing a critical and novel insight into the cell types involved in FSHD pathogenesis. In a further attempt to model rare, muscle-associated populations in vitro, a novel fibroadipogenic progenitor (FAP) cell isolation method was developed, which is then used to obtain FAPs from patient biopsies. Using this new isolation method, FAPs were isolated from FSHD patients, and analysis of this rare cell population revealed that FSHD FAPs did not express DUX4 and its target genes, and that patient FAPs remained functionally normal if derived from intact muscle. In addition to developing new methods for detecting rare cell populations in patient muscle, these studies use iPSC modeling to examine DUX4 in other rare cell types which are not accessible in standard patient biopsies. Using this in vitro modeling system this study replicated the myogenic and cardiac phenotypes seen in the patient and examined other rare and difficult-to-access cell types like mesenchymal stem/stromal cells (MSCs) and motor neurons to analyze DUX4 involvement in these cell types. Both MSCs and motor neurons showed evidence of DUX4 target gene expression at the terminal cell type. However, unexpectedly, robust DUX4 expression was seen during early neurogenesis with long-term, significant target gene response persisting through the motor neuron progenitor stage before it tapered off in the mature motor neuron. Analysis of early MSC differentiation showed a similar pattern of target gene expression, though the stage of DUX4 expression was not identified. In aggregate, these data demonstrate that transient activation of DUX4 during differentiation is enough to induce a long-lasting effect that lasts into some terminally differentiated cells.

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University of Minnesota Ph.D. dissertation.January 2021. Major: Molecular, Cellular, Developmental Biology and Genetics. Advisor: Michael Kyba. 1 computer file (PDF); x, 86 pages.

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Goloviznina, Natalya. (2021). MODELING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY USING PRIMARY AND PATIENT-DERIVED INDUCED PLURIPOTENT STEM CELLS. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/253720.

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