Author ORCID Identifier

0000-0001-5144-3635

Date of Graduation

8-2024

Document Type

Thesis (MS)

Program Affiliation

Biomedical Sciences

Degree Name

Masters of Science (MS)

Advisor/Committee Chair

Chunru Lin

Committee Member

Radbod Darabi

Committee Member

Hamed Jafar-Nejad

Committee Member

Xuelian Huang

Committee Member

Ali Azhdarinia

Abstract

Background: Muscular dystrophies are heterogeneous groups of inherited diseases leading to progressive muscular weakness and degeneration. In the case of Becker muscular dystrophy (BMD), non-disrupting mutations of the DMD gene reading frame is the causative defect. By the age of 30s, about 60 to 70% of BMD patients develop cardiomyopathy, which is often lethal. Unfortunately, there is currently no cure for BMD. Meanwhile, improvement of dystrophin stability by repressing polyubiquitination is a promising strategy. The application of human iPSCs in tissue regeneration also serves as a potential therapeutic strategy. This study aims to test the efficacy of selected drugs interfering with the dystrophin polyubiquitination using BMD iPSCs as the model system. The hypothesis is that myogenic differentiation of the BMD iPSC-derived myogenic progenitors and dystrophin expression and stability can be improved in vivo after the treatment with polyubiquitination inhibitors. Methods: The in vitro studies include evaluation of myogenic cell proliferation and differentiation using BMD and control iPSCs to identify affected phenotypes. These were done by measuring proliferation rate, myogenic marker quantification, apoptosis and senescence assay, gene expression profiling, and cell cycle analysis. The in vivo studies were done by transplanting BMD iPSC-derived myogenic progenitors into immunodeficient NSG mice with daily treatment of selected polyubiquitination inhibitors. iii Muscle cryosections were used for quantification of donor cell engraftment and dystrophin expression. Results & Progress: The in vitro data indicates reduced myogenic potential of BMD iPSCs, as well as defective cell cycle, proliferation, and differentiation, when compared to healthy iPSCs. Besides lower proliferation ability indicated by Ki67 staining and cell cycle FACS analysis, BMD iPSCs showed higher percentages of cells positive to apoptosis marker AnnexinV and senescence marker DPP4, as well as higher level of LDH releasing to the growth environment. In addition, muscle sections from the BMD-iPSC engrafted mice treated with the selected ubiquitination inhibitors demonstrated significantly higher percentage of human cell engraftment. The expression of human dystrophin in the acceptor mice muscles were also increased, which was measured by scoring expression patterns and fluorescence intensities, indicating the efficacy of the treatment. Conclusions: Inhibition of ubiquitination significantly improves the BMD iPSC-derived myogenic engraftment with improved dystrophin expression. The study provides a new therapeutic strategy that utilizes human iPSCs transplantation and molecular medicine.

Keywords

BMD, Dystrophin, iPSCs, Regenerative Medicine, Muscle Disease, Stem Cell

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