Development of a Pompe disease modeling system based on human skeletal muscle organoid
- 주제(키워드) Pompe disease , disease modeling , Pluripotent stem cell , skeletal muscle , organoid
- 주제(DDC) 570
- 발행기관 아주대학교 일반대학원
- 지도교수 Junghyun Jo
- 발행년도 2026
- 학위수여년월 2026. 8
- 학위명 석사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000036701
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Pompe disease （glycogen storage disease type II） is an inherited lysosomal storage disorder caused by deficiency of acid α-glucosidase （GAA）, leading to progressive glycogen accumulation and dysfunction of skeletal muscle, although enzyme replacement therapy has improved clinical outcomes, muscle pathology often persists and the mechanisms that shape disease progression and therapeutic response remain incompletely understood. Human, muscle-relevant experimental platforms that enable longitudinal and quantitative phenotyping are therefore needed. In this study, a human Pompe disease modeling system was established using isogenic wild-type （WT） and CRISPR-engineered GAA knockout （KO） human pluripotent stem cells （hPSCs）. Establishment of human skeletal muscle organoid （hSkMO） through stepwise myogenic induction and 3D culture. Organoids were characterized across developmental stages by morphology and myogenic marker expression, including early lineage specification markers and myogenic progenitor/differentiation markers. Disease- relevant endpoints were assessed through GAA enzymatic activity, glycogen quantification and lysosomal/autophagy-associated readouts, using immunostaining-based analyses of lysosomal markers and acidified compartments, and autophagy markers, alongside myofiber markers for tissue-context interpretation. GAA KO hSkMO reproducibly exhibited Pompe- associated biochemical and cellular phenotypes, including reduced GAA activity, increased glycogen burden, and altered lysosomal/autophagy-related signals compared with WT controls. Together, these data support that hPSC-derived skeletal muscle organoids provide a practical human 3D platform that captures key aspects of Pompe disease pathology while enabling controlled isogenic comparisons. This work establishes a scalable experimental framework for mechanistic interrogation of lysosome–autophagy dysfunction in Pompe disease and provides a foundation for future studies evaluating therapeutic interventions in a human skeletal muscle context. Keywords: Pompe disease, disease modeling, Pluripotent stem cell, skeletal muscle, organoid
more목차
I. Introduction １
II. Material and Methods ７
1. Culture of human pluripotent stem cells (hPSCs) ７
2. Generation of hSkMOs ７
3. Generation of GAA Knock out hPSC lines by CRISPR/Cas9 genome editing ８
4. RNA extraction and Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR) ８
5. GAA activity assay ９
6. Glycogen content assay １０
7. Immunohistochemistry １１
8. Western blotting １２
9. Substrate reduction therapy using MZ-101 １３
III. Results １４
1. Establishment and validation of an isogenic Pompe disease hPSC model １５
1.1 Overall experimental design for Pompe disease modeling using WT and GAA Knockout hPSC derived hSkMO １５
1.2 Generation of the GAA KO hPSC line using CRISPR/Cas9 system １７
1.3 Validation of the GAA knockout hPSC line １９
2. Differentiation & early myogenesis ２１
2.1 Stepwise 2D-to-3D differentiation protocol for generating hSkMO from hPSCs ２１
2.2 Morphological progression and size changes of WT and GAA KO hSkMO over time ２３
2.3 WT and GAA KO hSkMO at day 20 assessment of proliferating PAX7- positive populations ２５
2.4 Early lineage specification is induced during hSkMO differentiation in both WT and GAA KO lines ２７
2.5 Early myogenic progression in WT and GAA KO hSkMO at day 30 ２９
2.6 Progressive myogenic maturation at day 60 ３２
3. Structural maturation and long-term maintenance of hSkMO ３５
3.1 Structural and biochemical characterization ３５
3.2 Long-term culture and myofiber maturation ３８
4. Disease-specific phenotypes in GAA KO hSkMO ４０
4.1 Impaired GAA activity and glycogen accumulation ４０
4.2 Increased autophagy-associated protein expression in GAA KO hSkMO ４２
4.3 Lysosomal enlargement and dysfunction ４４
5. Therapeutic response evaluation in hSkMO ４６
5.1 MZ-101 treatment attenuates structural disruption in GAA KO hSkMO ４６
IV Discussion ５１
V. Reference ５６
