(183k) Derivation of Skeletal Myoblasts from Human Hutchinson-Gilford Progeria Syndrome iPSCs As a Model of Muscle Aging

Sarcopenia is the loss of muscle mass and strength in elderly, which leads to frailty. It arises from impaired regeneration of muscle. Satellite cells within the niche of skeletal muscle are responsible for muscle regeneration which deteriorate with aging. Hutchinson-Gilford Progeria Syndrome (HGPS) occurs as a result of a mutation in the LMNA gene that codes for Lamin A/C protein. This mutation leads to the expression of a toxic protein known as Progerin, which is accountable for dysregulation in several biological processes such as chromatin remodeling, transcriptional control, DNA repair, and nuclear membranes associated factors, similar to those involved in physiological aging. The patients of HGPS usually develop growth retardation, muscle loss, and atrophy of the skeletal muscle. While defects in growth are evident in skeletal muscle in HGPS, an in vitro model that mimics HGPS skeletal muscle for aging studies remains unexplored. Induced Pluripotent Stem Cell (iPSC) technology allowed to differentiate several cell types including neural progenitors, endothelial cells, fibroblasts, vascular smooth muscle cells, and mesenchymal stem cells from HGPS patient iPSCs. Previous studies reported that HGPS iPSC-derived cells reproduce disease characteristics such as nuclear abnormalities and increased DNA damage. These findings prompted us to differentiate HGPS iPSCs into skeletal muscle and examine the development of aging hallmarks during differentiation. This model can be used to study muscle aging and serve as a screening platform for the identification of possible anti-aging therapies. In this study, we derived myogenic cells from HGPS patient iPSCs as a model of aging for skeletal muscle. We used a three-stage differentiation protocol with two HGPS and two normal control cell lines using a commercially available differentiation medium. Four cell lines including two HGPS and two normal controls were subjected to this differentiation protocol. In order to examine myogenic differentiation, we have used RT-PCR and immunostaining at the end of each differentiation stage to assess Somite and myogenic markers. Apart from muscle differentiation markers, we also quantified senescence markers including DNA damage by immunostaining, Senescence-associated beta-galactosidase (SA-β-gal) by fluorogenic assay and Reactive Oxygen Species (ROS) by DCFDA staining. Furthermore, metabolism markers including Mitochondrial membrane potential and respiration was examined by TMRM staining and seahorse Mitostress test. In addition, glycolytic capacity was measured by seahorse Glycostress test. Our results showed that there was no significant difference in pluripotency genes between HGPS and non-HGPS iPSCs, however, we observed accumulation of Progerin and DNA damage in HGPS derived cells even in early stages of differentiation. In agreement, western blots showed low levels of Lamin A/C protein detectable and no Prelamin A and Progerin expression in HGPS iPSCs but high expression in myoblasts derived from these cells. This was accompanied by abnormal nuclear structure and nuclear blebbing, and higher senescence-associated β-galactosidase (SA-βgal) in the HGPS-iPSC-derived myogenic cells compared to controls. Additionally, HGPS myoblasts showed elevated levels of reactive oxygen species (ROS) and reduced mitochondrial membrane potential. HGPS myoblasts also exhibited impaired mitochondrial respiration and lower glycolytic capacity. Finally, HGPS iPSCs differentiated less efficiently into myotubes as indicated by reduced cell fusion and impaired expression of myogenic regulatory factors. In conclusion, we observed the onset of aging hallmarks in HGPS-derived myogenic cells, even during early stages of differentiation. This led to decreased myogenic differentiation potential and impaired myotube formation. These results form the basis for future studies to probe into the mechanisms of aging and employ this system as a platform for drug screening to alleviate aging hallmarks, potentially reducing reliance on animal models and enabling personalized approaches in drug discovery utilizing patient-derived cells.