Current autologous cell therapies for skeletal muscular diseases are inefficient and costly. For patients with Volumetric Muscle Loss (VML), treatments are unsuitable given patients’ time-sensitive nature. Using universal human pluripotent stem cells (UhPSCs), off-the-shelf options can be readily available due to immune “cloaking,” which allows UhPSCs to bypass the immune system by overexpressing 8 genes. In the study, we test UhPSCs for the ability to differentiate into skeletal muscle progenitor cells (SMPCs) and myofibers, in vitro and in vivo, using a humanized mouse model and diseased mouse model. In vitro, UhPSC-derived SMPCs efficiently formed myotubes and expressed myogenic genes, confirmed by qPCR. In vivo engraftment of UhPSC-derived SMPCs showed cell survival using bioluminescence luciferase assays. Immunostaining of engrafted tissues confirmed formation and maturity of human myofiber through positive expression of MYH genes. With these results, we will further test the maintenance of universal potential throughout different stages of development and test in other diseased mouse models such as Cachexia. Overall, UhPSCs are able to produce myofibers and maintain proliferative potential allowing them to be a potential allogeneic cell therapy alternative for muscular diseases.

Lung cancer remains the leading cause of cancer-related deaths worldwide, with non-small cell lung cancer (NSCLC) being the main histological subtype of lung cancer. PARP7 is a mono-ADP-ribosyltransferase that belongs to the PARP family, which consists of 17 members in humans. PARP7 has emerged as a promising therapeutic target for NSCLC due to its essential functions in tumor cell survival and immune regulation. Our previous data mining in the DepMap database suggests that 40% of NSCLC cell lines depend on PARP7 for survival. To assess whether this dependency is specific to PARP7, we compared the sensitivity of PARP7 inhibition to that of PARP family members, PARP1 and PARP2, by in vitro drug dose response studies. We treated a panel of human NSCLC cell lines with either PARP7 inhibitor RBN-2397, PARP1 inhibitor 3-aminobenzamide, or PARP1/2 inhibitor olaparib. Cell viability was measured via CellTiter-Glo, and percent survival was calculated. Several NSCLC cell lines, including NCIH1373 and NCIH2087, exhibited dose-dependent decreases in survival with RBN-2397, whereas most NSCLC lines tested were not inhibited by 3-aminobenzamide. Olaparib treatment led to limited cell death, although some lines showed reduced survival at the highest concentrations tested. Our findings reveal that NSCLC cell lines that are most sensitive to PARP7 inhibition are largely resistant to PARP1 or PARP2 inhibition, further supporting PARP7 as a specific therapeutic target in a subset of NSCLC.

Skeletal muscle regeneration is driven by satellite cells (SCs), a population of muscle stem cells that remain largely quiescent but activate in response to injury. Understanding how SCs regulate skeletal muscle growth requires the ability to observe their behavior in vivo over time. However, prior studies have relied on terminal imaging approaches, restricting analysis to single time points. To address this, we leverage advances in live imaging to visualize SCs in living mice, focusing on the panniculus carnosus muscle, which is a non-evolutionarily conserved muscle layer in the skin that enables repeated imaging over time. Using a tamoxifen-inducible Pax7CreER; R26mTmG mouse model in combination with two-photon microscopy, we successfully imaged SCs in the ear and paw. These preliminary observations revealed alignment of SCs along myofibers and elongated cell morphology. During imaging, we encountered significant motion artifacts due to muscle contractions, which initially limited the stability of the images. This challenge was overcome through the application of nifedipine, an L-type calcium channel blocker, enabling stable imaging. Future studies will quantify SC characteristics in postnatal development, in adulthood, and in response to injury. By establishing a system for repeated in vivo imaging, this work provides a framework to capture SC behavior longitudinally, offering a new way to look into the cellular mechanisms underlying muscle growth and regeneration.

Within the craniofacial skeleton, stem cells (SCs) support bone growth and repair. Cranial sutures, which are fibrous joints that separate the flat bones protecting the brain, house SCs responsible for maintaining cranial bones throughout postnatal life. However, in a common congenital anomaly called craniosynostosis, cranial sutures prematurely fuse, leading to the loss of SC populations and thereby impeding proper skull growth. While SC populations within these cranial sutures have been extensively investigated in adults, their embryonic and early postnatal counterparts – stages of robust bone growth and the onset of craniosynostosis – remain poorly understood. Using the embryonic suture mesenchyme marker Six2 to identify putative SCs at cranial sutures, I hypothesized that SCs are the primary method of bone growth at embryonic stages. I quantified lineage tracing in a Six2-CreERT mouse line and found broad labeling of the coronal suture. However, although labeling Six2+ cells at early stages revealed abundant traced osteoblasts, initiating labeling at later stages showed sparse traced osteoblasts. Thus, Six2+ cells may have dynamic contributions during development, and bone growth at later stages may be required in Six2-independent mechanisms. Consistent with this possibility, I found that osteoblasts at the osteogenic fronts are highly proliferative, although future studies are necessary to determine if osteogenic front cells have SC properties to sustain bone growth.

Rett syndrome (RTT) is an X-linked intellectual disability disorder caused by mutations in methyl-CpG binding protein 2 (MECP2). While traditionally characterized as a transcriptional repressor, loss of MECP2 results in surprisingly few differentially expressed genes (DEGs), suggesting a broader regulatory role. We performed proteomic and ChIP-seq analyses suggesting the presence of a master complex comprising MECP2, Poly(ADP-ribose) polymerase 1 (PARP1), topoisomerase 1 (TOP1) and 2B (TOP2B), and BRG1/BRM-associated factor (BAF) members. This complex appears to co-localize at transcription start sites (TSS) and regulate gene expression through TOP2B-mediated double-stranded breaks (DSBs). Using a modified BLESS-based approach to map DSBs, we generated MECP2-null neurons to test whether MECP2 is required to localize complex activity to the TSS. MECP2-deficient neurons exhibited a significant reduction in DSBs at the TSS, accompanied by a corresponding accumulation of breaks in quiescent regions. We also found that genes with more TSS-promoter DSBs generally showed higher expression than genes without DSBs, though it remains unclear whether this relationship is correlative or causal. Ongoing work aims to clarify this mechanism by examining gene expression before and after break induction. Together, these findings suggest that RTT phenotypes may be driven in part by mislocalization of the MECP2/topoisomerase/PARP1/BAF complex, leading to genomic instability and transcriptional dysregulation.