Skip to main content
2024 BMES Annual Meeting Main banner
Icon Legend
This session is not in your schedule.
This session is in your schedule. Click again to remove it.

Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session E

Poster V2 - Human Myoblast Injections into Skeletal Muscle Injuries Regenerate Differently Depending on Cell Passage

Saturday, October 26, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Urinary incontinence (UI), the involuntary release of urine, is a common and growing global concern. Both a financial and emotional burden, UI impacts patients’ daily lives. The etiology of UI can be understood in a few ways: myogenic, neurogenic, connective tissue, and hormonal. Here, we focus on the role of muscle, namely that the rhabdosphincter, a specialized skeletal muscle layer surrounding the urethra, functions as the primary gatekeeper, and maintains continence under increased intra-abdominal pressure. Muscle density in the rhabdosphincter can be lost with aging and childbirth, and older women account for a large proportion of cases. Current interventions for rhabdophincter-related UI include pelvic floor exercises, behavior modification, occlusive devices, and intravaginal supportive devices. Unfortunately, considerations for patient compliance and comfort often mean these are ineffective and uncomfortable for patients. These limitations underscore the urgent need for alternative treatment approaches for UI. To address these challenges, a pre-clinical rodent model was developed that allowed testing of the engraftment of human myoblasts into an injured muscle environment.

Materials and

Methods:  Immunocompromised male RNU rats were used for the following experiments. A muscle injury was created using a cardiotoxin injection into the tibialis anterior (TA) muscle. One day later, fluorescently labeled myoblasts were injected into the injury site. Either 100,000 or 500,000 cells were produced to passage three (P3) or passage six (P6) prior to cryopreservation and then injection. Myoblast engraftment was first assessed using an IVIS Spectrum Preclinical in vivo Imaging system and the bioluminescent signal determined localization and engraftment success rates. Muscle function was evaluated using an Aurora Scientific 1300 instrument where tetanic force and twitch force were measured. At 28 days post-injury, the muscle was harvested for both gene expression analysis and histology. Histology sections were prepared for immunostaining using antibodies against laminin, PAX7, Myosin Heavy Chain I, and Myosin Heavy Chain II to identify muscle fiber cross-sectional areas, fiber type, and satellite cell numbers. Human leukocyte antigen (HLA) antibody staining was used to determine whether engrafted myoblasts are integrated with host tissue. For gene expression analysis, total RNA was extracted using TriZol, and mRNA levels for PAX7, MYF5, MYOG, MYF6, MYH1, and MYH2 were quantified using standard curves created by known dilutions of muscle lysates normalized to Csnk2a2.

Results, Conclusions, and Discussions: 

Results: VivoTrack images showed that cells were localized to the injection site indicating successful engraftment. Muscle force testing showed that the 500,000 cell P3 (500KP3) group cells increase the time to force recovery following a cardiotoxin injection. Interestingly, P3 injections also reduced force output. P3 cell groups showed increases in early myogenic factor expression including PAX7, MYF5, and MYOG. Lower passage number cells also had a smaller fiber diameter compared to higher passages. P3 cells had increased Pax7-positive cells and showed significant increases in PAX7/HLA positive and MyHC-1/HLA positive cells indicating enhanced engraftment and the development of muscle fibers that integrated with the host muscle.

Discussion: Overall, muscle cell therapies that used P3 myoblasts changed the regenerative response following cardiotoxin injury when compared to P6. Lower passages enhanced the resident progenitor cell pool and increased the number of small regenerating fibers in recovering tissue. Cells remained local to the injection site indicating that engraftment of the cells was successful for any high and low passages. Further, the increase in gene expression of early myogenic markers in lower passage cells, particularly the 500KP3 group, correlated with histomorphometry data showing increased Pax7 and HLA-positive cells. Finally, reductions in muscle force output due to 500KP3 injections are likely due to the more immature state of the muscle where fibers are unable to produce as much force as a more mature fiber. Our data suggested that early passage cells contribute to muscle regeneration differently than later passages.

Conclusion: UI is a concerning problem that is yet to be effectively addressed for patients. These data show enhanced engraftment as well as an increase in early myogenic markers with injection of lower passage cells into a damaged TA muscle, and warrant further study to better understand how the passage number of a muscle cell therapy product may contribute to regeneration. This work will better inform future clinical studies to treat UI.

Acknowledgements (Optional):