Lydia Sibley, Shirley Ryan Ability Lab
Conference: Society for Neuroscience 2019, Chicago, IL
Abstract Title: Establishing a co-culture model of human induced pluripotent stem cell-derived motor neurons and primary stem cell-derived myotubes from patients with cerebral palsy
Abstract: Cerebral palsy (CP) is a non-progressive perinatal brain injury that causes progressive muscle weakness and impaired mobility. The brain injury disrupts the motor control centers and causes a miscommunication between the motor nerve system and skeletal muscles. Treatment therapies for muscle impairment focus on muscle stretching and strengthening, and while these therapies have been used for decades, they are minimally effective in preventing muscle weakness and wasting. Much of the biological research of CP is related to the impact the brain injury has on either the nervous system or the skeletal muscle. There is a large gap in knowledge where these two systems meet at the neuromuscular junction (NMJ) and the miscommunication between organ systems that is occurring in this disorder. Previous research has suggested that the NMJs are structurally dysmorphic in CP, but little is known about why this is or the downstream impact it has on changes in muscle growth patterns. Our goal is to establish a human in vitro assay to study NMJ formation, maintenance and physiology with cells derived from CP patients. To assess formation of NMJs in vitro, we co-cultured human primary stem cell-derived muscle fibers with human pluripotent stem cell (PSC)-derived motor neurons in a dish. The use of all human cell types ensures the patients phenotype is developed in the culture and will show any changes that are specific to CP patients. In our first set of experiments, we used cells from healthy patients to establish our model and to validate the functionality of the co-culture. Our first results showed that after 7 weeks of co-culture, there was evidence of NMJ development in vitro, as evidenced by staining with alpha-bungarotoxin, a marker for acetylcholine receptors. Field electrophysiology recordings using multi-well multielectrode arrays (Axion Biosystems, USA) also showed that co-cultures were actively producing action potentials, a sign that motor neurons could mature enough to electrically communicate with the muscle fibers. In our ongoing experiments, we are focused on optimizing the protocol for co-culturing motor neurons with muscle fibers and to test our system using cells derived from CP patients. We will be looking at structure, firing patterns, and health of the cells. Illuminating changes in NMJ formation and physiology could open a large field of new possibilities of treatments or drug testing to strengthen the connection between the nerve and muscle systems, and improve motor function in patients with CP.