(1402-C) Drastically Accelerated Directed Differentiation of Human Pluripotent Stem Cells into Motor Neurons for Disease Modeling and Screening Applications
Tuesday, February 6, 2024
12:00 PM – 1:00 PM EST
Location: Exhibit Halls AB
Abstract: Motor neurons are involved in the voluntary control of muscular contraction, and are the primary cell type affected in amyotrophic lateral sclerosis (ALS), an incurable and deadly neurological disease resulting in paralysis. Given their unlimited potential for self-renewal and differentiation, it has been a longstanding goal to model ALS patient-specifically using human pluripotent stem cell (hiPSC)- derived motor neurons for drug discovery to develop life-saving treatments for this disease. While innumerable methods have been published to differentiate motor neurons from hiPSCs, all protocols to date demonstrate substantial clone-to-clone and donor-to-donor variability, failing to produce motor neurons at high yields and purity from each hiPSC line. Many of these failings can be attributable to the protracted time in culture required to generate motor neurons, often extending beyond 30 days in vitro. Here we report a drastically accelerated, developmentally guided, directed differentiation protocol based on small molecules and growth factors to generate motor neurons in only 7 days. Owing to its speed, the protocol is highly efficient and reproducible, allowing for the for the manufacture of motor neurons of high purity and yield for several donor hiPSC lines tested. These resulting motor neurons express MNX1, ISLET1, and ChAT by immunocytochemistry and qPCR. To demonstrate utility for disease modeling, motor neurons were generated from a hiPSC line derived from an affected ALS donor with a TDP43 mutation. A small molecule screen was performed to induce mislocalization of TDP43 and results were compared to hiPSC-derived motor neurons from a healthy donor. Functional assays were also developed including co-cultures with human muscle using a microphysiological system and calcium imaging. Substrates and seeding density were optimized for long term culture on microelectrode arrays, and cultures show burst spiking within a week of maturation. In conclusion, we demonstrate a rapid protocol to generate motor neurons from a number of donor lines, along with their utility in a number of screening assays for ALS drug discovery.