Robust Generation of Ready-to-Use Cryopreserved Motor Neurons from Human Pluripotent Stem Cells for Disease Modeling.

Abstract

Human pluripotent stem cell (hPSC)-derived motor neurons (MNs) act as models for motor neuron diseases (MNDs), such as amyotrophic lateral sclerosis (ALS) or spinal muscular atrophy. However, the MN differentiation efficiency and viability following cryopreservation require further development for application in large-scale studies and drug screening. Here, we developed a robust protocol to convert hPSCs into MN cryopreservation stocks (hPSCs were converted into >92% motor neural progenitors and >91% MNs). Near-mature MNs were cryopreserved at a high thawing survival rate and 89% MN marker expression on day 32. Moreover, these MNs exhibited classical electrophysiological properties and neuromuscular junction (NMJ) formation ability within only 4-6 days after thawing. To apply this platform as an MND model, MN stocks were generated from SOD1G85R, SOD1G85G isogenic control, and sporadic ALS hPSC lines. The thawed ALS MNs expressed ALS-specific cytopathies, including SOD1 protein aggregation and TDP-43 redistribution. Thus, a stable and robust protocol was developed to generate ready-to-use cryopreserved MNs without further neuronal maturation processes for application in MND mechanistic studies, NMJ model establishment, and large-scale drug screening.