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.

Coating-Free Culture Medium for Establishing and Maintaining Human Induced Pluripotent Stem Cells

“…We steered the dissociated hPSCs differentiation to neural lineages by embryoid body (EB) formation 25 – 27 . In brief, the 1-2 × 10 5 /ml cells were cultured in Essential 6 medium (E6 medium, A1516401, Gibco) to form EB within the first 2 days.…”

Induced Pluripotent Stem Cells and Their Applications in Amyotrophic Lateral Sclerosis

“…However, how to transfer cell therapy from laboratory and experimental animals to clinical application is still a difficult problem that requires considerable efforts. In order to obtain accurate and repeatable data in future preclinical and clinical trials, it is necessary to establish a standardized cell preparation and transplantation scheme ( Figure 3 ) [ 105 , 106 ]. Although autologous iPSC therapy has potential benefits, it also has some limitations, such as higher medical costs and a long-time preparation.…”

Unlocking the Complexity of Neuromuscular Diseases: Insights from Human Pluripotent Stem Cell-Derived Neuromuscular Junctions

“…In zebrafish, the expression of the mutant SOD1 gene resulted in abnormal motor neuron branching and the defective formation of presynaptic nerve endings [67]. In a series of comprehensive investigations, researchers have used co-culture experiments to shed light on the complex mechanisms underlying ALS pathology [41,43,45,49,[68][69][70][71][72][73]. The use of co-culture models makes it possible to study the effects of a diseased presynaptic compartment on healthy muscles, or vice versa.…”