Abstract
Objectives
The FDA Modernization Act 2.0 invites alternatives to animal testing, to improve the translational success of clinical drug candidates. iPSC-derived neurons offer a more physiologically relevant model for pre-clinical assessment. To this end, iPSC-derived motor neurons from donors affected by ALS backgrounds (C9Orf72 expansion, TDP-43 A382T, SOD1 D109Y) and a donor unaffected by the disease, were used to develop quantitative phenotypic assays. Additionally, a more physiologically relevant triculture model of motor neurons, astrocytes and microglia was developed.
Method
Firing frequency and synchronisation of motor neurons was investigated, using a lentivirally-encoded calcium fluorophore driven from a synapsin 1 promoter (Incucyte® Neuroburst Orange Lentivirus, Sartorius). Morphology was intermittently assessed by phase contrast imaging on the Incucyte S3 (Sartorius). Expression of key markers was visualised by immunocytochemistry, with confocal imaging. Staining for puncta containing aggregated TDP-43 was also carried out.
Results
iPSC-derived motor neurons from donors affected by ALS demonstrate distinct morphology, patterns of firing frequency and synchronisation compared to those from donors unaffected by disease. The frequency and duration of burst firing in motor neurons is modified by the addition of astrocytes and microglia. This triculture model may increase confidence in the translation of compound efficacy from in vitro assays to the clinic.
Conclusions
iPSC-derived motor neurons and triculture systems provide a robust, physiologically relevant platform for assessing ALS phenotypes and compound effects. This approach supports more informed decision-making and strengthens the path toward identifying effective therapeutic candidates for ALS.
