Modelling ALS disease by 3D organoids culture from human-derived iPSC

Generation of cerebral organoids from human induced pluripotent stem cells (hiPSCs) has opened new possibilities to investigate brain development in vitro as regards not only neural cell differentiation, but also neuro-glial cell interactions and 3D organization. Recently, heterozygous loss-of-function (LOF) mutations in the mitotic protein kinase NEK1 gene were identified in ALS patients. However, the pathomechanisms whereby NEK1 LOF leads to neurodegeneration have not been investigated so far. Aim of our study was to study the biological effect of ALS-related NEK1 LOF mutations in hiPSCs-derived cerebral organoids. We used CRISPR/Cas9 to engineer a heterozygous LOF mutation in a healthy control hiPSCs line and generated organoids following a validated protocol for up to 140 days of differentiation. Immunofluorescence analysis performed on whole organoids at 26 days of differentiation revealed positivity for specific markers of pluripotency and proliferation (SOX2, NESTIN and Ki67), which progressively decreased at later differentiation time points (100-120-140 days). We observed the expression of markers typical of different neuronal maturation stages such as III-tubulin, double cortin, NeuN and MAP2 as well as the expression of the dopaminergic neuron marker TH. Interestingly, at day 100, organoids expressed also GFAP, marker of astrocytes differentiation. No differences were observed between the mutated NEK1 and the isogenic control organoids in the expression of these neuro-glial markers. Organoids have also been analyzed by electron microscopy to better determine their sub-cellular ultrastructure. The cerebral organoids we generated represent a suitable 3D disease model for ALS where the impact of NEK1 haploinsufficiency on different cell pathways associated to DNA damage repair, mitochondria functionality and ciliogenesis can be investigated. Moreover, this human 3D disease model can be exploited also for future pharmacological approaches.