Modelling ALS disease by 2D and 3D in vitro models of patient-derived iPSC

Objective: The study of Amyotrophic Lateral Sclerosis (ALS) is limited by the impossibility to obtain neuronal cells from alive patients in order to study pathomechanisms and to test pharmacological approaches. Induced Pluripotent Stem Cells (iPSCs), that can be obtained by reprogramming patient's peripheral blood cells and/or fibroblasts, represent an exceptional opportunity since they can be differentiated into different types of neurons and glial cells maintaining the patient’s genetic background. To model different forms of ALS in vitro. Materials and Methods: We generated, by Sendai virus reprogramming, iPSCs from patients with mutations in C9orf72 and TARDBP genes as well as isogenic lines and a loss-of-function NEK1 iPSC line. iPSCs were characterized for the expression of stem cell markers and differentiated into motor neurons, positive for specific markers as previously described (1). We analyzed the presence of ALS-associated pathological markers, such as aggregates of the TDP-43 protein and formation of RNA foci of mutant C9orf72 gene, as well as response to oxidative stress and to DNA damage. Since the 2D cultures of iPSC-motor neurons can be maintain in vitro for a limited time and do not allow to study non-cell autonomous mechanisms, we set up 3D cultures to follow the spontaneous development of cerebral ALS organoids and the spatial distribution of the different neuronal cell types. The organoids were generated following a validated protocol (2) and analyzed by immunofluorescence performed on whole organoids or cryostat sectioning slices. Results: iPSC-motor neurons from ALS patients showed the presence of all these defects compared to healthy controls, confirming the validity of this in vitro model. At 64 days of differentiation, the organoids showed positivity for specific markers of neuroepithelial cells, radial glial cells, and immature neurons, such as Doublecortin, Sox2, Nestin, NeuN, MAP2, and BetaIII-tubulin. On the other hand, no positivity was found for the astrocytic marker GFAP. At 73 days, no more positivity was observed for Nestin, demonstrating a maturation of neuronal cells over time, and no signs of necrosis were detected. Discussion and Conclusion: Considering these encouraging preliminary results, the organoids of ALS patients will be cultured for longer times, up to 7 months, in order to observe their evolution and in particular the appearance of markers of more mature neuroglial cells as well as their spatial distribution and number. We have obtained reliable patient-tailored in vitro platforms using iPSC in 2D and 3D cultures to study ALS pathomechanisms.