Motor neuron differentiation of iPSCs obtained from peripheral blood of a mutant TARDBP ALS patient

Abstract

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease, mainly affecting the motor neurons (MNs) and without effective therapy. Drug screening is hampered by the lack of satisfactory experimental and pre-clinical models. Induced pluripotent stem cells (iPSCs) could help to define disease mechanisms and therapeutic strategies as they could be differentiated into MNs, otherwise inaccessible from living humans. In this study, given the seminal role of TDP-43 in ALS pathophysiology, MNs were obtained from peripheral blood mononuclear cells-derived iPSCs of an ALS patient carrying a p.A382T TARDBP mutation and a healthy donor. Venous samples were preferred to fibroblasts for their ease of collection and no requirement for time consuming extended cultures before experimentation. iPSCs were characterized for expression of specific markers, spontaneously differentiated into primary germ layers and, finally, into MNs. No differences were observed between the mutated ALS patient and the control MNs with most of the cells displaying a nuclear localization of the TDP-43 protein. In conclusion, we here demonstrated for the first time that human TARDBP mutated MNs can be successfully obtained exploiting the reprogramming and differentiation ability of peripheral blood cells, an easily accessible source from any patient.

Reprogramming fibroblasts and peripheral blood cells from a C9ORF72 patient: A proof‐of‐principle study

“…Regarding another hallmark of ALS pathology, no pathological protein aggregates but a slight cytoplasmic mislocalization of the TDP‐43 protein were observed, in contrast to what is found in post mortem ALS patient MNs. Nevertheless, similar discrepancies between in vivo and in vitro cells were already been reported . Results from previous studies investigating the presence of TDP‐43 protein pathological phenotype in iPSC‐derived MNs vary considerably from one to the other.…”

“…Burkhardt et al identified nuclear aggregates in 3 out 24 reprogrammed skin fibroblasts from sporadic ALS patients. Similarly, two authors reported cytoplasmic pre‐inclusion‐like aggregates on MNs derived from TDP‐43‐mutated patients while no aggregation was observed in two other studies . No subcellular localization was observed for TDP‐43 protein in iPSCs and iPSC‐derived MNs from C9ORF72 ‐mutated patients while nuclear depletion and cytoplasmic accumulation of the protein were reported by Zhang et al The total amount of the TDP‐43 protein evaluated by WB was comparable between FB‐ and PB‐derived iPSCs and between iPSC‐derived MNs of both tissues, with a slight increase in MNs.…”

“…MNs were differentiated as previously described . Briefly, iPSCs were grown in 100 mm dishes until confluence, harvested and placed into low adhesion dishes to generate EBs.…”

The debated toxic role of aggregated TDP-43 in amyotrophic lateral sclerosis: a resolution in sight?

“…Nonetheless, recently one group did not find increased cytoplasmic localization for TDP-43 A382T in motor neurons from one ALS patient, at least during the time of their experiments (Bossolasco et al , 2018). However, they did not present data regarding the solubility of TDP-43 or cell viability.…”

Induction Of Chronic Stress Reveals An Interplay Of Stress Granules And TDP-43 Pathological Aggregates In Human ALS Fibroblasts And iPSC-Neurons

“…iPSC lines from 1 healthy control, 1 mutant TDP-43 and 1 mutant C9ORF72 ALS patient were obtained by reprogramming fibroblasts (Additional file 1: Table S1) with CytoTune-iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher Scientific, Waltham, MA) and differentiated into neurons, as previously described [28,29]. Briefly, iPSCs were grown in suspension in low adhesion cell culture dishes and embryoid bodies (EBs) formation was induced in 17 days by using different culture media supplemented with appropriate growth factors.…”