Optimized Protocol to Generate Spinal Motor Neuron Cells from Induced Pluripotent Stem Cells from Charcot Marie Tooth Patients

Pierre-Antoine Faye (1, 2) , Nicolas Vedrenne (3) , Federica Miressi (2) , Marion Rassat (2) , Sergii Romanenko (4) , Laurence Richard (2, 5) , Sylvie Bourthoumieu (2, 6) , Benoît Funalot (7, 8) , Franck Sturtz (1, 2) , Frederic Favreau (1, 2) , Anne-Sophie Lia (1, 2, 9)

1 Service de Biochimie et Génétique Moléculaire [CHU Limoges]
2 MMNP - Maintenance Myélinique et Neuropathies Périphériques
3 RMeS - Regenerative Medicine and Skeleton
4 Bogomoletz Institute of Physiology
5 Service de Neurologie [CHU Limoges]
6 Service d'Histologie, cytologie, cytogénétique, biologie cellulaire [CHU Limoges]
7 CHU Henri Mondor [Créteil]
8 UPEC UP12 - Université Paris-Est Créteil Val-de-Marne - Paris 12
9 UF de Bioinformatique, CHU de Limoges

Modelling rare neurogenetic diseases to develop new therapeutic strategies is highly challenging. The use of human-induced pluripotent stem cells (hiPSCs) is a powerful approach to obtain specialized cells from patients. For hereditary peripheral neuropathies, such as Charcot–Marie–Tooth disease (CMT) Type II, spinal motor neurons (MNs) are impaired but are very difficult to study. Although several protocols are available to differentiate hiPSCs into neurons, their efficiency is still poor for CMT patients. Thus, our goal was to develop a robust, easy, and reproducible protocol to obtain MNs from CMT patient hiPSCs. The presented protocol generates MNs within 20 days, with a success rate of 80%, using specifically chosen molecules, such as Sonic Hedgehog or retinoic acid. The timing and concentrations of the factors used to induce differentiation are crucial and are given hereby. We then assessed the MNs by optic microscopy, immunocytochemistry (Islet1/2, HB9, Tuj1, and PGP9.5), and electrophysiological recordings. This method of generating MNs from CMT patients in vitro shows promise for the further development of assays to understand the pathological mechanisms of CMT and for drug screening.

Mots clés: CMT; Charcot-Marie-Tooth; Cellular models; hiPSC; Induced pluripotent stem cells; Peripheral nervous system; Peripheral neuropathy; Spinal motor neurons

Domaines: Sciences du Vivant [q-bio]

Pierre-Antoine Faye, Nicolas Vedrenne, Federica Miressi, Marion Rassat, Sergii Romanenko, et al.. Optimized Protocol to Generate Spinal Motor Neuron Cells from Induced Pluripotent Stem Cells from Charcot Marie Tooth Patients. Brain Sciences, 2020, 10 (7), pp.407. ⟨10.3390/brainsci10070407⟩. ⟨hal-03269229⟩