Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

10.3791/59321 ◽  
2019 ◽  
Author(s):  
Maria G. Garone ◽  
Valeria de Turris ◽  
Alessandro Soloperto ◽  
Carlo Brighi ◽  
Riccardo De Santis ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Motor Neurons ◽  
Pluripotent Stem Cells ◽  
Induced Pluripotent

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

2020 ◽  
Vol 10 (7) ◽  
pp. 407
Author(s):  
Pierre-Antoine Faye ◽  
Nicolas Vedrenne ◽  
Federica Miressi ◽  
Marion Rassat ◽  
Sergii Romanenko ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Motor Neurons ◽  
Pluripotent Stem Cells ◽  
Charcot Marie Tooth ◽  
Charcot Marie Tooth Disease ◽  
Spinal Motor ◽  
Electrophysiological Recordings ◽  
Induced Pluripotent

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.

scholarly journals HDAC6 Inhibitors Rescued the Defective Axonal Mitochondrial Movement in Motor Neurons Derived from the Induced Pluripotent Stem Cells of Peripheral Neuropathy Patients with HSPB1 Mutation

2016 ◽  
Vol 2016 ◽  
pp. 1-14 ◽  
Author(s):  
Ji-Yon Kim ◽  
So-Youn Woo ◽  
Young Bin Hong ◽  
Heesun Choi ◽  
Jisoo Kim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Peripheral Neuropathy ◽  
Induced Pluripotent Stem Cells ◽  
Motor Neurons ◽  
Pluripotent Stem Cells ◽  
Patient Specific ◽  
Motor Neuropathy ◽  
Mitochondrial Movement ◽  
Induced Pluripotent

The Charcot-Marie-Tooth disease 2F (CMT2F) and distal hereditary motor neuropathy 2B (dHMN2B) are caused by autosomal dominantly inherited mutations of the heat shock 27 kDa protein 1 (HSPB1) gene and there are no specific therapies available yet. Here, we assessed the potential therapeutic effect of HDAC6 inhibitors on peripheral neuropathy with HSPB1 mutation using in vitro model of motor neurons derived from induced pluripotent stem cells (iPSCs) of CMT2F and dHMN2B patients. The absolute velocity of mitochondrial movements and the percentage of moving mitochondria in axons were lower both in CMT2F-motor neurons and in dHMN2B-motor neurons than those in controls, and the severity of the defective mitochondrial movement was different between the two disease models. CMT2F-motor neurons and dHMN2B-motor neurons also showed reduced α-tubulin acetylation compared with controls. The newly developed HDAC6 inhibitors, CHEMICAL X4 and CHEMICAL X9, increased acetylation of α-tubulin and reversed axonal movement defects of mitochondria in CMT2F-motor neurons and dHMN2B-motor neurons. Our results suggest that the neurons derived from patient-specific iPSCs can be used in drug screening including HDAC6 inhibitors targeting peripheral neuropathy.

scholarly journals Downregulation of VAPB expression in motor neurons derived from induced pluripotent stem cells of ALS8 patients

2011 ◽  
Vol 20 (18) ◽  
pp. 3642-3652 ◽  
Author(s):  
Miguel Mitne-Neto ◽  
Marcela Machado-Costa ◽  
Maria C.N. Marchetto ◽  
Mario H. Bengtson ◽  
Claudio A. Joazeiro ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Motor Neurons ◽  
Pluripotent Stem Cells ◽  
Induced Pluripotent

scholarly journals Derivation and Identification of Motor Neurons from Human Urine-Derived Induced Pluripotent Stem Cells

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Huan Yi ◽  
Bingbing Xie ◽  
Ben Liu ◽  
Xuan Wang ◽  
Li Xu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Small Molecules ◽  
Induced Pluripotent Stem Cells ◽  
Motor Neurons ◽  
Pluripotent Stem Cells ◽  
Neuromuscular Junctions ◽  
Therapeutic Development ◽  
Induced Pluripotent ◽  
Promising Source ◽  
First Time

Induced pluripotent stem cells (iPSCs) have provided new opportunities for motor neuron disease (MND) modeling, drug screening, and cellular therapeutic development. Among the various types of iPSCs, urine-derived iPSCs have become a promising source of stem cells because they can be safely and noninvasively isolated and easily reprogrammed. Here, for the first time, we differentiated urine-derived iPSCs (urine-iPSCs) into motor neurons (MNs) and compared the capacity of urine-iPSCs and cord-blood-derived iPSCs (B-iPSCs) to differentiate into MNs. With the use of small molecules, mature MNs were generated from urine-iPSCs as early as 26 days in culture. Furthermore, in coculture with muscle cells, MNs projected long axons and formed neuromuscular junctions (NMJs). Immunofluorescence and PCR confirmed the expression levels of both MN and NMJ markers. The comparison of the ratios of positive labeling for MN markers between urine-iPSCs and B-iPSCs demonstrated that the differentiation potentials of these cells were not significantly different. The abovementioned results indicate that urine-iPSCs are a new, promising source of stem cells for MND modeling and further cellular therapeutic development.

scholarly journals Split luciferase-based assay to detect botulinum neurotoxins using human motor neurons derived from induced pluripotent stem cell.

2021 ◽  
Author(s):  
Laurent Cotter ◽  
Feifan Yu ◽  
Juliette Duschene De Lamotte ◽  
Min Dong ◽  
Johannes Krupp ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Motor Neurons ◽  
Pluripotent Stem Cells ◽  
Detection System ◽  
Detection Sensitivity ◽  
Concanamycin A ◽  
Botulinum Neurotoxins ◽  
Induced Pluripotent

Abstract Botulinum neurotoxins (BoNTs) have been widely used clinically as a muscle relaxant. These toxins target motor neurons and cleave proteins essential for neurotransmitter release like Synaptosomal-associated protein of 25 kDa (SNAP-25). Most in vitro assays for BoNT testing use rodent cells or immortalized cell lines, which showed limitations in accuracy and physiological relevance. Here, we report a cell-based assay for detecting SNAP25-cleaving BoNTs by combining human induced Pluripotent Stem Cells (hiPSC)-derived motor neurons and a luminescent detection system based on split nanoluc luciferase. This assay is convenient, rapid, free-of-specialized antibodies, and can discriminate the potency of different BoNTs, with a detection sensitivity of femtomolar concentrations of toxin and can be used to study the different steps of BoNT intoxication. Abreviations: BoNT, Botulinum neurotoxin, SNAP-25, Synaptosomal-associated protein of 25 kDa, hiPSC, human induced Pluripotent Stem Cells, SNARE, soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor, SV2, synaptic vesicle proteins, MLB, mouse lethality bioassay, LD50, toxin’s dose lethal for half of the animal injected, CB-assay, cell-based assays, FRET, Förster resonance energy transfer, Concanamycin A, EC50, Half maximal effective concentration, MNs, motor neurons.

scholarly journals Cell freezing protocol suitable for ATAC-Seq on motor neurons derived from human induced pluripotent stem cells

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Pamela Milani ◽  
Renan Escalante-Chong ◽  
Brandon C. Shelley ◽  
Natasha L. Patel-Murray ◽  
Xiaofeng Xin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Motor Neurons ◽  
Pluripotent Stem Cells ◽  
Induced Pluripotent ◽  
Cell Freezing
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