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

2016 ◽  
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 ◽  
Muscular Atrophy ◽  
Cell Type ◽  
High Quality ◽  
Induced Pluripotent ◽  
Accessible Chromatin ◽  
Relevant Cell Type

In recent years, the assay for transposase-accessible chromatin using sequencing (ATAC-Seq) has become a fundamental tool of epigenomic research. However, it has proven difficult to perform this technique on frozen samples because freezing cells before extracting nuclei impairs nuclear integrity and alters chromatin structure. We describe a protocol for freezing cells that is compatible with ATAC-Seq, producing results that compare well with those generated from fresh cells. We found that while flash-frozen samples are not suitable for ATAC-Seq, the assay is successful with slow-cooled cryopreserved samples. Using this method, we were able to isolate high quality, intact nuclei, and we verified that epigenetic results from fresh and cryopreserved samples agree quantitatively. We developed our protocol on a disease-relevant cell type, namely motor neurons differentiated from induced pluripotent stem cells from a patient affected by spinal muscular atrophy.

scholarly journals Modeling the differential phenotypes of spinal muscular atrophy with high-yield generation of motor neurons from human induced pluripotent stem cells

Oncotarget ◽  
2017 ◽  
Vol 8 (26) ◽  
pp. 42030-42042 ◽  
Author(s):  
Xiang Lin ◽  
Jin-Jing Li ◽  
Wen-Jing Qian ◽  
Qi-Jie Zhang ◽  
Zhong-Feng Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Spinal Muscular Atrophy ◽  
Induced Pluripotent Stem Cells ◽  
Motor Neurons ◽  
Pluripotent Stem Cells ◽  
Muscular Atrophy ◽  
High Yield ◽  
Induced Pluripotent

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.

Red blood cell generation from human induced pluripotent stem cells of different donor cell type origin

2013 ◽  
Vol 41 (8) ◽  
pp. S27
Author(s):  
Isabel Dorn ◽  
Katharina Klich ◽  
Martina Radstaak ◽  
Katherina Psathaki ◽  
Marcos Arauzo-Bravo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Blood Cell ◽  
Induced Pluripotent Stem Cells ◽  
Red Blood Cell ◽  
Pluripotent Stem Cells ◽  
Donor Cell ◽  
Cell Generation ◽  
Cell Type ◽  
Induced Pluripotent ◽  
Donor Cell Type

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
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