scholarly journals A Monolayer System for the Efficient Generation of Motor Neuron Progenitors and Functional Motor Neurons from Human Pluripotent Stem Cells

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1127
Author(s):  
Alessandro Cutarelli ◽  
Vladimir A. Martínez-Rojas ◽  
Alice Tata ◽  
Ingrid Battistella ◽  
Daniela Rossi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Motor Neuron ◽  
Motor Neurons ◽  
Pluripotent Stem Cells ◽  
Disease Modelling ◽  
Disease Phenotypes ◽  
In Vitro Systems ◽  
Induced Pluripotent

Methods for the conversion of human induced pluripotent stem cells (hiPSCs) into motor neurons (MNs) have opened to the generation of patient-derived in vitro systems that can be exploited for MN disease modelling. However, the lack of simplified and consistent protocols and the fact that hiPSC-derived MNs are often functionally immature yet limit the opportunity to fully take advantage of this technology, especially in research aimed at revealing the disease phenotypes that are manifested in functionally mature cells. In this study, we present a robust, optimized monolayer procedure to rapidly convert hiPSCs into enriched populations of motor neuron progenitor cells (MNPCs) that can be further amplified to produce a large number of cells to cover many experimental needs. These MNPCs can be efficiently differentiated towards mature MNs exhibiting functional electrical and pharmacological neuronal properties. Finally, we report that MN cultures can be long-term maintained, thus offering the opportunity to study degenerative phenomena associated with pathologies involving MNs and their functional, networked activity. These results indicate that our optimized procedure enables the efficient and robust generation of large quantities of MNPCs and functional MNs, providing a valid tool for MNs disease modelling and for drug discovery applications.

In situ generation of human brain organoids on a micropillar array

Lab on a Chip ◽  
2017 ◽  
Vol 17 (17) ◽  
pp. 2941-2950 ◽  
Author(s):  
Yujuan Zhu ◽  
Li Wang ◽  
Hao Yu ◽  
Fangchao Yin ◽  
Yaqing Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Human Brain ◽  
Pluripotent Stem Cells ◽  
3D Culture ◽  
In Situ Generation ◽  
Induced Pluripotent ◽  
High Throughput Manner

We present a simple and high throughput manner to generate brain organoids in situ from human induced pluripotent stem cells on micropillar arrays and to investigate long-term brain organogenesis in 3D culture in vitro.

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 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 Generation of defined neural populations from pluripotent stem cells

2018 ◽  
Vol 373 (1750) ◽  
pp. 20170214 ◽  
Author(s):  
Sarah F. McComish ◽  
Maeve A. Caldwell
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Disease Modelling ◽  
Human Neural Stem Cells ◽  
Neural Populations ◽  
Human Disorders ◽  
Induced Pluripotent

Effective and efficient generation of human neural stem cells and subsequently functional neural populations from pluripotent stem cells has facilitated advancements in the study of human development and disease modelling. This review will discuss the established protocols for the generation of defined neural populations including regionalized neurons and astrocytes, oligodendrocytes and microglia. Early protocols were established in embryonic stem cells (ESC) but the discovery of induced pluripotent stem cells (iPSC) in 2006 provided a new platform for modelling human disorders of the central nervous system (CNS). The ability to produce patient- and disease-specific iPSC lines has created a new age of disease modelling. Human iPSC may be derived from adult somatic cells and subsequently patterned into numerous distinct cell types. The ability to derive defined and regionalized neural populations from iPSC provides a powerful in vitro model of CNS disorders. This article is part of the theme issue ‘Designer human tissue: coming to a lab near you’.

scholarly journals Human Pluripotent Stem Cell-Derived Cardiomyocytes as Research and Therapeutic Tools

2014 ◽  
Vol 2014 ◽  
pp. 1-14 ◽  
Author(s):  
Ivana Acimovic ◽  
Aleksandra Vilotic ◽  
Martin Pesl ◽  
Alain Lacampagne ◽  
Petr Dvorak ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Drug Testing ◽  
Embryonic Stem ◽  
Cell Types ◽  
Patient Specific ◽  
Disease Modelling ◽  
Therapeutic Tools ◽  
Induced Pluripotent

Human pluripotent stem cells (hPSCs), namely, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), with their ability of indefinite self-renewal and capability to differentiate into cell types derivatives of all three germ layers, represent a powerful research tool in developmental biology, for drug screening, disease modelling, and potentially cell replacement therapy. Efficient differentiation protocols that would result in the cell type of our interest are needed for maximal exploitation of these cells. In the present work, we aim at focusing on the protocols for differentiation of hPSCs into functional cardiomyocytesin vitroas well as achievements in the heart disease modelling and drug testing on the patient-specific iPSC-derived cardiomyocytes (iPSC-CMs).

scholarly journals Generating Diverse Spinal Motor Neuron Subtypes from Human Pluripotent Stem Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-13 ◽  
Author(s):  
Rickie Patani
Keyword(s):  
Stem Cells ◽  
Motor Neuron ◽  
Motor Neurons ◽  
Pluripotent Stem Cells ◽  
Muscular Atrophy ◽  
Human Pluripotent Stem Cells ◽  
Disease Models ◽  
Differential Vulnerability ◽  
Neuronal Diversification

Resolving the mechanisms underlying human neuronal diversification remains a major challenge in developmental and applied neurobiology. Motor neurons (MNs) represent a diverse pool of neuronal subtypes exhibiting differential vulnerability in different human neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). The ability to predictably manipulate MN subtype lineage restriction from human pluripotent stem cells (PSCs) will form the essential basis to establishing accurate, clinically relevantin vitrodisease models. I first overview motor neuron developmental biology to provide some context for reviewing recent studies interrogating pathways that influence the generation of MN diversity. I conclude that motor neurogenesis from PSCs provides a powerful reductionist model system to gain insight into the developmental logic of MN subtype diversification and serves more broadly as a leading exemplar of potential strategies to resolve the molecular basis of neuronal subclass differentiation within the nervous system. These studies will in turn permit greater mechanistic understanding of differential MN subtype vulnerability usingin vitrohuman disease models.

scholarly journals A library of ATTR amyloidosis patient-specific induced pluripotent stem cells for disease modelling and in vitro testing of novel therapeutics

Amyloid ◽  
2018 ◽  
Vol 25 (3) ◽  
pp. 148-155 ◽  
Author(s):  
Richard M. Giadone ◽  
Jessica D. Rosarda ◽  
Prithvi Reddy Akepati ◽  
Arianne C. Thomas ◽  
Batbold Boldbaatar ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Patient Specific ◽  
Disease Modelling ◽  
In Vitro Testing ◽  
Novel Therapeutics ◽  
Attr Amyloidosis ◽  
Induced Pluripotent

scholarly journals Motor Neuron Generation from iPSCs from Identical Twins Discordant for Amyotrophic Lateral Sclerosis

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 571 ◽  
Author(s):  
Emily R. Seminary ◽  
Stephanie Santarriaga ◽  
Lynn Wheeler ◽  
Marie Mejaki ◽  
Jenica Abrudan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Amyotrophic Lateral Sclerosis ◽  
Motor Neuron ◽  
Induced Pluripotent Stem Cells ◽  
Motor Neurons ◽  
Pluripotent Stem Cells ◽  
Identical Twins ◽  
Sporadic Als ◽  
Induced Pluripotent ◽  
Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disorder characterized by the loss of the upper and lower motor neurons. Approximately 10% of cases are caused by specific mutations in known genes, with the remaining cases having no known genetic link. As such, sporadic cases have been more difficult to model experimentally. Here, we describe the generation and differentiation of ALS induced pluripotent stem cells reprogrammed from discordant identical twins. Whole genome sequencing revealed no relevant mutations in known ALS-causing genes that differ between the twins. As protein aggregation is found in all ALS patients and is thought to contribute to motor neuron death, we sought to characterize the aggregation phenotype of the sporadic ALS induced pluripotent stem cells (iPSCs). Motor neurons from both twins had high levels of insoluble proteins that commonly aggregate in ALS that did not robustly change in response to exogenous glutamate. In contrast, established genetic ALS iPSC lines demonstrated insolubility in a protein- and genotype-dependent manner. Moreover, whereas the genetic ALS lines failed to induce autophagy after glutamate stress, motor neurons from both twins and independent controls did activate this protective pathway. Together, these data indicate that our unique model of sporadic ALS may provide key insights into disease pathology and highlight potential differences between sporadic and familial ALS.

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