hiPSC-Derived Neurons Provide a Robust and Physiologically Relevant In Vitro Platform to Test Botulinum Neurotoxins

Botulinum neurotoxins (BoNTs) are zinc metalloproteases that block neurotransmitter release at the neuromuscular junction (NMJ). Their high affinity for motor neurons combined with a high potency have made them extremely effective drugs for the treatment of a variety of neurological diseases as well as for aesthetic applications. Current in vitro assays used for testing and developing BoNT therapeutics include primary rodent cells and immortalized cell lines. Both models have limitations concerning accuracy and physiological relevance. In order to improve the translational value of preclinical data there is a clear need to use more accurate models such as human induced Pluripotent Stem Cells (hiPSC)-derived neuronal models. In this study we have assessed the potential of four different human iPSC-derived neuronal models including Motor Neurons for BoNT testing. We have characterized these models in detail and found that all models express all proteins needed for BoNT intoxication and showed that all four hiPSC-derived neuronal models are sensitive to both serotype A and E BoNT with Motor Neurons being the most sensitive. We showed that hiPSC-derived Motor Neurons expressed authentic markers after only 7 days of culture, are functional and able to form active synapses. When cultivated with myotubes, we demonstrated that they can innervate myotubes and induce contraction, generating an in vitro model of NMJ showing dose-responsive sensitivity BoNT intoxication. Together, these data demonstrate the promise of hiPSC-derived neurons, especially Motor Neurons, for pharmaceutical BoNT testing and development.

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

10.21203/rs.3.rs-730824/v1 ◽

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.

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Honing the Double-Edged Sword: Improving Human iPSC-Microglia Models

Frontiers in Immunology ◽

10.3389/fimmu.2020.614972 ◽

2020 ◽

Vol 11 ◽

Author(s):

Anne Hedegaard ◽

Szymon Stodolak ◽

William S. James ◽

Sally A. Cowley

Keyword(s):

Extracellular Matrix ◽

Stem Cell ◽

Pluripotent Stem Cell ◽

Induced Pluripotent Stem Cell ◽

Media Composition ◽

Physiological Relevance ◽

Key Features ◽

Induced Pluripotent ◽

Human Ipsc

Human induced Pluripotent Stem Cell (hiPSC) models are a valuable new tool for research into neurodegenerative diseases. Neuroinflammation is now recognized as a key process in neurodegenerative disease and aging, and microglia are central players in this. A plethora of hiPSC-derived microglial models have been published recently to explore neuroinflammation, ranging from monoculture through to xenotransplantation. However, combining physiological relevance, reproducibility, and scalability into one model is still a challenge. We examine key features of the in vitro microglial environment, especially media composition, extracellular matrix, and co-culture, to identify areas for improvement in current hiPSC-microglia models.

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Establishment of a Human Induced Pluripotent Stem Cell-Derived Neuromuscular Co-Culture Under Optogenetic Control

10.1101/2020.04.10.036400 ◽

2020 ◽

Cited By ~ 1

Author(s):

Elliot W. Swartz ◽

Greg Shintani ◽

Jijun Wan ◽

Joseph S. Maffei ◽

Sarah H. Wang ◽

...

Keyword(s):

Motor Neurons ◽

Induced Pluripotent Stem Cell ◽

Cell Types ◽

Calcium Flux ◽

Electrode Arrays ◽

Spinal Motor Neurons ◽

Culture Model ◽

Skeletal Myotubes ◽

Induced Pluripotent

SummaryThe failure of the neuromuscular junction (NMJ) is a key component of degenerative neuromuscular disease, yet how NMJs degenerate in disease is unclear. Human induced pluripotent stem cells (hiPSCs) offer the ability to model disease via differentiation toward affected cell types, however, the re-creation of an in vitro neuromuscular system has proven challenging. Here we present a scalable, all-hiPSC-derived co-culture system composed of independently derived spinal motor neurons (MNs) and skeletal myotubes (sKM). In a model of C9orf72-associated disease, co-cultures form functional NMJs that can be manipulated through optical stimulation, eliciting muscle contraction and measurable calcium flux in innervated sKM. Furthermore, co-cultures grown on multi-electrode arrays (MEAs) permit the pharmacological interrogation of neuromuscular physiology. Utilization of this co-culture model as a tunable, patient-derived system may offer significant insights into NMJ formation, maturation, repair, or pathogenic mechanisms that underlie NMJ dysfunction in disease.

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

Brain Sciences ◽

10.3390/brainsci10070407 ◽

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.

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Identification of Molecular Signatures in Neural Differentiation and Neurological Diseases Using Digital Color-Coded Molecular Barcoding

Stem Cells International ◽

10.1155/2020/8852313 ◽

2020 ◽

Vol 2020 ◽

pp. 1-9

Author(s):

Debora Salerno ◽

Alessandro Rosa

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Neural Differentiation ◽

Disease Modeling ◽

Neurological Diseases ◽

Embryonic Stem ◽

Molecular Signatures ◽

Molecular Barcoding ◽

Induced Pluripotent

Human pluripotent stem cells (PSCs), including embryonic stem cells and induced pluripotent stem cells, represent powerful tools for disease modeling and for therapeutic applications. PSCs are particularly useful for the study of development and diseases of the nervous system. However, generating in vitro models that recapitulate the architecture and the full variety of subtypes of cells that make the complexity of our brain remains a challenge. In order to fully exploit the potential of PSCs, advanced methods that facilitate the identification of molecular signatures in neural differentiation and neurological diseases are highly demanded. Here, we review the literature on the development and application of digital color-coded molecular barcoding as a potential tool for standardizing PSC research and applications in neuroscience. We will also describe relevant examples of the use of this technique for the characterization of the heterogeneous composition of the brain tumor glioblastoma multiforme.

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Mitochondrial Abnormalities in Induced Pluripotent Stem Cells-Derived Motor Neurons from Patients with Riboflavin Transporter Deficiency

Antioxidants ◽

10.3390/antiox9121252 ◽

2020 ◽

Vol 9 (12) ◽

pp. 1252

Author(s):

Fiorella Colasuonno ◽

Enrico Bertini ◽

Marco Tartaglia ◽

Claudia Compagnucci ◽

Sandra Moreno

Keyword(s):

Motor Neurons ◽

Neurodegenerative Disorder ◽

Ion Beam ◽

Sensorineural Deafness ◽

Microscopic Analysis ◽

Patient Specific ◽

Neuronal Marker ◽

Transporter Deficiency ◽

Induced Pluripotent

Riboflavin transporter deficiency (RTD) is a childhood-onset neurodegenerative disorder characterized by sensorineural deafness and motor neuron degeneration. Since riboflavin plays key functions in biological oxidation-reduction reactions, energy metabolism pathways involving flavoproteins are affected in RTD. We recently generated induced pluripotent stem cell (iPSC) lines from affected individuals as an in vitro model of the disease and documented mitochondrial impairment in these cells, dramatically impacting cell redox status. This work extends our study to motor neurons (MNs), i.e., the cell type most affected in patients with RTD. Altered intracellular distribution of mitochondria was detected by confocal microscopic analysis (following immunofluorescence for superoxide dismutase 2 (SOD2), as a dual mitochondrial and antioxidant marker), and βIII-Tubulin, as a neuronal marker. We demonstrate significantly lower SOD2 levels in RTD MNs, as compared to their healthy counterparts. Mitochondrial ultrastructural abnormalities were also assessed by focused ion beam/scanning electron microscopy. Moreover, we investigated the effects of combination treatment using riboflavin and N-acetylcysteine, which is a widely employed antioxidant. Overall, our findings further support the potential of patient-specific RTD models and provide evidence of mitochondrial alterations in RTD-related iPSC-derived MNs—emphasizing oxidative stress involvement in this rare disease. We also provide new clues for possible therapeutic strategies aimed at correcting mitochondrial defects, based on the use of antioxidants.

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The Src/c-Abl pathway is a potential therapeutic target in amyotrophic lateral sclerosis

Science Translational Medicine ◽

10.1126/scitranslmed.aaf3962 ◽

2017 ◽

Vol 9 (391) ◽

pp. eaaf3962 ◽

Cited By ~ 90

Author(s):

Keiko Imamura ◽

Yuishin Izumi ◽

Akira Watanabe ◽

Kayoko Tsukita ◽

Knut Woltjen ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Motor Neuron ◽

Motor Neurons ◽

New Drugs ◽

Small Interfering Rnas ◽

Altered Expression ◽

Sporadic Als ◽

Induced Pluripotent ◽

Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS), a fatal disease causing progressive loss of motor neurons, still has no effective treatment. We developed a phenotypic screen to repurpose existing drugs using ALS motor neuron survival as readout. Motor neurons were generated from induced pluripotent stem cells (iPSCs) derived from an ALS patient with a mutation in superoxide dismutase 1 (SOD1). Results of the screen showed that more than half of the hits targeted the Src/c-Abl signaling pathway. Src/c-Abl inhibitors increased survival of ALS iPSC-derived motor neurons in vitro. Knockdown of Src or c-Abl with small interfering RNAs (siRNAs) also rescued ALS motor neuron degeneration. One of the hits, bosutinib, boosted autophagy, reduced the amount of misfolded mutant SOD1 protein, and attenuated altered expression of mitochondrial genes. Bosutinib also increased survival in vitro of ALS iPSC-derived motor neurons from patients with sporadic ALS or other forms of familial ALS caused by mutations in TAR DNA binding protein (TDP-43) or repeat expansions in C9orf72. Furthermore, bosutinib treatment modestly extended survival of a mouse model of ALS with an SOD1 mutation, suggesting that Src/c-Abl may be a potentially useful target for developing new drugs to treat ALS.

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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

Stem Cells International ◽

10.1155/2016/9475981 ◽

2016 ◽

Vol 2016 ◽

pp. 1-14 ◽

Cited By ~ 20

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.

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From “Directed Differentiation” to “Neuronal Induction”: Modeling Neuropsychiatric Disease

Biomarker Insights ◽

10.4137/bmi.s20066 ◽

2015 ◽

Vol 10s1 ◽

pp. BMI.S20066 ◽

Cited By ~ 4

Author(s):

Seok-Man Ho ◽

Aaron Topol ◽

Kristen J. Brennand

Keyword(s):

Psychiatric Disorders ◽

Neurological Diseases ◽

Disease Onset ◽

Induced Pluripotent Stem Cell ◽

Directed Differentiation ◽

Neuropsychiatric Disease ◽

Somatic Cell Reprogramming ◽

Human Neurons ◽

Induced Pluripotent

Aberrant behavior and function of neurons are believed to be the primary causes of most neurological diseases and psychiatric disorders. Human postmortem samples have limited availability and, while they provide clues to the state of the brain after a prolonged illness, they offer limited insight into the factors contributing to disease onset. Conversely, animal models cannot recapitulate the polygenic origins of neuropsychiatric disease. Novel methods, such as somatic cell reprogramming, deliver nearly limitless numbers of pathogenic human neurons for the study of the mechanism of neuropsychiatric disease initiation and progression. First, this article reviews the advent of human induced pluripotent stem cell (hiPSC) technology and introduces two major methods, “directed differentiation” and “neuronal induction,” by which it is now possible to generate neurons for modeling neuropsychiatric disease. Second, it discusses the recent applications, and the limitations, of these technologies to in vitro studies of psychiatric disorders.

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“Axonal Length Determines distinct homeostatic phenotypes in human iPSC derived motor neurons on a bioengineered platform”

10.1101/2021.08.30.458271 ◽

2021 ◽

Author(s):

Cathleen Hagemann ◽

Carmen Moreno Gonzalez ◽

Ludovica Guetta ◽

Giulia Tyzack ◽

Ciro Chiappini ◽

...

Keyword(s):

Motor Neurons ◽

Systematic Investigation ◽

Threshold Size ◽

Spinal Motor Neurons ◽

Mitochondrial Homeostasis ◽

Conventional Culture ◽

Human Ipsc ◽

First Time

AbstractStem cell-based experimental platforms for neuroscience can effectively model key mechanistic aspects of human development and disease. However, conventional culture systems often overlook the engineering constraints that cells face in vivo. This is particularly relevant for neurons covering long range connections such as spinal motor neurons (MNs). The axons of these neurons extend up to 1m in length and require a complex interplay of mechanisms to maintain cellular homeostasis. It follows that shorter axons in conventional cultures may not faithfully capture important aspects of their longer counterparts. Here we directly address this issue by establishing a bioengineered platform to assemble arrays of human axons ranging from micrometers to centimeters, permitting systematic investigation of the effects of length on human axonal biology for the first time. With this approach, we reveal a link between length and metabolism in human MNs in vitro, where axons above a “threshold” size induce specific molecular adaptations in cytoskeleton composition, functional properties, local translation and mitochondrial homeostasis. Our findings specifically demonstrate the existence of a length-dependent mechanism that switches homeostatic processes within human MNs in order to sustain long axons. Our findings have critical implications for in vitro modelling of several neurodegenerative disorders and reinforce the importance of modelling cell shape and biophysical constraints with fidelity and precision in vitro.

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