scholarly journals An integrated multi-omic analysis of iPSC-derived motor neurons from C9ORF72 ALS patients

2020 ◽  
Author(s):  
◽  
Loren Ornelas ◽  
Emilda Gomez ◽  
Lindsay Panther ◽  
Aaron Frank ◽  
...  
Keyword(s):  
Motor Neuron ◽  
Motor Neurons ◽  
Rna Splicing ◽  
Induced Pluripotent Stem Cell ◽  
Patient Specific ◽  
Data Independent Acquisition ◽  
Induced Pluripotent ◽  
Als Patients ◽  
And Control

SummaryNeurodegenerative diseases present a challenge for systems biology, due to the lack of reliable animal models and the difficulties in obtaining samples from patients at early stages of disease, when interventions might be most effective. Studying induced pluripotent stem cell (iPSC)-derived neurons could overcome these challenges and dramatically accelerate and broaden therapeutic strategies. Here we undertook a network-based multi-omic characterization of iPSC-derived motor neurons from ALS patients carrying genetically dominant hexanucleotide expansions in C9orf72 to gain a deeper understanding of the relationship between DNA, RNA, epigenetics and protein in the same pool of tissue. ALS motor neurons showed the expected C9orf72-related alterations to specific nucleoporins and production of dipeptide repeats. RNA-seq, ATAC-seq and data-independent acquisition mass-spectrometry (DIA-MS) proteomics were then performed on the same motor neuron cultures. Using integrative computational methods that combined all of the omics, we discovered a number of novel dysregulated pathways including biological adhesion and extracellular matrix organization and disruption in other expected pathways such as RNA splicing and nuclear transport. We tested the relevance of these pathways in vivo in a C9orf72 Drosophila model, analyzing the data to determine which pathways were causing disease phenotypes and which were compensatory. We also confirmed that some pathways are altered in late-stage neurodegeneration by analyzing human postmortem C9 cervical spine data. To validate that these key pathways were integral to the C9 signature, we prepared a separate set of C9orf72 and control motor neuron cultures using a different differentiation protocol and applied the same methods. As expected, there were major overall differences between the differentiation protocols, especially at the level of in individual omics data. However, a number of the core dysregulated pathways remained significant using the integrated multiomic analysis. This new method of analyzing patient specific neural cultures allows the generation of disease-related hypotheses with a small number of patient lines which can be tested in larger cohorts of patients.

Abstract 518: In Vitro and in vivo Disease Modeling Using Patient Derived iPSCs to Characterize the Calcification Phenotype in Arterial Calcification Due to Deficiency of CD73

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Cynthia St. Hilaire ◽  
Hui Jin ◽  
Yuting Huang ◽  
Dan Yang ◽  
Alejandra Negro ◽  
...  
Keyword(s):  
Vascular Calcification ◽  
Disease Modeling ◽  
Induced Pluripotent Stem Cell ◽  
Dermal Fibroblasts ◽  
In Vivo Studies ◽  
Arterial Calcification ◽  
Patient Specific ◽  
And Control

Objective: The objective of this study was to develop a patient-specific induced pluripotent stem cell (iPSC)-based disease model to understand the process by which CD73-deficiency leads to vascular calcification in the disease, Arterial Calcification due to Deficiency of CD73 (ACDC). Approach & Results: ACDC is an autosomal recessive disease resulting from mutations in the gene encoding for CD73, which converts extracellular AMP to adenosine. CD73-deficiency manifests with tortuosity and vascular calcification of the medial layer of lower-extremity arteries, a pathology associated with diabetes and chronic kidney disease. We previously identified that dermal fibroblasts isolated from ACDC patients calcify in vitro, however in vivo studies of the vasculature are limited, as murine models of CD73 deficiency do not recapitulate the human disease phenotype. Thus, we created iPSCs from ACDC patients and control fibroblasts. ACDC and Control iPSCs form teratomas when injected in immune-compromised mice, however ACDC iPSC teratomas exhibit extensive calcifications. Control and ACDC iPSCs were differentiated down the mesenchymal lineage (MSC) and while there was no difference in chondrogenesis and adipogenesis, ACDC iMSCs underwent osteogenesis sooner than control iPSC, have higher activity of tissue-nonspecific alkaline phosphatase (TNAP), and lower levels of extracellular adenosine. During osteogenic simulation, TNAP activity in ACDC cells significantly increased adenosine levels, however, not to levels needed for functional compensatory stimulation of the adenosine receptors. Inhibition of TNAP with levimisole ablates this increase in adenosine. Treatment with an A2b adenosine receptor (AR) agonist drastically reduced TNAP activity in vitro, and calcification in ACDC teratomas, as did treatment with etidronate, which is currently being tested in a clinical trial on ACDC patients. Conclusions: These results illustrate a pro-osteogenic phenotype in CD73-deficient cells whereby TNAP activity attempts to compensate for CD73 deficiency, but subsequently induces calcification that can be reversed by activation of the A2bAR. The iPSC teratoma model may be used to screen other potential therapeutics for calcification disorders.

scholarly journals AAV9-mediated delivery of miR-23a reduces disease severity in Smn2B/−SMA model mice

2019 ◽  
Vol 28 (19) ◽  
pp. 3199-3210 ◽  
Author(s):  
Kevin A Kaifer ◽  
Eric Villalón ◽  
Benjamin S O'Brien ◽  
Samantha L Sison ◽  
Caley E Smith ◽  
...  
Keyword(s):  
Motor Neuron ◽  
Motor Neurons ◽  
Molecular Mechanisms ◽  
Viral Vector ◽  
Muscular Atrophy ◽  
Induced Pluripotent Stem Cell ◽  
Survival Motor Neuron ◽  
Virus Serotype

Abstract Spinal muscular atrophy (SMA) is a neuromuscular disease caused by deletions or mutations in survival motor neuron 1 (SMN1). The molecular mechanisms underlying motor neuron degeneration in SMA remain elusive, as global cellular dysfunction obscures the identification and characterization of disease-relevant pathways and potential therapeutic targets. Recent reports have implicated microRNA (miRNA) dysregulation as a potential contributor to the pathological mechanism in SMA. To characterize miRNAs that are differentially regulated in SMA, we profiled miRNA levels in SMA induced pluripotent stem cell (iPSC)-derived motor neurons. From this array, miR-23a downregulation was identified selectively in SMA motor neurons, consistent with previous reports where miR-23a functioned in neuroprotective and muscle atrophy-antagonizing roles. Reintroduction of miR-23a expression in SMA patient iPSC-derived motor neurons protected against degeneration, suggesting a potential miR-23a-specific disease-modifying effect. To assess this activity in vivo, miR-23a was expressed using a self-complementary adeno-associated virus serotype 9 (scAAV9) viral vector in the Smn2B/− SMA mouse model. scAAV9-miR-23a significantly reduced the pathology in SMA mice, including increased motor neuron size, reduced neuromuscular junction pathology, increased muscle fiber area, and extended survival. These experiments demonstrate that miR-23a is a novel protective modifier of SMA, warranting further characterization of miRNA dysfunction in SMA.

scholarly journals Application of Patient-Specific iPSCs for Modelling and Treatment of X-Linked Cardiomyopathies

2021 ◽  
Vol 22 (15) ◽  
pp. 8132
Author(s):  
Jennifer Zhang ◽  
Oscar Hou-In Chou ◽  
Yiu-Lam Tse ◽  
Kwong-Man Ng ◽  
Hung-Fat Tse
Keyword(s):  
Drug Testing ◽  
Control Cell ◽  
Induced Pluripotent Stem Cell ◽  
Patient Specific ◽  
Mortality And Morbidity ◽  
Danon Disease ◽  
Disease Phenotypes ◽  
Induced Pluripotent ◽  
And Control

Inherited cardiomyopathies are among the major causes of heart failure and associated with significant mortality and morbidity. Currently, over 70 genes have been linked to the etiology of various forms of cardiomyopathy, some of which are X-linked. Due to the lack of appropriate cell and animal models, it has been difficult to model these X-linked cardiomyopathies. With the advancement of induced pluripotent stem cell (iPSC) technology, the ability to generate iPSC lines from patients with X-linked cardiomyopathy has facilitated in vitro modelling and drug testing for the condition. Nonetheless, due to the mosaicism of the X-chromosome inactivation, disease phenotypes of X-linked cardiomyopathy in heterozygous females are also usually more heterogeneous, with a broad spectrum of presentation. Recent advancements in iPSC procedures have enabled the isolation of cells with different lyonisation to generate isogenic disease and control cell lines. In this review, we will summarise the current strategies and examples of using an iPSC-based model to study different types of X-linked cardiomyopathy. The potential application of isogenic iPSC lines derived from a female patient with heterozygous Danon disease and drug screening will be demonstrated by our preliminary data. The limitations of an iPSC-derived cardiomyocyte-based platform will also be addressed.

scholarly journals Mutation-specific differences in arrhythmias and drug responses in CPVT patients: simultaneous patch clamp and video imaging of iPSC derived cardiomyocytes

2019 ◽  
Vol 47 (2) ◽  
pp. 1067-1077 ◽  
Author(s):  
R. P. Pölönen ◽  
H. Swan ◽  
K. Aalto-Setälä
Keyword(s):  
Disease Modeling ◽  
Induced Pluripotent Stem Cell ◽  
Polymorphic Ventricular Tachycardia ◽  
Patient Specific ◽  
Electrophysiological Properties ◽  
Electrophysiological Measurements ◽  
Skin Biopsies ◽  
Induced Pluripotent ◽  
Almost All ◽  
And Control

AbstractCatecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited cardiac disease characterized by arrhythmias under adrenergic stress. Mutations in the cardiac ryanodine receptor (RYR2) are the leading cause for CPVT. We characterized electrophysiological properties of CPVT patient-specific induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying different mutations in RYR2 and evaluated effects of carvedilol and flecainide on action potential (AP) and contractile properties of hiPSC-CMs. iPSC-CMs were generated from skin biopsies of CPVT patients carrying exon 3 deletion (E3D) and L4115F mutation in RYR2. APs and contractile movement were recorded simultaneously from the same hiPSC-CMs. Differences in AP properties of ventricular like CMs were seen in CPVT and control CMs: APD90 of both E3D (n = 20) and L4115F (n = 25) CPVT CMs was shorter than in control CMs (n = 15). E3D-CPVT CMs had shortest AP duration, lowest AP amplitude, upstroke velocity and more depolarized diastolic potential than controls. Adrenaline had positive and carvedilol and flecainide negative chronotropic effect in all hiPSC CMs. CPVT CMs had increased amount of delayed after depolarizations (DADs) and early after depolarizations (EADs) after adrenaline exposure. E3D CPVT CMs had the most DADs, EADs, and tachyarrhythmia. Discordant negatively coupled alternans was seen in L4115F CPVT CMs. Carvedilol cured almost all arrhythmias in L4115F CPVT CMs. Both drugs decreased contraction amplitude in all hiPSC CMs. E3D CPVT CMs have electrophysiological properties, which render them more prone to arrhythmias. iPSC-CMs provide a unique platform for disease modeling and drug screening for CPVT. Combining electrophysiological measurements, we can gain deeper insight into mechanisms of arrhythmias.

Abstract P445: A Combinatorial Approach Comprehensively Improves The Maturation Of Human Induced Pluripotent Stem Cell Derived Atrial Cardiomyocytes

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Olivia T Ly ◽  
Grace Brown ◽  
Hanna Chen ◽  
Liang Hong ◽  
Xinge Wang ◽  
...  
Keyword(s):  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Patient Specific ◽  
Combinatorial Approach ◽  
Atrial Cardiomyocytes ◽  
Myocardial Substrate ◽  
Induced Pluripotent

Introduction: The limited success of pharmacological approaches to atrial fibrillation ( AF ) is due to limitations of in vitro and in vivo models and inaccessibility of human atrial tissue. Patient-specific induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs) are a robust platform to model the heterogeneous myocardial substrate of AF, but their immaturity limits their fidelity. Objective: We hypothesized that a combinatorial approach of biochemical (triiodothyronine [ T3 ], insulin-like growth factor-1 [ IGF-1 ], and dexamethasone; collectively TID ), bioenergetic (fatty acids [ FA ]), and electrical stimulation ( ES ) will enhance electrophysiological ( EP ), structural, and metabolic maturity of iPSC- a CMs. Methods: We assessed maturation with whole cell patch clamping, calcium transients, immunofluorescence (IF), Seahorse Analyzer, contractility assay, RT-PCR, Western Blotting, and RNA sequencing (RNAseq). Using a time series with RNAseq we identified signaling pathways and transcriptional regulation that drive EP, structural, and metabolic atrial development and compared iPSC-aCM maturity with human aCMs (haCMs) obtained from the same patient. Results: TID+FA+ES significantly improved structural organization and cell morphology ( Fig. 1a ), enhanced membrane potential stability and improved depolarization ( Fig. 1b ), improved Ca 2+ kinetics with faster and increased Ca 2+ release from sarcoplasmic reticulum ( Fig. 1c ), and increased expression of Na + , Ca 2+ , and K + channels, markers of structural maturity, FA metabolism, and oxidative phosphorylation ( Fig. 1d ). There was no difference in each parameter between TID+FA+ES iPSC-aCMs and haCMs from the same patient. Conclusion: Our optimized, combinatorial TID+FA+ES approach markedly enhanced EP, structural, and metabolic maturity of human iPSC-aCMs, which will be useful for elucidating the genetic basis of AF developing precision drug therapies.

scholarly journals Motor neurons from ALS patients with mutations in C9ORF72 and SOD1 exhibit distinct transcriptional landscapes

2019 ◽  
Vol 28 (16) ◽  
pp. 2799-2810 ◽  
Author(s):  
Ching-On Wong ◽  
Kartik Venkatachalam
Keyword(s):  
Cell Adhesion ◽  
Motor Neurons ◽  
Induced Pluripotent Stem Cell ◽  
Data Sets ◽  
Spinal Motor Neurons ◽  
Expression Of Genes ◽  
Sporadic Als ◽  
Elevated Expression ◽  
Induced Pluripotent ◽  
Als Patients

Abstract Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease that culminates in paralysis and death. Here, we present our analyses of publicly available multiOMIC data sets generated using motor neurons from ALS patients and control cohorts. Functional annotation of differentially expressed genes in induced pluripotent stem cell (iPSC)-derived motor neurons generated from patients with mutations in C9ORF72 (C9-ALS) suggests elevated expression of genes that pertain to extracellular matrix (ECM) and cell adhesion, inflammation and TGFβ targets. On the other end of the continuum, we detected diminished expression of genes repressed by quiescence-promoting E2F4/DREAM complex. Proteins whose abundance was significantly altered in C9-ALS neurons faithfully recapitulated the transcriptional aberrations. Importantly, patterns of gene expression in spinal motor neurons dissected from C9-ALS or sporadic ALS patients were highly concordant with each other and with the C9-ALS iPSC neurons. In contrast, motor neurons from patients with mutations in SOD1 exhibited dramatically different signatures. Elevated expression of gene sets such as ECM and cell adhesion genes occurs in C9 and sporadic ALS but not SOD1-ALS. These analyses indicate that despite the similarities in outward manifestations, transcriptional and proteomic signatures in ALS motor neurons can vary significantly depending on the identity of the causal mutations.

scholarly journals Substantial somatic genomic variation and selection for BCOR mutations in human induced pluripotent stem cells

2021 ◽  
Author(s):  
Foad J Rouhani ◽  
Xueqing Zou ◽  
Petr Danecek ◽  
Tauanne Dias Amarante ◽  
Gene Koh ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Induced Pluripotent Stem Cell ◽  
Specific Model ◽  
Genomic Variation ◽  
Patient Specific ◽  
Sequencing Data ◽  
Induced Pluripotent

SummaryHuman Induced Pluripotent Stem Cells (hiPSC) are an established patient-specific model system where opportunities are emerging for cell-based therapies. We contrast hiPSCs derived from different tissues, skin and blood, in the same individual. We show extensive single-nucleotide mutagenesis in all hiPSC lines, although fibroblast-derived hiPSCs (F-hiPSCs) are particularly heavily mutagenized by ultraviolet(UV)-related damage. We utilize genome sequencing data on 454 F-hiPSCs and 44 blood-derived hiPSCs (B-hiPSCs) to gain further insights. Across 324 whole genome sequenced(WGS) F-hiPSCs derived by the Human Induced Pluripotent Stem Cell Initiative (HipSci), UV-related damage is present in ~72% of cell lines, sometimes causing substantial mutagenesis (range 0.25-15 per Mb). Furthermore, we find remarkable genomic heterogeneity between independent F-hiPSC clones derived from the same reprogramming process in the same donor, due to oligoclonal populations within fibroblasts. Combining WGS and exome-sequencing data of 452 HipSci F-hiPSCs, we identify 272 predicted pathogenic mutations in cancer-related genes, of which 21 genes were hit recurrently three or more times, involving 77 (17%) lines. Notably, 151 of 272 mutations were present in starting fibroblast populations suggesting that more than half of putative driver events in F-hiPSCs were acquired in vivo. In contrast, B-hiPSCs reprogrammed from erythroblasts show lower levels of genome-wide mutations (range 0.28-1.4 per Mb), no UV damage, but a strikingly high prevalence of acquired BCOR mutations of ~57%, indicative of strong selection pressure. All hiPSCs had otherwise stable, diploid genomes on karyotypic pre-screening, highlighting how copy-number-based approaches do not have the required resolution to detect widespread nucleotide mutagenesis. This work strongly suggests that models for cell-based therapies require detailed nucleotide-resolution characterization prior to clinical application.

scholarly journals Human motor units in microfluidic devices are impaired by FUS mutations and improved by HDAC6 inhibition

2020 ◽  
Author(s):  
Katarina Stoklund Dittlau ◽  
Emily N. Krasnow ◽  
Laura Fumagalli ◽  
Tijs Vandoorne ◽  
Pieter Baatsen ◽  
...  
Keyword(s):  
Motor Neuron ◽  
Neurite Outgrowth ◽  
Motor Neurons ◽  
Neuromuscular Junctions ◽  
Induced Pluripotent Stem Cell ◽  
Microfluidic Devices ◽  
Motor Units ◽  
Human Motor ◽  
Induced Pluripotent

AbstractNeuromuscular junctions (NMJs) ensure proper communication between motor neurons and muscle through the release of neurotransmitters. In motor neuron disorders, such as amyotrophic lateral sclerosis (ALS), NMJs degenerate resulting in muscle atrophy, paralysis and respiratory failure. The aim of this study was to establish a versatile and reproducible in vitro model of a human motor unit to study the effect of ALS-causing mutations. Therefore, we generated a co-culture of human induced pluripotent stem cell-derived motor neurons and human primary mesoangioblast-derived myotubes in microfluidic devices. A chemotactic and volumetric gradient facilitated the growth of motor neuron neurites through microgrooves resulting in the interaction with myotubes and the formation of NMJs. We observed that ALS-causing FUS mutations resulted in a reduced neurite outgrowth and in a decreased NMJ number. Interestingly, the selective HDAC6 inhibitor, Tubastatin A, improved the neurite outgrowth and the NMJ morphology of FUS-ALS co-cultures, further prompting HDAC6 inhibition as a potential therapeutic strategy for ALS.

scholarly journals Screening for axon regeneration promoting compounds with human iPSC-derived motor neurons

2021 ◽  
Author(s):  
Tammy Szu-Yu Ho ◽  
J. Tabitha Hees ◽  
Zhuqiu Xu ◽  
Riki Kawaguchi ◽  
Natalia P Biscola ◽  
...  
Keyword(s):  
Nerve Injury ◽  
Motor Neurons ◽  
Large Scale ◽  
Axon Regeneration ◽  
Induced Pluripotent Stem Cell ◽  
Screening Strategy ◽  
Therapeutic Interventions ◽  
Induced Pluripotent ◽  
Human Ipsc

CNS neurons do not regenerate after injury, leading to permanent functional deficits. Although sensory and motor neuron axons do regrow after peripheral nerve injury, functional outcome is limited due to the incomplete and slow regrowth. The lack of human-relevant assays suitable for large-scale drug screens has limited neuro-repair therapy discovery. To address this we developed a phenotypic screening strategy using human induced pluripotent stem cell-derived motor neurons to identify axon-regeneration promoting compounds and targets. The screens involve both re-plating human motor neurons on chondroitin sulfate proteoglycans and measuring regeneration responses to laser axotomy in spot cultures, and from them we identified multiple hits that promote injured axon regrowth. The top hit blebbistatin, a non-muscle myosin II inhibitor, accelerated axon regeneration and functional recovery after sciatic nerve injury in vivo. Human injury in a dish assays are suitable, therefore, to screen for therapeutic interventions that can induce or accelerate axon regeneration.

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