A reference human induced pluripotent stem cell line for large-scale collaborative studies

Cell stem cell, Journal Year: 2022, Volume and Issue: 29(12), P. 1685 - 1702.e22

Published: Dec. 1, 2022

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between makes it challenging to replicate key findings integrate data across research groups. To address this issue, we sub-cloned candidate human iPSC deeply characterized their genetic properties using whole genome sequencing, genomic stability upon CRISPR-Cas9-based gene editing, including differentiation commonly used types. These studies identified KOLF2.1J as an all-around well-performing line. We then shared with groups around world who tested its performance in head-to-head comparisons own preferred diverse range of protocols functional assays. On strength these findings, have made gene-edited derivative clones readily accessible promote standardization required large-scale collaborative science field.

Language: Английский

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Single-Cell Transcriptomics of Parkinson’s Disease Human In Vitro Models Reveals Dopamine Neuron-Specific Stress Responses

Cell Reports, Journal Year: 2020, Volume and Issue: 33(2), P. 108263 - 108263

Published: Oct. 1, 2020

The advent of induced pluripotent stem cell (iPSC)-derived neurons has revolutionized Parkinson's disease (PD) research, but single-cell transcriptomic analysis suggests unresolved cellular heterogeneity within these models. Here, we perform the largest study human iPSC-derived dopaminergic to elucidate gene expression dynamics in response cytotoxic and genetic stressors. We identify multiple neuronal subtypes with transcriptionally distinct profiles differential sensitivity stress, highlighting dopamine vitro validate this model by showing robust PD GWAS genes overlap postmortem adult substantia nigra neurons. Importantly, stress signatures are ameliorated using felodipine, an FDA-approved drug. Using isogenic SNCA-A53T mutants, find perturbations glycolysis, cholesterol metabolism, synaptic signaling, ubiquitin-proteasomal degradation. Overall, our reveals type-specific neurons, which will further understanding have implications for replacement therapies.

Language: Английский

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Modeling Cardiac Disease Mechanisms Using Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Progress, Promises and Challenges

International Journal of Molecular Sciences, Journal Year: 2020, Volume and Issue: 21(12), P. 4354 - 4354

Published: June 19, 2020

Cardiovascular diseases (CVDs) are a class of disorders affecting the heart or blood vessels. Despite progress in clinical research and therapy, CVDs still represent leading cause mortality morbidity worldwide. The hallmarks cardiac include dysfunction cardiomyocyte death, inflammation, fibrosis, scar tissue, hyperplasia, hypertrophy, abnormal ventricular remodeling. loss cardiomyocytes is an irreversible process that leads to fibrosis formation, which, turn, induce failure with progressive dramatic consequences. Both genetic environmental factors pathologically contribute development CVDs, but precise causes trigger their progression largely unknown. lack reliable human model systems for such has hampered unraveling underlying molecular mechanisms cellular processes involved at initial stage during progression. Over past decade, significant scientific advances field stem cell biology have literally revolutionized study disease vitro. Remarkably, possibility generate disease-relevant types from induced pluripotent cells (iPSCs) developed into unprecedented powerful opportunity achieve long-standing ambition investigate level, uncovering mechanisms, finally translate bench discoveries potential new therapeutic strategies. This review provides update on previous current iPSC-driven cardiovascular modeling, aim underlining stem-cell biology-based approaches elucidation pathophysiology these life-threatening diseases.

Language: Английский

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SORL1 deficiency in human excitatory neurons causes APP-dependent defects in the endolysosome-autophagy network

Cell Reports, Journal Year: 2021, Volume and Issue: 35(11), P. 109259 - 109259

Published: June 1, 2021

Dysfunction of the endolysosomal-autophagy network is emerging as an important pathogenic process in Alzheimer's disease. Mutations sorting receptor-encoding gene SORL1 cause autosomal-dominant disease, and variants increase risk for late-onset AD. To understand contribution mutations to AD pathogenesis, we analyze effects a truncating mutation on protein levels endolysosome function human neurons. We find that results haploinsufficiency enlarged endosomes Analysis isogenic wild-type, heterozygous, homozygous null neurons demonstrates that, whereas endosome dysfunction, complete loss leads additional defects lysosome autophagy. Neuronal endolysosomal dysfunction caused by relieved extracellular antisense oligonucleotide-mediated reduction APP protein, demonstrating PSEN1, APP, act common pathway regulating system, which becomes dysfunctional

Language: Английский

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Pioneer factor ASCL1 cooperates with the mSWI/SNF complex at distal regulatory elements to regulate human neural differentiation

Genes & Development, Journal Year: 2023, Volume and Issue: 37(5-6), P. 218 - 242

Published: March 1, 2023

Pioneer transcription factors are thought to play pivotal roles in developmental processes by binding nucleosomal DNA activate gene expression, though mechanisms through which pioneer remodel chromatin remain unclear. Here, using single-cell transcriptomics, we show that endogenous expression of neurogenic factor ASCL1, considered a classical factor, defines transient population progenitors human neural differentiation. Testing ASCL1's function knockout model define the unbound state, found ASCL1 drives progenitor differentiation

Language: Английский

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Dynein and dynactin move long-range but are delivered separately to the axon tip

The Journal of Cell Biology, Journal Year: 2024, Volume and Issue: 223(5)

Published: Feb. 6, 2024

Axonal transport is essential for neuronal survival. This driven by microtubule motors including dynein, which transports cargo from the axon tip back to cell body. function requires its cofactor dynactin and regulators LIS1 NDEL1. Due difficulties imaging dynein at a single-molecule level, it unclear how this motor coordinate along length of axon. Here, we use neuron-inducible human stem line (NGN2-OPTi-OX) endogenously tag components visualize them near-single molecule regime. In retrograde direction, find that can move entire (>500 µm). Furthermore, NDEL1 also undergo long-distance movement, despite being mainly implicated with initiation transport. Intriguingly, in anterograde dynein/LIS1 moves faster than dynactin/NDEL1, consistent on different cargos. Therefore, neurons ensure efficient holding dynein/dynactin cargos over long distances but keeping separate until required.

Language: Английский

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Proximity proteomics reveals UCH-L1 as an essential regulator of NLRP3-mediated IL-1β production in human macrophages and microglia

Cell Reports, Journal Year: 2024, Volume and Issue: 43(5), P. 114152 - 114152

Published: April 25, 2024

Activation of the NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome complex is an essential innate immune signaling mechanism. To reveal how human NLRP3 assembly activation are controlled, in particular by components ubiquitin system, proximity labeling, affinity purification, RNAi screening approaches were performed. Our study provides intricate time-resolved molecular map different phases activation. Also, we show that C-terminal hydrolase 1 (UCH-L1) interacts with NACHT domain NLRP3. Downregulation UCH-L1 decreases pro-interleukin-1β (IL-1β) levels. chemical inhibition small molecules interfered puncta formation ASC oligomerization, leading to altered IL-1β cleavage secretion, particularly microglia cells, which exhibited elevated expression as compared monocytes/macrophages. Altogether, profiled dynamics highlight important modulator NLRP3-mediated production, suggesting a pharmacological inhibitor may decrease inflammation-associated pathologies.

Language: Английский

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A functional genetic toolbox for human tissue-derived organoids

eLife, Journal Year: 2021, Volume and Issue: 10

Published: Oct. 6, 2021

Human organoid systems recapitulate key features of organs offering platforms for modelling developmental biology and disease. Tissue-derived organoids have been widely used to study the impact extrinsic niche factors on stem cells. However, they are rarely endogenous gene function due lack efficient manipulation tools. Previously, we established a human foetal lung system (Nikolić et al., 2017). Here, using this as an example, systematically developed optimised complete genetic toolbox use in tissue-derived organoids. This includes 'Organoid Easytag', our workflow targeting all types loci through CRISPR-mediated homologous recombination followed by flow cytometry enriching correctly targeted Our also incorporates conditional knockdown or overexpression tightly inducible CRISPR interference activation which is first application these techniques These tools will facilitate perturbation studies facilitating disease providing functional counterpart many ongoing descriptive studies, such Cell Atlas Project.

Language: Английский

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Complement receptor 1 is expressed on brain cells and in the human brain

Glia, Journal Year: 2023, Volume and Issue: 71(6), P. 1522 - 1535

Published: Feb. 24, 2023

Abstract Genome wide association studies (GWAS) have highlighted the importance of complement cascade in pathogenesis Alzheimer's disease (AD). Complement receptor 1 (CR1; CD35) is among top GWAS hits. The long variant CR1 associated with increased risk for AD; however, roles brain health and are poorly understood. A critical confounder that expression controversial; failure to demonstrate has provoked suggestion peripherally expressed influences AD risk. We took a multi‐pronged approach establish whether brain. Expression at protein mRNA level was assessed human microglial lines, induced pluripotent stem cell (iPSC)‐derived microglia from two sources tissue control donors. detected lines iPSC‐derived expressing different variants when immunostained validated panel CR1‐specific antibodies; extracts were positive mRNA. brains, co‐localizing astrocytes microglia, significantly compared controls. all samples tested; AD. data unequivocally transcript ex vivo on situ brain; findings support hypothesis affect by directly impacting glial functions.

Language: Английский

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Loss of CNTNAP2 Alters Human Cortical Excitatory Neuron Differentiation and Neural Network Development

Biological Psychiatry, Journal Year: 2023, Volume and Issue: 94(10), P. 780 - 791

Published: March 29, 2023

Loss-of-function mutations in the contactin-associated protein-like 2 (CNTNAP2) gene are causal for neurodevelopmental disorders, including autism, schizophrenia, epilepsy, and intellectual disability. CNTNAP2 encodes CASPR2, a single-pass transmembrane protein that belongs to neurexin family of cell adhesion molecules. These proteins have variety functions developing neurons, connecting presynaptic postsynaptic mediating signaling across synapse.To study effect loss function on human cerebral cortex development, how this contributes pathogenesis we generated induced pluripotent stem cells from one neurotypical control donor null full-length CNTNAP2, modeling cortical development neurogenesis through neural network formation vitro.CNTNAP2 is particularly highly expressed first two populations early-born excitatory shifted relative proportions these neuronal types. Live imaging growth showed reduced neurite branching overall complexity. At level, networks had complex changes activity compared with isogenic controls: an initial period relatively controls, followed by lengthy hyperexcitability, then further switch activity.Complete disorders several aspects neuron culminate aberrant function.

Language: Английский

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