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1,247 results for “human pluripotent stem cell”
Efficient embryoid-based method to improve generation of optic vesicles from human induced pluripotent stem cells data
<p>Animal models have provided many insights into ocular development and disease, but they remain suboptimal for understanding human oculogenesis. Eye development requires spatiotemporal gene expression patterns and disease phenotypes can differ significantly between humans and animal models, with patient-associated mutations causing embryonic lethality reported in some animal models. The emergence of human induced pluripotent stem cell (hiPSC) technology has provided a new resource for dissecting the complex nature of early eye morphogenesis through the generation of three-dimensional (3D) cellular models. By using patient-specific hiPSCs to generate <em>in vitro </em>optic vesicle-like models, we can enhance the understanding of early developmental eye disorders and provide a pre-clinical platform for disease modelling and therapeutics testing. A major challenge of <em>in vitro </em>optic vesicle generation is the low efficiency of differentiation in 3D cultures. To address this, we adapted a previously published protocol of retinal organoid differentiation to improve embryoid body formation using a microwell plate. Established morphology, upregulated transcript levels of known early eye-field transcription factors and protein expression of standard retinal progenitor markers confirmed the optic vesicle/presumptive optic cup identity of <em>in vitro </em>models between day 20 and 50 of culture. This adapted protocol is relevant to researchers seeking a physiologically relevant model of early human ocular development and disease with a view to replacing animal models.</p>
Generation of beta-like cell subtypes from differentiated human induced pluripotent stem cells in 3D spheroids
<p>This repository contains single-cell RNA-sequencing data files (raw FASTQ files generated from Illumina HiSeq sequencing) related to the article entitled "Generation of beta-like cell subtypes from differentiated human induced pluripotent stem cells in 3D spheroids" by Lisa Morisseau et al. (2023) published in the Molecular Omics journal (DOI: 10.1039/d3mo00050h).</p> <p> </p>
Underlying and Extended Data for Refined and benchmarked homemade media for cost-effective, weekend-free human pluripotent stem cell culture
<p>Extended Data and raw data for the manuscript "Refined home-brew media for cost-effective, weekend-free hiPSC culture and genetic engineering"</p> <p> </p> <p dir="ltr">Extended Data 1.zip - protocol for preparation of the supplement for hE8 and B8+ media</p> <p dir="ltr">Extended Data 2.zip - Gene counts, supporting files, and output results of bulk analyses</p> <p dir="ltr">Extended Data 3.zip - Images of iPS cells adapted to cE8, hE8 and B8+ taken 24, 48 and 72 hours after passage. Contains raw .tiff files for each image and a .pdf with a compiled figure</p> <p dir="ltr">Extended Data 4.zip - Results of miloR analysis on the differences in the distribution of cells adapted to cE8, hE8 and B8+ to assigned monocle clusters </p> <p dir="ltr">Manuscript Data.zip – Raw data underlying the Figures 1, 2, 4, 5, 6 and 7.</p> <p>Bulk_RNA_seq_archive – archived source code used for generating results in Figure 3</p>
GC-MS raw data_Figure 6E_Lysophosphatidic Acid Shifts Metabolic and Transcriptional Landscapes to Induce a Distinct Cellular State in Human Pluripotent Stem Cells
<p><strong>Sample name</strong></p> <p>hESCs (H1 cells) were given treatments for two days and then collected for GC-MS analysis.</p> <p>E8: E8 medium</p> <p>AX: E8 + 1.6% AlbuMAX;</p> <p>BSA: E8 + 1% albumin;</p> <p>BSA+hCDL: E8 + 1% albumin + 0.1% hCDL;</p> <p>LPA+BSA: E8 + 1 μM LPA + 1% albumin;</p> <p>LPA+BSA+hCDL: E8 + 1 μM LPA + 1% albumin + 0.1% hCDL</p> <p>STD: standard lipids mixture used as reference</p> <p><strong>Extraction and Methylation</strong></p> <p>Sample preparation was conducted according to the previously reported method (Araujo et al., 2008) with the modification. Briefly, spent medium was removed, and cells were rinsed with 1 mL/well 0.9% (w/v) saline twice. Then 0.5 mL/well -80°C 80% methanol was added to quench the metabolism. Five wells of cells (from 6-well plate) were scrapped off into a glass screw-cap tube. Then 4 mL heptadecanoate containing chloroform (4 μg/mL, internal standard for fatty acids) was added into the tube. Vortex, and then centrifuge at 2000 rpm for 5 min. Cellular debris was carefully removed, and nitrogen blow the solution till dry. Add 1.5 mL hexane and 1.5 mL 14% boron trifluoride (BF<sub>3</sub>)/methanol solution. Seal the tube with nitrogen gas, heat it at 100°C for 1 h using MK200-2 dry bath incubator (Aosheng), and then cool down to room temperature. Add 1 mL water into the tube, vortex and then centrifuge at 3000 rpm for 10 min. The upper layer was transferred into a new 1.5-mL eppendorf tube and evaporated by nitrogen gas. The residue was re-dissolved in 100 μL hexane for GC-MS analysis.</p> <p><strong>GC-MS method</strong></p> <p>Samples were analyzed using an Agilent GC-MS system (Agilent) consisting of a 6890 gas chromatography and a 5973 mass spectrometer. Fatty acid methyl esters were separated by an Omegawax™ 250 fused silica capillary column (30 m × 0.25 mm i.d., 0.25 μm film thickness, Supelco, Bellefonte, PA). The optimized oven temperature program was: initial temperature set at 180°C and held for 3 min; ramped to 206°C at 2°C/min and held at 206°C for 25 min, then, ramped to 240°C at 10°C/min and held for 5 min. Overall, the total run time was 50 min. Carrier gas was high-purity helium at a flow rate of 1.5 mL/min. Injector temperature was set at 250°C. Injection volume was 2 μL with a split ratio of 1:15. The mass spectrometer was operated in electron-impact (EI) mode at 70 eV ionization energy. The temperatures of quadrupole and ionization source were set at 150°C and 280°C, respectively. The spectra from 3 to 50 min were acquired with the <em>m/z</em> range of 35–550 at a scan rate of 0.34 s per scan.</p>
LC-MS raw data_Lysophosphatidic Acid Shifts Metabolic and Transcriptional Landscapes to Induce a Distinct Cellular State in Human Pluripotent Stem Cells
<p><strong>LC-MS/MS analysis</strong></p> <p><strong>Metabolite extraction</strong></p> <p>For LC-MS/MS quantification, cell sample preparation was conducted as described in the previous literatures (Ying, Kimmelman et al. 2012, Zhang, Badur et al. 2016). Briefly, the spent medium was removed, and cells were rinsed with 1 mL/well 0.9% (w/v) saline twice. Then 0.5 mL/well -80°C 0.2 μg/mL norvaline containing 80% methanol was added to quench the metabolism. Cells were scraped off into 1.5-mL eppendorf tube and stored in -80℃ overnight. The mixtures were vortexed and then centrifuged 12500 × <em>g </em>for 15 min at 4℃. The supernatant was used for LC-MS analysis.</p> <p><strong>LC-MS/MS method</strong></p> <p>Waters Xevo TQD coupled with Waters Acquity UPLC system was used for quantification. Acquity UPLC BEH HILIC column (2.1 × 100 mm, 1.7 μm), Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm), and Acquity UPLC BEH amide column (2.1 × 100 mm, 1.7 μm) were used for the separation of metabolites. Column temperature was set at 40 °C.</p> <p>For the quantification of norvaline, amino acids, GSH, GSSG, SAH, SAM, ascorbic acid and myo-inositol, amide column was used for the separation. Acetonitrile with 0.1% formic acid (A) and water with 0.1% formic acid (B) were used as mobile phases. The gradient setting is: 0-4 min, 99% A to 90% A; 4-10 min, 90% A to 67% A; 10-13 min, 67% A to 1% A; 13-15 min, 1% A; 15-16.5 min, 1% A to 99% A; 16.5-20 min, 99% A. Flowrate was set as 0.4 mL/min.</p> <p>For the quantification of metabolites involved in TCA cycle, energy related and ribonucleotides, an amide column was used for the separation. Acetonitrile with 0.1% formic acid (A) and water with 0.1% formic acid (B) were used as mobile phases. The gradient setting is: 0-2 min, 80% A; 2-3 min, 80% A to 20% A; 3-5 min, 20% A; 5-6 min, 20% A to 80% A; 6-10 min, 80% A. Flowrate was set as 0.4 mL/min.</p> <p>For the quantification of acetate, acetyl-CoA and metabolites involved in glycolysis and pentose phosphate pathway, HILIC column was used for the separation. Acetonitrile (A) and 10 mM ammonium bicarbonate were used as mobile phases. The gradient setting is: 0-2 min, 10% A; 2-5 min, 10% A to 5% A; 5-6 min, 5% A to 10% A; 6-10 min, 10% A. Flowrate was set as 0.2 mL/min.</p> <p>For the quantification of LPA, LPC and PC, HILIC column was used for the separation. Acetonitrile (A) and 10 mM ammonium bicarbonate aqueous solution (B) were used as mobile phases. The gradient setting is: 0-2 min, 95% A; 2-4 min, 95% A to 10% A; 4-7 min, 10% A; 7-9 min, 10% A to 95% A; 9-15 min, 95% A. Flowrate was set as 0.2 mL/min.</p> <p>For the quantification of CDL lipids, C18 column was used for the separation. 98% Acetonitrile aqueous solution (A) and 10 mM ammonium acetate 90% acetonitrile aqueous solution (B) were used as mobile phases. The gradient setting is: 0-5 min, 0.1% A; 5-6 min, 0.1% A to 99.9% A; 6-11 min, 99.9% A; 11-12 min, 99.9% A to 0.1% A; 12-15 min, 0.1% A. Flowrate was set as 0.4 mL/min.</p> <p>Argon was used as source gas, capillary voltage was 3500 V, and desolvation temperature was 500 °C. Multiple reaction monitoring (MRM) was conducted, and the ion transitions are listed in the supplemental Table S2. Selected ion recording (SIR) was conducted for the detection of CDL-related lipids, and the setting is listed in the supplemental Table S3.</p> <p>Standard solutions of TCA metabolites (100 μg/mL) and intermediates of glycolysis and pentose phosphate pathway (10 μg/mL) were prepared to confirm the retention time. Peak intensity of product ion was used for the quantification. Data analysis was performed by TargetLynx software (Waters) with statistical analysis in Graphpad Prism (version 8.4.0) and R.</p>
2D trajectories of human pluripotent stem cell colonies
<p>This dataset is a part of the following manuscript submitted for publication in Life (MDPI):</p> <p>"Human Pluripotent Stem Cell Colony Migration is Related to Culture Environment and Morphological Phenotype"<br>by Vitaly V. Gursky, Alina S. Chabina, Olga A. Krasnova, Anastasiya A. Kovaleva, Daria V. Kriger, Michail S. Zadorsky, Konstantin N. Kozlov, and Irina E. Neganova</p> <p>A trajectory in the csv file is an array of the 2D coordinates {{x1,y1}, {x2,y2}, ...} (in µm; centered at {x,y}={0,0}), which represent the migration history of the center of one cell colony. Trajectories were obtained from the time-lapse bright-field images, using the Manual Tracking tool in ImageJ (Fiji). The data were collected for colonies from three human pluripotent stem cell lines (human induced pluripotent stem cell line AD3, patient specific human induced pluripotent stem cell line HPCASRi002-A (CaSR), and human embryonic stem cell line H9), grown under two culture conditions (media/matrix = mTESR1/MG or E8/GT). The colonies were phenotyped according to their morphological characteristics associated with the pluripotency status ("good" and "bad" phenotype); cells with the "bad" phenotype showed signs of incipient differentiation. The time interval between adjacent coordinates is 15 min.</p> <p>The data were obtained under the financial supprot of the Russian Science Foundation, grant number 21-75-20132.</p>
RNA-seq - Definitive endoderm differentiation of human pluripotent stem cells in G1 phase
<p>Backup copy of the processed RNA-seq data at http://ngs.sanger.ac.uk/production/endoderm/</p>
Transcriptomic characterization of 2D and 3D human induced pluripotent stem cell-based in vitro models as New Approach Methodologies for developmental neurotoxicity testing
<p><strong>Abstract:</strong> The safety and developmental neurotoxicity (DNT) potential of chemicals remain critically understudied due to limitations of current in vivo testing guidelines, which are low throughput, resource-intensive, and hindered by species differences that limit their relevance to human health. To address these issues, robust new approach methodologies (NAMs) using deeply characterized cell models are essential. This study presents the comprehensive transcriptomic characterization of two advanced human-induced pluripotent stem cell (hiPSC)-derived models: a 2D adherent and a 3D neurosphere model of human neural progenitor cells (hiNPCs) differentiated up to 21 days. Using high-throughput RNA sequencing, we compared gene expression profiles of 2D and 3D models at three developmental stages (3, 14, and 21 days of differentiation). Both models exhibit maturation towards post-mitotic neurons, with the 3D model maturing faster and showing a higher prevalence of GABAergic neurons, while the 2D model is enriched with glutamatergic neurons. Both models demonstrate broad applicability domains, including excitatory and inhibitory neurons, astrocytes, and key endocrine and especially the understudied cholinergic receptors. Comparison with human fetal brain samples confirms their physiological relevance. This study provides novel in-depth applicability insights into the temporal and dimensional aspects of hiPSC-derived neural models for DNT testing. The complementary use of these two models is highlighted: the 2D model excels in synaptogenesis assessment, while the 3D model is particularly suited for neural network formation as observed as well in previous functional studies with these models. This research marks a significant advancement in developing human-relevant, high-throughput DNT assays for regulatory purposes.</p> <p><strong>This data sets contains:</strong></p> <p><strong>Tab. S1</strong> - Significant genes results</p> <p><strong>Tab. S2</strong> - Enriched pathways_GO_Biological Processes</p> <p><strong>Tab. S3</strong> - Enriched pathways_GO_Cellular Components</p> <p><strong>Tab. S4</strong> - Enriched pathways_GO_Molecular Function</p> <p><strong>Tab. S5</strong> - Enriched pathways_KEGG</p> <p><strong>Tab. S6</strong> - EnrichEnriched pathways_Panther</p> <p><strong>Tab. S7</strong> - Enriched pathways_Reactome</p> <p><strong>Tab. S8</strong> - Gene counts</p> <p><strong>Tab. S9</strong> - Gene selection for targeted analysis</p>
Differentiation of parathyroid hormone expressing cells from human pluripotent stem cells
Abstract Differentiation of pluripotent stem cells into functional parathyroid-like cells would accelerate development of important therapeutic options for subjects with parathyroid-related disorders, from the design and screening of novel pharmaceutical agents to the development of durable cellular therapies. We have established a highly reproducible directed differentiation approach leading to parathyroid hormone (PTH) expressing cells from human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC). We accomplished this through the comparison of multiple different basal media, the inclusion of the CDK inhibitor PD0332991 in both definitive endoderm (DE) and anterior foregut endoderm (AFE) stages, and a 2-stage pharyngeal endoderm (PE) series. This is the first protocol to reproducibly establish PTH expressing-cells from human pluripotent stem cells and represents a first step towards the development of functional parathyroid cells with broad applicability for medicinal and scientific investigation.
A targeted metabolomics-based assay using human induced pluripotent stem cell-derived cardiomyocytes identifies structural and functional cardiotoxicity potential
<p>Implementing screening assays that identify functional and structural cardiotoxicity earlier in the drug development pipeline has the potential to improve safety and the cost and time required to bring new drugs to market. In this study, a metabolic biomarker-based assay was developed that predicts the cardiotoxicity potential of a drug based on changes in the metabolism and viability of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). Assay development and testing was conducted in two phases: (1) biomarker identification and (2) targeted assay development. In the first phase, metabolomic data from hiPSC-CM spent media following exposure to 66 drugs was used to identify biomarkers that identified both functional and structural cardiotoxicants. Four metabolites that represent different metabolic pathways (arachidonic acid, lactic acid, 2'-deoxycytidine, and thymidine) were identified as indicators of cardiotoxicity. In phase two, a targeted, exposure-based biomarker assay was developed that measured these metabolites and hiPSC-CM viability across an eight-point concentration curve. Metabolite-specific predictive thresholds for identifying the cardiotoxicity potential of a drug were established and optimized for balanced accuracy or sensitivity. When predictive thresholds were optimized for balanced accuracy, the assay predicted the cardiotoxicity potential of 81 drugs with 86% balanced accuracy, 83% sensitivity, and 90% specificity. Alternatively, optimizing the thresholds for sensitivity yields a balanced accuracy of 85%, 90% sensitivity, and 79% specificity. This new hiPSC-CM-based assay provides a paradigm that can identify structural and functional cardiotoxic drugs that could be used in conjunction with other endpoints to provide a more comprehensive evaluation of a drug's cardiotoxicity potential.</p>
In situ monitoring reveals cellular environmental instabilities in human pluripotent stem cell culture
<p>Mammalian cell cultures are a keystone resource in biomedical research, but the results of published experiments often suffer from reproducibility challenges. This has led to a focus on the influence of cell culture conditions on cellular responses and reproducibility of experimental findings. Here, we perform frequent in situ monitoring of dissolved O<sub>2</sub> and CO<sub>2</sub> with optical sensor spots and contemporaneous evaluation of cell proliferation and medium pH in standard batch cultures of three widely used human somatic and pluripotent stem cell lines. We collate data from the literature to demonstrate that standard cell cultures consistently exhibit environmental instability, indicating that this may be a pervasive issue affecting experimental findings. Our results show that <i>in vitro</i> cell cultures consistently undergo large departures of environmental parameters during standard batch culture. These findings should catalyze further efforts to increase the relevance of experimental results to the in vivo physiology and enhance reproducibility.</p>
Dataset with values of morphological parameters and phenotypes of cells and colonies from three human pluripotent stem cell lines
<p>The dataset is a part of the following manuscript submitted for publication in International Journal of Molecular Sciences (MDPI):</p> <p>"Prognostic Analysis of Human Pluripotent Stem Cells Based on their Morphological Portrait and Expression of Pluripotent Markers" by Olga A. Krasnova, Vitaly V. Gursky, Alina S. Chabina, K. A. Kulakova, L. L. Alekseenko, Alexandra V. Panova, Sergey L. Kiselev and Irina E. Neganova</p> <p>The files contain values of several morphological parameters and phenotypes obtained for cells and colonies from hESC line H9, hiPSC line AD3, and hiPSC line CaSR. The phenotypic information is presented in three forms: four possible classes of colonies according to the visually assessed phenotype ('bad', 'average', 'good', and 'excellent'), three possible classes (previous 'good' and 'excellent' combined in one 'good' class), and two possible classes (previous 'bad' and 'average' combined in one 'bad' class, and previous 'good' and 'excellent' combined in one 'good' class).</p>
ATAC-seq - Definitive endoderm differentiation of human pluripotent stem cells in G1 phase
<p>Backup copy of the processed ATAC-seq data at http://ngs.sanger.ac.uk/production/endoderm/</p>
Generation of human induced pluripotent stem cells-derived cortical neurons for high throughput imaging of neurite morphology and neuron maturation
<p>Figure 5: Whole cell patch clamping showed the differentiated neurons are functional.</p>
Generation of human induced pluripotent stem cells-derived cortical neurons for high throughput imaging of neurite morphology and neuron maturation
<p>Figure 3 - Neurite outgrowth dataset. Comparing neurite outgrowth at Day1 and 15 post-seeding cortical neural progenitors. </p>
Safety and Efficacy of Induced Pluripotent Stem Cell-derived Engineered Human Myocardium as Biological Ventricular Assist Tissue in Terminal Heart Failure
ClinicalTrials.gov study NCT04396899. IPD Sharing: Not stated. Countries: 1. Publications: 0.
Generation of Cancer Antigen-Specific T-cells From Human Induced Pluripotent Stem Cells (iPSC) for Research and Potential FutureTherapy
ClinicalTrials.gov study NCT03407040. IPD Sharing: Not stated. Countries: 1. Publications: 3.
Derivation of Induced Pluripotent Stem Cells From an Existing Collection of Human Somatic Cells
ClinicalTrials.gov study NCT00801333. IPD Sharing: Not stated. Countries: 1. Publications: 1.
In situ monitoring reveals cellular environmental instabilities in human pluripotent stem cell culture
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Differentiation of parathyroid hormone expressing cells from human pluripotent stem cells
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