scholarly journals A fully automated FAIMS-DIA proteomic pipeline for high-throughput characterization of iPSC-derived neurons

2021 ◽  
Author(s):  
Luke Reilly ◽  
Lirong Peng ◽  
Erika Lara ◽  
Daniel Ramos ◽  
Michael Fernandopulle ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Induced Pluripotent Stem Cell ◽  
Steady Increase ◽  
Parental Line ◽  
Proteomic Profiling ◽  
Data Independent Acquisition ◽  
Biological Insight ◽  
Data Browsing ◽  
Induced Pluripotent

Fully automated proteomic pipelines have the potential to achieve deep coverage of cellular proteomes with high throughput and scalability. However, it is important to evaluate performance, including both reproducibility and ability to provide meaningful levels of biological insight. Here, we present an approach combining high field asymmetric waveform ion mobility spectrometer (FAIMS) interface and data independent acquisition (DIA) proteomics approach developed as part of the induced pluripotent stem cell (iPSC) Neurodegenerative Disease Initiative (iNDI), a large-scale effort to understand how inherited diseases may manifest in neuronal cells. Our FAIMS-DIA approach identified more than 8000 proteins per mass spectrometry (MS) acquisition as well as superior total identification, reproducibility, and accuracy compared to other existing DIA methods. Next, we applied this approach to perform a longitudinal proteomic profiling of the differentiation of iPSC-derived neurons from the KOLF2.1J parental line used in iNDI. This analysis demonstrated a steady increase in expression of mature cortical neuron markers over the course of neuron differentiation. We validated the performance of our proteomics pipeline by comparing it to single cell RNA-Seq datasets obtained in parallel, confirming expression of key markers and cell type annotations. An interactive webapp of this temporal data is available for aligned-UMAP visualization and data browsing (https://share.streamlit.io/anant-droid/singlecellumap). In summary, we report an extensively optimized and validated proteomic pipeline that will be suitable for large-scale studies such as iNDI.

scholarly journals Proteomic profiling dataset of chemical perturbations in multiple biological backgrounds

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Deborah O. Dele-Oni ◽  
Karen E. Christianson ◽  
Shawn B. Egri ◽  
Alvaro Sebastian Vaca Jacome ◽  
Katherine C. DeRuff ◽  
...  
Keyword(s):  
Large Scale ◽  
Cell Model ◽  
Cellular Responses ◽  
Proteomic Profiling ◽  
Reduced Representation ◽  
Link Type ◽  
Original Dataset ◽  
Quality Control Metrics ◽  
Biological Insight ◽  
Chromatin Profiling

AbstractWhile gene expression profiling has traditionally been the method of choice for large-scale perturbational profiling studies, proteomics has emerged as an effective tool in this context for directly monitoring cellular responses to perturbations. We previously reported a pilot library containing 3400 profiles of multiple perturbations across diverse cellular backgrounds in the reduced-representation phosphoproteome (P100) and chromatin space (Global Chromatin Profiling, GCP). Here, we expand our original dataset to include profiles from a new set of cardiotoxic compounds and from astrocytes, an additional neural cell model, totaling 5300 proteomic signatures. We describe filtering criteria and quality control metrics used to assess and validate the technical quality and reproducibility of our data. To demonstrate the power of the library, we present two case studies where data is queried using the concept of “connectivity” to obtain biological insight. All data presented in this study have been deposited to the ProteomeXchange Consortium with identifiers PXD017458 (P100) and PXD017459 (GCP) and can be queried at https://clue.io/proteomics.

High-throughput screening of human induced pluripotent stem cell-derived brain organoids

2020 ◽  
Vol 335 ◽  
pp. 108627 ◽  
Author(s):  
Madel Durens ◽  
Jonathan Nestor ◽  
Madeline Williams ◽  
Kevin Herold ◽  
Robert F. Niescier ◽  
...  
Keyword(s):  
Stem Cell ◽  
High Throughput ◽  
High Throughput Screening ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Induced Pluripotent

scholarly journals CNTN5−/+orEHMT2−/+iPSC-Derived Neurons from Individuals with Autism Develop Hyperactive Neuronal Networks

2018 ◽  
Author(s):  
Eric Deneault ◽  
Muhammad Faheem ◽  
Sean H. White ◽  
Deivid C. Rodrigues ◽  
Song Sun ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Large Scale ◽  
De Novo ◽  
Electrode Array ◽  
Induced Pluripotent Stem Cell ◽  
Autism Spectrum ◽  
High Throughput Phenotyping ◽  
Family Based ◽  
Induced Pluripotent ◽  
Relationship Of

AbstractInduced pluripotent stem cell (iPSC)-derived cortical neurons are increasingly used as a model to study developmental aspects of Autism Spectrum Disorder (ASD), which is clinically and genetically heterogeneous. To study the complex relationship of rare (penetrant) variant(s) and common (weaker) polygenic risk variant(s) to ASD, “isogenic” iPSC-derived neurons from probands and family-based controls, for modeling, is critical. We developed a standardized set of procedures, designed to control for heterogeneity in reprogramming and differentiation, and generated 53 different iPSC-derived glutamatergic neuronal lines from 25 participants from 12 unrelated families with ASD (14 ASD-affected individuals, 3 unaffected siblings, 8 unaffected parents). Heterozygousde novo(7 families; 16p11.2,NRXN1,DLGAP2,CAPRIN1,VIP,ANOS1,THRA) and rare-inherited (2 families;CNTN5,AGBL4) presumed-damaging variants were characterized in ASD risk genes/loci. In three additional families, functional candidates for ASD (SET), and combinations of putative etiologic variants (GLI3/KIF21AandEHMT2/UBE2Icombinations in separate families), were modeled. We used a large-scale multi-electrode array (MEA) as our primary high-throughput phenotyping assay, followed by patch clamp recordings. Our most compelling new results revealed a consistent spontaneous network hyperactivity in neurons deficient forCNTN5orEHMT2.Our biobank of iPSC-derived neurons and accompanying genomic data are available to accelerate ASD research.

scholarly journals Chromatin compartment dynamics in a haploinsufficient model of cardiac laminopathy

2019 ◽  
Author(s):  
Alessandro Bertero ◽  
Paul A. Fields ◽  
Alec S. T. Smith ◽  
Andrea Leonard ◽  
Kevin Beussman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Large Scale ◽  
Induced Pluripotent Stem Cell ◽  
Lamin A ◽  
Chromosome Conformation ◽  
Human Cardiomyocytes ◽  
Nuclear Lamins ◽  
Genome Wide ◽  
Induced Pluripotent ◽  
Gene Expression Alterations

AbstractPathogenic mutations in A-type nuclear lamins cause dilated cardiomyopathy, which is postulated to result from dysregulated gene expression due to changes in chromatin organization into active and inactive compartments. To test this, we performed genome-wide chromosome conformation analyses (Hi-C) in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with a haploinsufficient mutation for lamin A/C. Compared to gene-corrected cells, mutant hiPSC-CMs have marked electrophysiological and contractile alterations, with modest gene expression changes. While large-scale changes in chromosomal topology are evident, differences in chromatin compartmentalization are limited to a few hotspots that escape inactivation during cardiogenesis. These regions exhibit upregulation of multiple non-cardiac genes including CACNA1A, encoding for neuronal P/Q-type calcium channels. Pharmacological inhibition of the resulting current partially mitigates the electrical alterations. On the other hand, A/B compartment changes do not explain most gene expression alterations in mutant hiPSC-CMs. We conclude that global errors in chromosomal compartmentation are not the primary pathogenic mechanism in heart failure due to lamin A/C haploinsufficiency.SummaryBertero et al. observe that lamin A/C haploinsufficiency in human cardiomyocytes markedly alters electrophysiology, contractility, gene expression, and chromosomal topology. Contrary to expectations, however, changes in chromatin compartments involve just few regions, and most dysregulated genes lie outside these hotspots.Condensed titleGenomic effects of lamin A/C haploinsufficiency

scholarly journals A high-throughput screening platform for pigment regulating agents using pluripotent stem cell derived melanocytes

2020 ◽  
Author(s):  
Valentin Parat ◽  
Brigitte Onteniente ◽  
Julien Maruotti
Keyword(s):  
Stem Cell ◽  
High Throughput ◽  
High Throughput Screening ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Light Scatter ◽  
Chemical Treatments ◽  
Human Melanocytes ◽  
Screening Platform ◽  
Induced Pluripotent

AbstractIn this study, we describe a simple and straight-forward assay using induced pluripotent stem cell derived melanocytes and high-throughput flow cytometry, to screen and identify pigment regulating agents. The assays is based on the correlation between forward light-scatter characteristics and melanin content, with pigmented cells displaying high light absorption/low forward light-scatter, while the opposite is true for lowly pigmented melanocytes, as a result of genetic background or chemical treatments. Orthogonal validation is then performed by regular melanin quantification. Such approach was validated using a set of 80 small molecules, and yielded a confirmed hit. The assay described in this study may prove a useful tool to identify modulators of melanogenesis in human melanocytes.

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