ASSURED-optimized CRISPR protocol for knockout/SNP knockin in hiPSCs

CRISPR-Cas9 technology coupled with human induced pluripotent stem cells allows precise disease modeling in pluripotent cells and subsequently derived specialized cell types. Here, we present an optimized CRISPR-Cas9 pipeline, ASSURED (affordable, successful, specific, user-friendly, rapid, efficient, and deliverable), to produce gene-modified single-cell-derived knockout or single-nucleotide-polymorphism-modified knockin hiPSCs clones. We describe steps for analyzing targeted genomic sequence and designing guide RNAs and homology repair template. We then detail the CRISPR-Cas9 delivery workflow, evaluation of editing efficiency, and automated cell isolation followed by clone screening

Preliminary evaluation of the CellFinder literature curation pipeline for gene expression in kidney cells and anatomical parts

Biomedical literature curation is the process of automatically and/or manually deriving knowledge from scientific publications and recording it into specialized databases for structured delivery to users. It is a slow, error-prone, complex, costly and, yet, highly important task. Previous experiences have proven that text mining can assist in its many phases, especially, in triage of relevant documents and extraction of named entities and biological events. Here, we present the curation pipeline of the CellFinder database, a repository of cell research, which includes data derived from literature curation and microarrays to identify cell types, cell lines, organs and so forth, and especially patterns in gene expression. The curation pipeline is based on freely available tools in all text mining steps, as well as the manual validation of extracted data. Preliminary results are presented for a data set of 2376 full texts from which >4500 gene expression events in cell or anatomical part have been extracted. Validation of half of this data resulted in a precision of ~50% of the extracted data, which indicates that we are on the right track with our pipeline for the proposed task. However, evaluation of the methods shows that there is still room for improvement in the named-entity recognition and that a larger and more robust corpus is needed to achieve a better performance for event extraction. Database URL: http://www.cellfinder.org

CellFinder: a cell data repository

CellFinder (http://www.cellfinder.org) is a comprehensive one-stop resource for molecular data characterizing mammalian cells in different tissues and in different development stages. It is built from carefully selected data sets stemming from other curated databases and the biomedical literature. To date, CellFinder describes 3394 cell types and 50 951 cell lines. The database currently contains 3055 microscopic and anatomical images, 205 whole-genome expression profiles of 194 cell/tissue types from RNA-seq and microarrays and 553 905 protein expressions for 535 cells/tissues. Text mining of a corpus of >2000 publications followed by manual curation confirmed expression information on ∼900 proteins and genes. CellFinder's data model is capable to seamlessly represent entities from single cells to the organ level, to incorporate mappings between homologous entities in different species and to describe processes of cell development and differentiation. Its ontological backbone currently consists of 204 741 ontology terms incorporated from 10 different ontologies unified under the novel CELDA ontology. CellFinder's web portal allows searching, browsing and comparing the stored data, interactive construction of developmental trees and navigating the partonomic hierarchy of cells and tissues through a unique body browser designed for life scientists and clinicians

CELDA - an ontology for the comprehensive representation of cells in complex systems

BACKGROUND: The need for detailed description and modeling of cells drives the continuous generation of large and diverse datasets. Unfortunately, there exists no systematic and comprehensive way to organize these datasets and their information. CELDA (Cell: Expression, Localization, Development, Anatomy) is a novel ontology for the association of primary experimental data and derived knowledge to various types of cells of organisms. RESULTS: CELDA is a structure that can help to categorize cell types based on species, anatomical localization, subcellular structures, developmental stages and origin. It targets cells in vitro as well as in vivo. Instead of developing a novel ontology from scratch, we carefully designed CELDA in such a way that existing ontologies were integrated as much as possible, and only minimal extensions were performed to cover those classes and areas not present in any existing model. Currently, ten existing ontologies and models are linked to CELDA through the top-level ontology BioTop. Together with 15.439 newly created classes, CELDA contains more than 196.000 classes and 233.670 relationship axioms. CELDA is primarily used as a representational framework for modeling, analyzing and comparing cells within and across species in CellFinder, a web based data repository on cells (http://cellfinder.org). CONCLUSIONS: CELDA can semantically link diverse types of information about cell types. It has been integrated within the research platform CellFinder, where it exemplarily relates cell types from liver and kidney during development on the one hand and anatomical locations in humans on the other, integrating information on all spatial and temporal stages. CELDA is available from the CellFinder website: http://cellfinder.org/about/ontology

Generation of iPSC lines with SLC16A2:G401R or SLC16A2 knock out

The X-linked Allan-Herndon-Dudley syndrome (AHDS) is characterized by severely impaired psychomotor development and is caused by mutations in the SLC16A2 gene encoding the thyroid hormone transporter MCT8 (monocarboxylate transporter 8). By targeting exon 3 of SLC16A2 using CRISPR/Cas9 with single-stranded oligodeoxynucleotides as homology-directed repair templates, we introduced the AHDS patient missense variant G401R and a novel knock-out deletion variant (F400Sfs*17) into the male healthy donor hiPSC line BIHi001-B. We successfully generated cerebral organoids from these genome-edited lines, demonstrating the utility of the novel lines for modelling the effects of MCT8-deficency on human neurodevelopment

Methods for automated single cell isolation and sub-cloning of human pluripotent stem cells

Advances in human pluripotent stem cell (hPSC) techniques have led them to become a widely used and powerful tool for a vast array of applications, including disease modeling, developmental studies, drug discovery and testing, and emerging cell-based therapies. hPSC workflows that require clonal expansion from single cells, such as CRISPR/Cas9-mediated genome editing, face major challenges in terms of efficiency, cost, and precision. Classical sub-cloning approaches depend on limiting dilution and manual colony picking, which are both time-consuming and labor-intensive, and lack a real proof of clonality. Here we describe the application of three different automated cell isolation and dispensing devices that can enhance the single-cell cloning process for hPSCs. In combination with optimized cell culture conditions, these devices offer an attractive alternative compared to manual methods. We explore various aspects of each device system and define protocols for their practical application. Following the workflow described here, single cell-derived hPSC sub-clones from each system maintain pluripotency and genetic stability. Furthermore, the workflows can be applied to uncover karyotypic mosaicism prevalent in bulk hPSC cultures. Our robust automated workflow facilitates high-throughput hPSC clonal selection and expansion, urgently needed in the operational pipelines of hPSC applications

Teratoma formation of human embryonic stem cells in three-dimensional perfusion culture bioreactors

Teratoma formation in mice is today the most stringent test for pluripotency that is available for human pluripotent cells, as chimera formation and tetraploid complementation cannot be performed with human cells. The teratoma assay could also be applied for assessing the safety of human pluripotent cell-derived cell populations intended for therapeutic applications. In our study we examined the spontaneous differentiation behaviour of human embryonic stem cells (hESCs) in a perfused 3D multi-compartment bioreactor system and compared it with differentiation of hESCs and human induced pluripotent cells (hiPSCs) cultured in vitro as embryoid bodies and in vivo in an experimental mouse model of teratoma formation. Results from biochemical, histological/immunohistological and ultrastuctural analyses revealed that hESCs cultured in bioreactors formed tissue-like structures containing derivatives of all three germ layers. Comparison with embryoid bodies and the teratomas revealed a high degree of similarity of the tissues formed in the bioreactor to these in the teratomas at the histological as well as transcriptional level, as detected by comparative whole-genome RNA expression profiling. The 3D culture system represents a novel in vitro model that permits stable long-term cultivation, spontaneous multi-lineage differentiation and tissue formation of pluripotent cells that is comparable to in vivo differentiation. Such a model is of interest, e.g. for the development of novel cell differentiation strategies. In addition, the 3D in vitro model could be used for teratoma studies and pluripotency assays in a fully defined, controlled environment, alternatively to in vivo mouse models. Copyright (c) 2012 John Wiley & Sons, Ltd

FRC-QE: a robust and comparable 3D microscopy image quality metric for cleared organoids

Three-dimensional stem-cell-derived organoids are a powerful tool for studying cellular processes in tissue-like structures, enabling in vitro experiments in an organ-specific context. While organoid research has been closely linked to advances in fluorescence microscopy, capturing cellular structures within their global context in an organoid often remains challenging due to the organoid’s dense structure and opacity. The development of optical clearing methods has provided a solution for fixed organoids but optimizing clearing protocols for a given sample type and staining can be challenging. Importantly, quantitative measures for assessing image quality throughout cleared fluorescent samples are missing. Here, we propose Fourier ring correlation quality estimation (FRC-QE) as a new metric for automated 3D image quality estimation in cleared organoids. We show that FRC-QE robustly captures differences in clearing efficiency within an organoid, across replicates and clearing protocols, as well as for different microscopy modalities. FRC-QE is open-source, written in ImgLib2 and provided as an easy-to-use and macro-scriptable plugin for the popular Fiji software. We therefore envision FRC-QE to fill the gap of providing a reliable quality metric for testing, optimizing and comparing optical clearing methods

Modeling chemotherapy induced neurotoxicity with human induced pluripotent stem cell (iPSC)-derived sensory neurons

Chemotherapy-induced peripheral neuropathy (CIPN) is a frequent, potentially irreversible adverse effect of cytotoxic chemotherapy often leading to a reduction or discontinuation of treatment which negatively impacts patients' prognosis. To date, however, neither predictive biomarkers nor preventive treatments for CIPN are available, which is partially due to a lack of suitable experimental models. We therefore aimed to evaluate whether sensory neurons derived from induced pluripotent stem cells (iPSC-DSN) can serve as human disease model system for CIPN. Treatment of iPSC-DSN for 24 h with the neurotoxic drugs paclitaxel, bortezomib, vincristine and cisplatin led to axonal blebbing and a dose dependent decline of cell viability in clinically relevant IC(50) ranges, which was not observed for the non-neurotoxic compounds doxorubicin and 5-fluorouracil. Paclitaxel treatment effects were less pronounced after 24 h but prominent when treatment was applied for 72 h. Global transcriptome analyses performed at 24 h, i.e. before paclitaxel-induced cell death occurred, revealed the differential expression of genes of neuronal injury, cellular stress response, and sterol pathways. We further evaluated if known neuroprotective strategies can be reproduced in iPSC-DSN and observed protective effects of lithium replicating findings from rodent dorsal root ganglia cells. Comparing sensory neurons derived from two different healthy donors, we found preliminary evidence that these cell lines react differentially to neurotoxic drugs as expected from the variable presentation of CIPN in patients. In conclusion, iPSC-DSN are a promising platform to study the pathogenesis of CIPN and to evaluate neuroprotective treatment strategies. In the future, the application of patient-specific iPSC-DSN could open new avenues for personalized medicine with individual risk prediction, choice of chemotherapeutic compounds and preventive treatments

Generation of an NCS1 gene knockout human induced pluripotent stem cell line using CRISPR/Cas9

NCS1 (Neuronal calcium sensor protein 1) encodes a highly conserved calcium binding protein abundantly expressed in neurons. It modulates intracellular calcium homeostasis, calcium-dependent signaling pathways as well as neuronal transmission and plasticity. Here, we generated a NCS1 knockout human induced pluripotent stem cell (hiPSC) line using CRISPR-Cas9 genome editing. It shows regular expression of pluripotent markers, normal iPSC morphology and karyotype as well as no detectable off-target effects on top 6 potentially affected genes. This newly generated cell line constitutes a valuable tool for studying the role of NCS1 in the pathophysiology of various neuropsychiatric disorders and non-neurological disease