A novel approach for studying mast cell-driven disorders: Mast cells derived from induced pluripotent stem cells.

BACKGROUND: Mast cells (MCs) are considered the main effectors in allergic reactions and well known for their contribution to the pathogenesis of various inflammatory diseases, urticaria, and mastocytosis. To study their functions in vitro, human primary MCs are isolated directly from several tissues or differentiated from hematopoietic progenitors. However, these techniques bear several disadvantages and challenges including low proliferation capacity, donor-dependent heterogeneity, and the lack of a continuous cell source. OBJECTIVE: To address this, we developed a novel strategy for the rapid and efficient differentiation of MCs from human-induced pluripotent stem cells (hiPSCs). METHODS: A 4-step protocol for the generation of hiPSC-derived MCs, based on the use of 3 hiPSC lines, was established and validated by comparison with human skin MCs and peripheral hematopoietic stem cell-derived MCs. RESULTS: hiPSC-MCs share phenotypic and functional characteristics of human skin MCs and peripheral hematopoietic stem cell-derived MCs. They display stable expression of the MC-associated receptors CD117, FcεRIα, and Mas-related G protein-coupled receptor X2 and degranulate in response to IgE/anti-IgE and substance P. CONCLUSIONS: This novel hiPSC-based approach provides a sustainable and homogeneous source for a rapid and highly productive generation of phenotypically mature, functional MCs, and its principle allows for the investigation of disease- and patient-specific MC populations.

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

SUMMARY: Here, we propose Fourier ring correlation-based quality estimation (FRC-QE) as a new metric for automated image quality estimation in 3D fluorescence microscopy acquisitions of cleared organoids that yields comparable measurements across experimental replicates, clearing protocols and works for different microscopy modalities. AVAILABILITY AND IMPLEMENTATION: FRC-QE is written in ImgLib2/Java and provided as an easy-to-use and macro-scriptable plugin for Fiji. Code, documentation, sample images and further information can be found under https://github.com/PreibischLab/FRC-QE. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

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 IC50 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.

Parallel generation of easily selectable multiple nephronal cell types from human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) provide a source for the generation of defined kidney cells and renal organoids applicable in regenerative medicine, disease modeling, and drug screening. These applications require the provision of hPSC-derived renal cells by reproducible, scalable, and efficient methods. We established a chemically defined protocol by application of Activin A, BMP4, and Retinoic acid followed by GDNF, which steered hPSCs to the renal lineage and resulted in populations of SIX2+/CITED1+ metanephric mesenchyme- (MM) and of HOXB7+/GRHL2+ ureteric bud (UB)-like cells already by 6 days. Transcriptome analysis corroborated that the PSC-derived cell types at day 8 resemble their renal vesicle and ureteric epithelial counterpart in vivo, forming tubular and glomerular renal cells 6 days later. We demonstrate that starting from hPSCs, our in vitro protocol generates a pool of nephrogenic progenitors at the renal vesicle stage, which can be further directed into specialized nephronal cell types including mesangial-, proximal tubular-, distal tubular, collecting duct epithelial cells, and podocyte precursors after 14 days. This simple and rapid method to produce renal cells from a common precursor pool in 2D culture provides the basis for scaled-up production of tailored renal cell types, which are applicable for drug testing or cell-based regenerative therapies.

Removing Critical Gaps in Chemical Test Methods by Developing New Assays for the Identification of Thyroid Hormone System-Disrupting Chemicals-The ATHENA Project.

The test methods that currently exist for the identification of thyroid hormone system-disrupting chemicals are woefully inadequate. There are currently no internationally validated in vitro assays, and test methods that can capture the consequences of diminished or enhanced thyroid hormone action on the developing brain are missing entirely. These gaps put the public at risk and risk assessors in a difficult position. Decisions about the status of chemicals as thyroid hormone system disruptors currently are based on inadequate toxicity data. The ATHENA project (Assays for the identification of Thyroid Hormone axis-disrupting chemicals: Elaborating Novel Assessment strategies) has been conceived to address these gaps. The project will develop new test methods for the disruption of thyroid hormone transport across biological barriers such as the blood-brain and blood-placenta barriers. It will also devise methods for the disruption of the downstream effects on the brain. ATHENA will deliver a testing strategy based on those elements of the thyroid hormone system that, when disrupted, could have the greatest impact on diminished or enhanced thyroid hormone action and therefore should be targeted through effective testing. To further enhance the impact of the ATHENA test method developments, the project will develop concepts for better international collaboration and development in the area of thyroid hormone system disruptor identification and regulation.

Regenerative Medicine/Cardiac Cell Therapy: Pluripotent Stem Cells.

For more than 20 years, tremendous efforts have been made to develop cell-based therapies for treatment of heart failure. However, the results of clinical trials using somatic, nonpluripotent stem or progenitor cells have been largely disappointing in both cardiology and cardiac surgery scenarios. Surgical groups were among the pioneers of experimental and clinical myocyte transplantation ("cellular cardiomyoplasty"), but little translational progress was made prior to the development of cellular reprogramming for creation of induced pluripotent stem cells (iPSC). Ever since, protocols have been developed which allow for the derivation of large numbers of autologous cardiomyocytes (CMs) from patient-specific iPSC, moving translational research closer toward clinical pilot trials. However, compared with somatic cell therapy, the technology required for safe and efficacious pluripotent stem cell (PSC)-based therapies is extremely complex and requires tremendous resources and close interactions between basic scientists and clinicians. This review summarizes PSC sources, strategies to derive CMs, current cardiac tissue engineering approaches, concerns regarding immunogenicity and cellular maturity, and highlights the contributions made by surgical groups.

hPSCreg--the human pluripotent stem cell registry.

The human pluripotent stem cell registry (hPSCreg), accessible at http://hpscreg.eu, is a public registry and data portal for human embryonic and induced pluripotent stem cell lines (hESC and hiPSC). Since their first isolation the number of hESC lines has steadily increased to over 3000 and new iPSC lines are generated in a rapidly growing number of laboratories as a result of their potentially broad applicability in biomedicine and drug testing. Many of these lines are deposited in stem cell banks, which are globally established to store tens of thousands of lines from healthy and diseased donors. The Registry provides comprehensive and standardized biological and legal information as well as tools to search and compare information from multiple hPSC sources and hence addresses a translational research need. To facilitate unambiguous identification over different resources, hPSCreg automatically creates a unique standardized name for each cell line registered. In addition to biological information, hPSCreg stores extensive data about ethical standards regarding cell sourcing and conditions for application and privacy protection. hPSCreg is the first global registry that holds both, manually validated scientific and ethical information on hPSC lines, and provides access by means of a user-friendly, mobile-ready web application.

Semantic Body Browser: graphical exploration of an organism and spatially resolved expression data visualization.

UNLABELLED: Advancing technologies generate large amounts of molecular and phenotypic data on cells, tissues and organisms, leading to an ever-growing detail and complexity while information retrieval and analysis becomes increasingly time-consuming. The Semantic Body Browser is a web application for intuitively exploring the body of an organism from the organ to the subcellular level and visualising expression profiles by means of semantically annotated anatomical illustrations. It is used to comprehend biological and medical data related to the different body structures while relying on the strong pattern recognition capabilities of human users. AVAILABILITY AND IMPLEMENTATION: The Semantic Body Browser is a JavaScript web application that is freely available at http://sbb.cellfinder.org. The source code is provided on https://github.com/flekschas/sbb.

Selective cell targeting and lineage tracing of human induced pluripotent stem cells using recombinant avian retroviruses.

Human induced pluripotent stem cells (hiPSC) differentiate into multiple cell types. Selective cell targeting is often needed for analyzing gene function by overexpressing proteins in a distinct population of hiPSC-derived cell types and for monitoring cell fate in response to stimuli. However, to date, this has not been possible, as commonly used viruses enter the hiPSC via ubiquitously expressed receptors. Here, we report for the first time the application of a heterologous avian receptor, the tumor virus receptor A (TVA), to selectively transduce TVA(+) cells in a mixed cell population. Expression of the TVA surface receptor via genetic engineering renders cells susceptible for infection by avian leucosis virus (ALV). We generated hiPSC lines with this stably integrated, ectopic TVA receptor gene that expressed the receptor while retaining pluripotency. The undifferentiated hiPSC(TVA+) as well as their differentiating progeny could be infected by recombinant ALV (so-called RCAS virus) with high efficiency. Due to incomplete receptor blocking, even sequential infection of differentiating or undifferentiated TVA(+) cells was possible. In conclusion, the TVA/RCAS system provides an efficient and gentle gene transfer system for hiPSC and extends our possibilities for selective cell targeting and lineage tracing studies.

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.

MGFM: a novel tool for detection of tissue and cell specific marker genes from microarray gene expression data.

BACKGROUND: Identification of marker genes associated with a specific tissue/cell type is a fundamental challenge in genetic and cell research. Marker genes are of great importance for determining cell identity, and for understanding tissue specific gene function and the molecular mechanisms underlying complex diseases. RESULTS: We have developed a new bioinformatics tool called MGFM (Marker Gene Finder in Microarray data) to predict marker genes from microarray gene expression data. Marker genes are identified through the grouping of samples of the same type with similar marker gene expression levels. We verified our approach using two microarray data sets from the NCBI's Gene Expression Omnibus public repository encompassing samples for similar sets of five human tissues (brain, heart, kidney, liver, and lung). Comparison with another tool for tissue-specific gene identification and validation with literature-derived established tissue markers established functionality, accuracy and simplicity of our tool. Furthermore, top ranked marker genes were experimentally validated by reverse transcriptase-polymerase chain reaction (RT-PCR). The sets of predicted marker genes associated with the five selected tissues comprised well-known genes of particular importance in these tissues. The tool is freely available from the Bioconductor web site, and it is also provided as an online application integrated into the CellFinder platform ( http://cellfinder.org/analysis/marker ). CONCLUSIONS: MGFM is a useful tool to predict tissue/cell type marker genes using microarray gene expression data. The implementation of the tool as an R-package as well as an application within CellFinder facilitates its use.

Generation of two human induced pluripotent stem cell lines with BAX and BAK1 double knock-out using CRISPR/Cas9.

Bcl-2-associated X protein (BAX) and Blc-2 homologous antagonist killer 1 (BAK) are two pro-apoptotic members of BCL2 family. Here, two BAX/BAK double knock-out human induced pluripotent stem cell lines (iPSC) we generated using CRISPR-Cas9 to generate apoptosis incompetent cell lines. The resulting cell lines were karyotypically normal, had typical morphology and expressed typical markers for the undifferentiated state.

Generation of THRB-GS(E125G_G126S) and THRB-KO human iPSC lines to study noncanonical thyroid hormone signalling.

THRB is a nuclear receptor, regulating gene expression dependent on thyroid hormone (TH) binding. The same receptor mediates signaling pathway activation in the cytosol. The challenge is to distinguish which of the two mechanisms is responsible for physiological effects of TH. We established an iPSC cell line with two mutations (E125G_G126S) in the THRB DNA-binding domain, which abrogates nuclear action and, thus, allows to study signaling pathway activation exclusively. We also generated a THRB knockout cell line to abolish all THRB effects. Comparison of WT and these two cell lines allows attribution of thyroid hormone effects to the underlying mechanism.

Spatiotemporal expression of thyroid hormone transporter MCT8 and THRA mRNA in human cerebral organoids recapitulating first trimester cortex development.

Thyroid hormones (TH) play critical roles during nervous system development and patients carrying coding variants of MCT8 (monocarboxylate transporter 8) or THRA (thyroid hormone receptor alpha) present a spectrum of neurological phenotypes resulting from perturbed local TH action during early brain development. Recently, human cerebral organoids (hCOs) emerged as powerful in vitro tools for disease modelling recapitulating key aspects of early human cortex development. To begin exploring prospects of this model for thyroid research, we performed a detailed characterization of the spatiotemporal expression of MCT8 and THRA in developing hCOs. Immunostaining showed MCT8 membrane expression in neuronal progenitor cell types including early neuroepithelial cells, radial glia cells (RGCs), intermediate progenitors and outer RGCs. In addition, we detected robust MCT8 protein expression in deep layer and upper layer neurons. Spatiotemporal SLC16A2 mRNA expression, detected by fluorescent in situ hybridization (FISH), was highly concordant with MCT8 protein expression across cortical cell layers. FISH detected THRA mRNA expression already in neuroepithelium before the onset of neurogenesis. THRA mRNA expression remained low in the ventricular zone, increased in the subventricular zone whereas strong THRA expression was observed in excitatory neurons. In combination with a robust up-regulation of known T3 response genes following T3 treatment, these observations show that hCOs provide a promising and experimentally tractable model to probe local TH action during human cortical neurogenesis and eventually to model the consequences of impaired TH function for early cortex development.

Establishment of a high-content compatible platform to assess effects of monocyte-derived factors on neural stem cell proliferation and differentiation.

During neuroinflammation, monocytes that infiltrate the central nervous system (CNS) may contribute to regenerative processes depending on their activation status. However, the extent and mechanisms of monocyte-induced CNS repair in patients with neuroinflammatory diseases remain largely unknown, partly due to the lack of a fully human assay platform that can recapitulate monocyte-neural stem cell interactions within the CNS microenvironment. We therefore developed a human model system to assess the impact of monocytic factors on neural stem cells, establishing a high-content compatible assay for screening monocyte-induced neural stem cell proliferation and differentiation. The model combined monocytes isolated from healthy donors and human embryonic stem cell derived neural stem cells and integrated both cell-intrinsic and -extrinsic properties. We identified CNS-mimicking culture media options that induced a monocytic phenotype resembling CNS infiltrating monocytes, while allowing adequate monocyte survival. Monocyte-induced proliferation, gliogenic fate and neurogenic fate of neural stem cells were affected by the conditions of monocytic priming and basal neural stem cell culture as extrinsic factors as well as the neural stem cell passage number as an intrinsic neural stem cell property. We developed a high-content compatible human in vitro assay for the integrated analysis of monocyte-derived factors on CNS repair.

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.

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.

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.

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.

Early-set POMC methylation variability is accompanied by increased risk for obesity and is addressable by MC4R agonist treatment.

Increasing evidence points toward epigenetic variants as a risk factor for developing obesity. We analyzed DNA methylation of the POMC (pro-opiomelanocortin) gene, which is pivotal for satiety regulation. We identified sex-specific and nongenetically determined POMC hypermethylation associated with a 1.4-fold (confidence interval, 1.03 to 2.04) increased individual risk of developing obesity. To investigate the early embryonic establishment of POMC methylation states, we established a human embryonic stem cell (hESC) model. Here, hESCs (WA01) were transferred into a naïve state, which was associated with a reduction of DNA methylation. Naïve hESCs were differentiated via a formative state into POMC-expressing hypothalamic neurons, which was accompanied by re-establishment of DNA methylation patterning. We observed that reduced POMC gene expression was associated with increased POMC methylation in POMC-expressing neurons. On the basis of these findings, we treated POMC-hypermethylated obese individuals (n = 5) with an MC4R agonist and observed a body weight reduction of 4.66 ± 2.16% (means ± SD) over a mean treatment duration of 38.4 ± 26.0 weeks. In summary, we identified an epigenetic obesity risk variant at the POMC gene fulfilling the criteria for a metastable epiallele established in early embryonic development that may be addressable by MC4R agonist treatment to reduce body weight.