Name: human pluripotent stem cell based developmental toxicity assays for chemical safety screening and systems biology data generation
Uploaded: 2015-06-17T13:00:00.000+00:00
Duration: 17 min 28 s
Description: Watch this Scientific Journal Video about Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation at JoVE.com

We thank M. Kapitza, Margit Henry, Tamara Rotshteyn, Susan Rohani and Cornelia B&#246;ttinger for excellent technical support. This work was supported by grants from the German Research Foundation (RTG 1331) and the German Ministry for Research (BMBF).

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The authors have nothing to disclose.

Traditional approaches to toxicological testing involve extensive animal studies thus making testing costly and time-consuming. Moreover, due to the interspecies differences the preclinical animal safety studies are not always valid to predict toxicity effects of potential drugs relevant for humans. Although non-human primates are most predictable, still strong ethical, and socioeconomical demands are rapidly raising by modern societies for developing sensitive and robust in vitro test system relevant t...

The ability of human embryonic stem cells (hESC) to differentiate into various types of cells opened up a new era of in vitro toxicity testing1, disease modelling and regenerative medicine2. The stem cells are endowed with the capacity to self-replicate, to keep their pluripotent state, and to differentiate into specialized cells3,4. The properties of hESC (capacity to differentiate to all major cell types) are also found in other human pluripotent stem cells, such as human induced pluripotent stem cells (hiPSC) or cells generated by nuclear transfer5. For instance, many different hESC lines have been differentiated into neurons6, renal cells7, neural crest cells8, cardiomyocytes9-12, or hepatocytes like cells13,14. Moreover, hESC can spontaneously differentiate into cells of all three germ layers15-18 in embryoid bodies (EBs)19,20. Early embryonic development is regulated by differential expression of various genes related to the different germ layers which has been captured at mRNA level by transcriptomics using microarray technology15. These efforts resulted in the establishment of organ specific toxicological models based on hESC/hiPSC and transcriptomics analysis (for review see 21,22). These models have advantages over the traditional use of laboratory animals for toxicological studies, as preclinical studies using laboratory animals are not always predictive of human safety. The drug induced toxicities encountered in patients are often related to metabolic or signaling processes that differ between humans and experimental animals. The species difference has prevented the reliable early detection of developmental toxicity in humans, and for instance drugs such as thalidomide23,24 and diethylstilbestrol25,26 were withdrawn from the market due to teratogenicity. Thalidomide has not shown any developmental toxicity in rats or mice. Environmental chemicals such as methyl mercury27 resulted in prenatal developmental toxicity with respect to the nervous system in various species, but human manifestations have been hard to model in animals. To address the problem of species specificity issues, scientists working under different projects based on stem cells like ReProTect, ESNATS, DETECTIVE etc. are engaged in the development of different models for embryonic toxicity, neurotoxicity, cardiotoxicity, hepatotoxicity and nephrotoxicity using human toxicants suspected to affect humans. Under the European consortium project &#39;Embryonic Stem cell-based Novel Alternative Testing Strategies (ESNATS)&#39; five test systems have been established. One test system the so called UKK (Universit&#228;tsklinikum K&#246;ln) test system partially captures early human embryonic development. In this system human embryonic H9 cells are differentiated in to three germ layers (ectoderm, endoderm and mesoderm)15 and germ layer specific signatures have been captured by transcriptomics profile using the Affymetrix microarray platform. Various developmental toxicants like thalidomide28, valproic acid, methyl mercury16,17, or cytosine arabinoside15 have been tested in this system, and toxicant specific gene signatures have been obtained. In a second test system, the so called the UKN1 (University of Konstanz) test system 1, H9 cells are differentiated to neuroectodermal progenitor cells (NEP) for 6 days. This is evidenced by high expression of neural gene markers such as PAX6 and OTX2. During differentiation for 6 days, NEP cells have been exposed to developmental neuro-toxicants such as VPA, methyl mercury. Toxicant-specific de-regulated transcriptomics profiles have been obtained as well by using the Affymetrix microarray platform16,29.
The new vision for toxicology of the 21st century envisages that test systems do not only yield phenotypic descriptions like histopathology in vivo, or transcriptome changes at the end of long-term toxicant incubations. It rather suggests that assays provide mechanistic information3, and that this information can be mapped to so-called adverse outcome pathways (AOP) that provide a scientific rationale for hazardous effects30. To provide such information, the test systems applied have to be highly quality controlled31, as for instance documented by robust standard operation procedures. Moreover, time-dependent changes need to be mapped with high resolution. This requires test systems with synchronized changes32. The UKN1 and UKK test systems described here have been optimized for these requirements.

<table><tbody><tr><td>DMEM/F-12</td><td>Life Technologies</td><td>11320082</td><td>Dulbecco&#039;s Modified Eagle Medium:Nutrient Mixture F-12</td></tr><tr><td>KOSR</td><td>Life Technologies</td><td>10828028</td><td>Knockout Serum Replacement</td></tr><tr><td>GlutaMAX</td><td>Life Technologies</td><td>35050061</td><td>GlutaMAX supplement</td></tr><tr><td>NEAA</td><td>Life Technologies</td><td>11140050</td><td>MEM Nonessential Amino Acids Solution</td></tr><tr><td>DPBS</td><td>Life Technologies</td><td>14190-0144</td><td>Dulbecco&#039;s Phosphate-Buffered Saline, without calcium, without magnesium</td></tr><tr><td>mTeSR medium</td><td>Stemcell Technologies</td><td>5850</td><td></td></tr><tr><td>Pluronic F-127</td><td>Sigma</td><td>P2443-250G</td><td></td></tr><tr><td>V bottom plate</td><td>VWR</td><td>734-0483</td><td>Plate,Microwell,V BTTM,96 Well,Sterile 1 * 50 ST</td></tr><tr><td>V bottom plate lid</td><td>VWR</td><td>634-0011</td><td>Lid, Microtitre plates, Cond. Ring 1 * 50 ST</td></tr><tr><td>Pen/Strep</td><td>Life Technologies</td><td>15140-122</td><td>Penicillin-Streptomycin, Liquid</td></tr><tr><td>Distilled Water</td><td>Life Technologies</td><td>15230-089.</td><td>Sterile Distilled Water</td></tr><tr><td>Human FGF-2 (bFGF)</td><td>Millipore</td><td>GF003AF-100UG</td><td>Fibroblast Growth Factor basic, human recombinant, animal-free</td></tr><tr><td>Filter 0.22 &amp;mu;m</td><td>Millipore</td><td>SCGPU02RE</td><td>Stericup-GP, 0.22 &amp;mu;m, polyethersulfone, 250 ml, radio-sterilized</td></tr><tr><td>StemPro EZPassage&lt;sup&gt;TM&lt;/sup&gt; Disposablte</td><td>Invitrogen</td><td>23181010</td><td></td></tr><tr><td>BD MatrigelTM, hESC qualified Matrix</td><td>Stemcell Technologies</td><td>354277</td><td>5 ml vial</td></tr><tr><td>DMSO</td><td>Sigma</td><td>D-2650</td><td></td></tr><tr><td>RNAlater Stabilization Solution</td><td>Life Technologies</td><td>AM7020</td><td>It stabilizes and protect the RNA integrity in unfrozen samples.</td></tr><tr><td>70 &amp;mu;m Cell Strainer</td><td>Becton Dickinson</td><td>352350</td><td>Cell strainer with 70 &amp;mu;m Nylon mesh</td></tr><tr><td>35 &amp;mu;m Lid cell strainer, 5&amp;nbsp;ml tube</td><td>Becton Dickinson</td><td>352235</td><td>5 ml polystyrene round bottom test tube, with a cell strainer cap (35&amp;nbsp;&amp;mu;m)</td></tr><tr><td>50 ml sterile Polypropylene tube</td><td>Greiner Bio-One</td><td>227261</td><td>50 ml Polypropylene tube with conical bottom, Sterile</td></tr><tr><td>T75 flask</td><td>Greiner Bio-One</td><td>658175</td><td>CELLSTAR Filter Cap Cell Culture 75 cm&lt;sup&gt;2&lt;/sup&gt; Flasks</td></tr><tr><td>TRIzol</td><td>Life Technologies</td><td>10296010</td><td></td></tr><tr><td>96 well optical bottom plates</td><td>Thermo Scientific</td><td>165305</td><td></td></tr><tr><td>CellTiter-Blue</td><td>Promega</td><td>G8081</td><td></td></tr><tr><td>Accutase</td><td>PAA</td><td>L11-007</td><td></td></tr><tr><td>Apotransferin</td><td>Sigma-Aldrich</td><td>T-2036</td><td></td></tr><tr><td>Dispase</td><td>Worthington Biochemicals</td><td>LS002104</td><td></td></tr><tr><td>Dorsomorphin</td><td>Tocris Bioscience</td><td>3093</td><td></td></tr><tr><td>EDTA</td><td>Roth</td><td>8043.2</td><td></td></tr><tr><td>FBS</td><td>PAA</td><td>A15-101</td><td></td></tr><tr><td>FGF-2</td><td>R&amp;amp;D Systems</td><td>233-FB</td><td></td></tr><tr><td>Gelatine</td><td>Sigma-Aldrich</td><td>G1890-100G</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G7021-100G</td><td></td></tr><tr><td>GlutaMAX</td><td>Gibco Invitrogen</td><td>35050-038</td><td></td></tr><tr><td>HEPES</td><td>Gibco Invitrogen</td><td>15630-056</td><td></td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>I-6634</td><td></td></tr><tr><td>Knockout DMEM</td><td>Gibco Invitrogen</td><td>10829-018</td><td></td></tr><tr><td>Matrigel</td><td>BD Biosciences</td><td>354234</td><td></td></tr><tr><td>Noggin</td><td>R&amp;amp;D Systems</td><td>719-NG</td><td></td></tr><tr><td>PBS</td><td>Biochrom AG</td><td>L1825</td><td></td></tr><tr><td>Progesteron</td><td>Sigma-Aldrich</td><td>P7556</td><td></td></tr><tr><td>Putrescine</td><td>Sigma-Aldrich</td><td>P-5780</td><td></td></tr><tr><td>ROCK inhibitor Y-27632</td><td>Tocris Biosciences</td><td>1254</td><td></td></tr><tr><td>SB431542</td><td>Tocris Biosciences</td><td>1614</td><td></td></tr><tr><td>SDS</td><td>Bio-Rad</td><td>161-0416</td><td></td></tr><tr><td>Selenium</td><td>Sigma-Aldrich</td><td>S-5261</td><td></td></tr><tr><td>&amp;beta;-Mercaptoethanol</td><td>Gibco Invitrogen</td><td>31350-010</td><td></td></tr><tr><td colspan="4">[header]</td></tr><tr><td colspan="4">&lt;strong&gt;List of Kits&lt;/strong&gt;</td></tr><tr><td>RNeasy Mini Kit (250)</td><td>QIAGEN</td><td>74106</td><td></td></tr><tr><td>GeneChip Hybridization, Wash, and Stain Kit</td><td>Affymetrix</td><td>900721, 22, 23</td><td>This kit provides all reagents required for hybridization wash and staining of microarrays.</td></tr><tr><td>Rnase-Free DNase Set</td><td>QIAGEN</td><td>79254</td><td></td></tr><tr><td colspan="4">[header]</td></tr><tr><td colspan="4">&lt;strong&gt;List of equipment&lt;/strong&gt;</td></tr><tr><td>Inverted microscope</td><td>Olympus</td><td></td><td>IX71</td></tr><tr><td>Genechip Hybridisation Oven - 645</td><td>Affymetrix</td><td></td><td></td></tr><tr><td>Genechip Fluidics Station-450</td><td>Affymetrix</td><td></td><td></td></tr><tr><td>Affymetrix Gene-Chip Scanner-3000-7 G</td><td>Affymetrix</td><td></td><td></td></tr><tr><td>Spectramax M5</td><td>Molecular Devices</td><td></td><td>&lt;br /&gt;&amp;#160;&lt;br /&gt;&amp;#160;&lt;br /&gt;&amp;#160;</td></tr><tr><td colspan="4">[header]</td></tr><tr><td colspan="4">&lt;strong&gt;List of softwares&lt;/strong&gt;</td></tr><tr><td>Prism 4</td><td></td><td></td><td></td></tr><tr><td>Affymetrix GCOS</td><td></td><td></td><td></td></tr><tr><td>Partek Genomic Suite 6.25</td><td></td><td></td><td></td></tr><tr><td>Online tools for Functional annotation&lt;br/&gt; DAVID&lt;br/&gt; Onto-tools Intelligent Systems and Bioinformatics Laboratory</td><td></td><td></td><td></td></tr></tbody></table>

human pluripotent stem cell based developmental toxicity assays for chemical safety screening and systems biology data generation

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in vitro toxicity testing of potential drugs. To provide a solid scientific basis for such assays, it will be important to gain quantitative information on the time course of development and on the underlying regulatory mechanisms by systems biology approaches. Two assays have therefore been tuned here for these requirements. In the UKK test system, human embryonic stem cells (hESC) (or other pluripotent cells) are left to spontaneously differentiate for 14 days in embryoid bodies, to allow generation of cells of all three germ layers. This system recapitulates key steps of early human embryonic development, and it can predict human-specific early embryonic toxicity/teratogenicity, if cells are exposed to chemicals during differentiation. The UKN1 test system is based on hESC differentiating to a population of neuroectodermal progenitor (NEP) cells for 6 days. This system recapitulates early neural development and predicts early developmental neurotoxicity and epigenetic changes triggered by chemicals. Both systems, in combination with transcriptome microarray studies, are suitable for identifying toxicity biomarkers. Moreover, they may be used in combination to generate input data for systems biology analysis. These test systems have advantages over the traditional toxicological studies requiring large amounts of animals. The test systems may contribute to a reduction of the costs for drug development and chemical safety evaluation. Their combination sheds light especially on compounds that may influence neurodevelopment specifically.

Methyl mercury exposure in UKK test system
The cytotoxicity assay was performed with H9 EBs to obtain an IC10 value (reduction of viability by 10%) for the cytotoxicity of methyl mercury (Figure 1). We also performed a microarray based (affymetrix platform) biomarker study. The H9 EBs have been exposed to methyl mercury (0.25 and 1 &#181;M) for 14 days. On day 14, samples have been collected using TRIzol and RNA was isolated. Transcriptional profiling was performed ...

Watch this Scientific Journal Video about Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation at JoVE.com

Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation

The protocols describe two in vitro developmental toxicity test systems (UKK and UKN1) based on human embryonic stem cells and transcriptome studies. The test systems predict human developmental toxicity hazard, and may contribute to reduce animal studies, costs and the time required for chemical safety testing.

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in ...

human-pluripotent-stem-cell-based-developmental-toxicity-assays-for

Research

JoVE Journal

Developmental Biology

12.5K Views.  University of Cologne.  De protocollen beschrijven twee in vitro ontwikkelingstoxiciteit testsystemen (UKK en UKN1) op basis van menselijke embryonale stamcellen en transcriptoom studies. De testsystemen voorspellen mens ontwikkelingsstoornissen giftigheidsgevaar, en kunnen bijdragen aan dierproeven, de kosten en de tijd die nodig is voor de chemische veiligheid testen te verminderen. 

Video: Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

Applying toxicity testing of chemicals in higher order organisms, such as mice or rats, is time-consuming and expensive, due to their long lifespan and maintenance issues. On the contrary, the nematode Caenorhabditis elegans (C. elegans) has advantages to make it an ideal choice for toxicity testing: a short lifespan, easy cultivation, and efficient reproduction. Here, we describe a protocol for the automatic phenotypic profiling of C. elegans in a 384-well plate. The nematode worms are cultured in a 384-well plate with liquid medium and chemical treatment, and videos are taken of each well to quantify the chemical influence on 33 worm features. Experimental results demonstrate that the quantified phenotype features can classify and predict the acute toxicity for different chemical compounds and establish a priority list for further traditional chemical toxicity assessment tests in a rodent model.

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

A quantitative method has been developed to identify and predict the acute toxicity of chemicals by automatically analyzing the phenotypic profiling of Caenorhabditis elegans. This protocol describes how to treat worms with chemicals in a 384-well plate, capture videos, and quantify toxicological related phenotypes.

Applying toxicity testing of chemicals in higher order organisms, such as mice or rats, is time-consuming and expensive, due to their long lifespan and ...

Environment

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

Semi-automated Production of Hepatocyte Like Cells from Pluripotent Stem Cells

Human pluripotent stem cells represent a renewable source of human tissue. Our research is focused on generating human liver tissue from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). Current differentiation procedures generate human hepatocyte-like cells (HLCs) displaying a mixture of fetal and adult traits. To improve cell phenotype, we have fully defined our differentiation procedure and the cell niche, resulting in the generation of cell populations which display improved gene expression and&#160;function. While these studies mark progress, the ability to generate large quantities of multi well plates for screening has been limited by labour intensive procedures and batch to batch variation. To tackle this issue, we have developed a semi-automated platform to differentiate pluripotent stem cells into HLCs. Stem cell seeding and differentiation were performed using liquid handling and automatic pipetting systems in&#160;96-well plate format. Following the differentiation, cell phenotype was analyzed using automated microscopy and a multi well luminometer.

This protocol describes a semi-automated approach to produce hepatocyte-like cells from human pluripotent stem cells in a 96 well plate format. This process is rapid and cost-effective, allowing the production of quality assured batches of hepatocyte-like cells for basic and applied human research.

Human pluripotent stem cells represent a renewable source of human tissue. Our research is focused on generating human liver tissue from human embryonic ...

A Neurite Outgrowth Assay and Neurotoxicity Assessment with Human Neural Progenitor Cell-Derived Neurons

Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides reliable analysis of neuronal morphology together with quantitative measurements of modifications on neurite length and synaptic protein localization and abundance upon treatment with small molecule compounds. In addition to the application of the presented method in neurite outgrowth studies, neurotoxicity assessment can be performed to assess, distinguish and rank commercial chemical compounds based on their potential developmental neurotoxicity effect.
Even though cell lines are nowadays widely used in compound screening assays in neuroscience, they often differ genetically and phenotypically from their tissue origin. Primary cells, on the other hand, maintain important markers and functions observed in vivo. Therefore, due to the translation potential and physiological relevance that these cells could offer neurite outgrowth assay and neurotoxicity assessment can considerably benefit from using human neural progenitor cells (hNPCs) as the primary human cell model.
The presented method herein can be utilized to screen for the ability of compounds to induce neurite outgrowth and neurotoxicity by taking advantage of the human neural progenitor cell-derived neurons, a cell model closely representing human biology.&#34;

The presented protocol describes a method for a neurite outgrowth assay and neurotoxicity assessment of small molecule compounds.

Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides ...

Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical studies are substantial, therefore it is essential that more predictive toxicology screens are developed and deployed early on in drug development (Greenhough et al 2010). Human hepatocytes represent the current gold standard model for evaluating drug toxicity, but are a limited resource that exhibit variable function. Therefore, the use of immortalised cell lines and animal tissue models are routinely employed due to their abundance. While both sources are informative, they are limited by poor function, species variability and/or instability in culture (Dalgetty et al 2009). Pluripotent stem cells (PSCs) are an attractive alternative source of human hepatocyte like cells (HLCs) (Medine et al 2010). PSCs are capable of self renewal and differentiation to all somatic cell types found in the adult and thereby represent a potentially inexhaustible source of differentiated cells. We have developed a procedure that is simple, highly efficient, amenable to automation and yields functional human HLCs (Hay et al 2008 ; Fletcher et al 2008 ; Hannoun et al 2010 ; Payne et al 2011 and Hay et al 2011). We believe our technology will lead to the scalable production of HLCs for drug discovery, disease modeling, the construction of extra-corporeal devices and possibly cell based transplantation therapies.

This article will focus on the generation of human hepatic endoderm from human embryonic stem cell populations.

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical ...

Biology

Toxicity Screens in Human Retinal Organoids for Pharmaceutical Discovery

Organoids provide a promising platform to study disease mechanism and treatments, directly in the context of human tissue with the versatility and throughput of cell culture. Mature human retinal organoids are utilized to screen potential pharmaceutical treatments for the age-related retinal degenerative disease macular telangiectasia type 2 (MacTel).
We have recently shown that MacTel can be caused by elevated levels of an atypical lipid species, deoxysphingolipids (deoxySLs). These lipids are toxic to the retina and may drive the photoreceptor loss that occurs in MacTel patients. To screen drugs for their ability to prevent deoxySL photoreceptor toxicity, we generated human retinal organoids from a non-MacTel induced pluripotent stem cell (iPSC) line and matured them to a post-mitotic age where they develop all of the neuronal lineage-derived cells of the retina, including functionally mature photoreceptors. The retinal organoids were treated with a deoxySL metabolite and apoptosis was measured within the photoreceptor layer using immunohistochemistry. Using this toxicity model, pharmacological compounds that prevent deoxySL-induced photoreceptor death were screened. Using a targeted candidate approach, we determined that fenofibrate, a drug commonly prescribed for the treatment of high cholesterol and triglycerides, can also prevent deoxySL toxicity in the cells of the retina.
The toxicity screen successfully identified an FDA-approved drug that can prevent photoreceptor death. This is a directly actionable finding owing to the highly disease-relevant model tested. This platform can be easily modified to test any number of metabolic stressors and potential pharmacological interventions for future treatment discovery in retinal diseases.

Here we present a step-by-step protocol to generate mature human retinal organoids and utilize them in a photoreceptor toxicity assay to identify pharmaceutical candidates for the age-related retinal degenerative disease macular telangiectasia type 2 (MacTel).

Organoids provide a promising platform to study disease mechanism and treatments, directly in the context of human tissue with the versatility and ...

Bioengineering

Hybrid Cell Analysis System to Assess Structural and Contractile Changes of Human iPSC-Derived Cardiomyocytes for Preclinical Cardiac Risk Evaluation

Cardiac contractility assessment is of immense importance for the development of new therapeutics and their safe transition into clinical stages. While human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold promise to serve as a human-relevant model in preclinical phases of drug discovery and safety pharmacology, their maturity is still controversial in the scientific community and under constant development. We present a hybrid contractility and impedance/extracellular field potential (EFP) technology, adding significant pro-maturation features to an industry-standard 96-well platform.
The impedance/EFP system monitors cellular functionality in real-time. Besides the beat rate of contractile cells, the electrical impedance spectroscopy readouts detect compound-induced morphological changes like cell density and integrity of the cellular monolayer. In the other component of the hybrid cell analysis system, the cells are cultured on bio-compliant membranes that mimic the mechanical environment of real heart tissue. This physiological environment supports the maturation of hiPSC-CMs in vitro, leading to more adult-like contractile responses including positive inotropic effects after treatment with isoproterenol, S-Bay K8644, or omecamtiv mecarbil. Parameters such as the amplitude of contraction force (mN/mm2) and beat duration also reveal downstream effects of compounds with influence on electrophysiological properties and calcium handling.
The hybrid system provides the ideal tool for holistic cell analysis, allowing preclinical cardiac risk assessment beyond the current perspectives of human-relevant cell-based assays.

The analysis of changes in contractile function and cellular integrity of human iPSC-derived cardiomyocytes is of immense importance for nonclinical drug development. A hybrid 96-well cell analysis system addresses both parameters in a real-time and physiological manner for reliable, human-relevant results, necessary for a safe transition into clinical stages.

Cardiac contractility assessment is of immense importance for the development of new therapeutics and their safe transition into clinical stages. While ...

High-Throughput Cardiotoxicity Screening Using Mature Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Monolayers

Human induced stem cell-derived cardiomyocytes (hiPSC-CMs) are used to replace and reduce the dependence on animals and animal cells for preclinical cardiotoxicity testing. In two-dimensional monolayer formats, hiPSC-CMs recapitulate the structure and function of the adult human heart muscle cells when cultured on an optimal extracellular matrix (ECM). A human perinatal stem cell-derived ECM (maturation-inducing extracellular matrix-MECM) matures the hiPSC-CM structure, function, and metabolic state in 7 days after plating. 
Mature hiPSC-CM monolayers also respond as expected to clinically relevant medications, with a known risk of causing arrhythmias and cardiotoxicity. The maturation of hiPSC-CM monolayers was an obstacle to the widespread adoption of these valuable cells for regulatory science and safety screening, until now. This article presents validated methods for the plating, maturation, and high-throughput, functional phenotyping of hiPSC-CM electrophysiological and contractile function. These methods apply to commercially available purified cardiomyocytes, as well as stem cell-derived cardiomyocytes generated in-house using highly efficient, chamber-specific differentiation protocols. 
High-throughput electrophysiological function is measured using either voltage-sensitive dyes (VSDs; emission: 488 nm), calcium-sensitive fluorophores (CSFs), or genetically encoded calcium sensors (GCaMP6). A high-throughput optical mapping device is used for optical recordings of each functional parameter, and custom dedicated software is used for electrophysiological data analysis. MECM protocols are applied for medication screening using a positive inotrope (isoprenaline) and human Ether-a-go-go-related gene (hERG) channel-specific blockers. These resources will enable other investigators to successfully utilize mature hiPSC-CMs for high-throughput, preclinical cardiotoxicity screening, cardiac medication efficacy testing, and cardiovascular research.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer an alternative to using animals for preclinical cardiotoxicity screening. A limitation to the widespread adoption of hiPSC-CMs in preclinical toxicity screening is the immature, fetal-like phenotype of the cells. Presented here are protocols for robust and rapid maturation of hiPSC-CMs.

Human induced stem cell-derived cardiomyocytes (hiPSC-CMs) are used to replace and reduce the dependence on animals and animal cells for preclinical ...

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents (HPHC)

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it is challenging to assess the contributions of individual constituents to its overall toxicity. Toxicological profiles of individual constituents as well as mixtures can be however established in vitro, by applying high through-put screening tools, which enable the profiling of Harmful and Potentially Harmful Constituents (HPHCs) of tobacco smoke, as defined by the U.S. Food and Drug Administration (FDA).2
For an initial assessment, an impedance-based instrument was used for a real-time, label-free assessment of the compound&#39;s toxicity. The instrument readout relies on cell adhesion, viability and morphology that all together provide an overview of the cell status. A dimensionless parameter, named cell index, is used for quantification. A set of different staining protocols was developed for a fluorescence imaging-based investigation and a HCS platform was used to gain more in-depth information on the kind of cytotoxicity elicited by each HPHC.
Of the 15 constituents tested, only five were selected for HCS-based analysis as they registered a computable LD50 (&#60; 20 mM). These included 1-aminonaphtalene, Arsenic (V), Chromium (VI), Crotonaldehyde and Phenol. Based on their effect in the HCS, 1-aminonaphtalene and Phenol could be identified to induce mitochondrial dysfunction, and, together with Chromium (VI) as genotoxic based on the increased histone H2AX phosphorylation. Crotonaldehyde was identified as an oxidative stress inducer and Arsenic as a stress kinase pathway activator.
This study demonstrates that a combination of impedance-based and HCS technologies provides a robust tool for in vitro assessment of CS constituents.

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents HPHC

The objective of the study was to assess the biological impact of 15 cigarette smoke constituents using a combination of an impedance-based real time cell analyzer and a high-content screening (HCS)-based platform for toxicological assessment in vitro. This study provides information on effective doses, toxicity and modes of action of the tested compounds.

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it ...

Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation

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