Toxicol. Sci. 2013 Kameoka Toxsci Kft239

Toxicological Sciences ToxSci Advance Access published October 23, 2013
A high-throughput screen for teratogens using human pluripotent stem cells

Downloaded from http://toxsci.oxfordjournals.org/ at National University of Singapore on November 30, 2013
Journal:
Manuscript ID:
Manuscript Type:
Date Submitted by the Author:
Complete List of Authors:
Toxicological Sciences
TOXSCI-13-0670.R1
Research Article
09-Oct-2013
Kameoka, Sei; Hoffmann-La Roche, Non-Clinical Safety Babiarz, Josh ; Hoffmann-La Roche, Non-Clinical Safety Kolaja, Kyle; Hoffmann-La Roche, Non-Clinical Safety Chiao, Eric; Hoffmann-La Roche, Non-Clinical Safety
In Vitro and Altenatives, alternatives to animal testing < In Vitro and Altenatives, embryonic stem cells < In Vitro and Altenatives, Developmental/Teratology < Reproductive & Developmental Toxicology
In Vitro and Alternative Methods [114]
Key Words:
Society of Toxicology Specialty Section Subject Area:
Page 1 of 34
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Title Page A high-throughput screen for teratogens using human pluripotent stem cells Sei Kameoka, Joshua Babiarz, Kyle Kolaja, and Eric Chiao*.
*Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, Nutley, New Jersey 07110, USA * To whom correspondence should be addressed. Express delivery address: Eric Chiao, Biogen Idec, 14 Cambridge Center, Bld. 6, Cambridge, MA 02142
Page 2 of 34
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Abstract There is need in the pharmaceutical and chemical industries for high-throughput human cell-based assays for identifying hazardous chemicals, thereby reducing the overall reliance on animal studies for predicting the risk of toxic responses in humans. Despite instances of human-specific teratogens such as thalidomide, the use of human cell-teratogenicity assays has just started to be explored. Herein, a human Pluripotent Stem cell Test (hPST) for identifying teratogens is described, benchmarking the in vitro findings to traditional preclinical toxicology teratogenicity studies and when available to teratogenic outcomes in humans. The hPST method employs a 3-day monolayer directed differentiation of human embryonic stem cells. The teratogenic risk of a compound is gauged by measuring the reduction in nuclear translocation of the transcription factor SOX17 in mesendodermal cells. Decreased nuclear SOX17 in the hPST model was strongly correlated with in vivo teratogenicity. Specifically, 71 drug-like compounds with known in vivo effects, including thalidomide, were examined in the hPST. A threshold of 5 M demonstrated 94% accuracy (97% sensitivity and 92% specificity). Furthermore, 15 environmental toxicants with physicochemical properties distinct from small molecule pharmaceutical agents were examined and a similarly strong concordance with teratogenicity outcomes from in vivo studies was observed. Finally, to assess the suitability of the hPST for high-throughput screens, a small library of 300 kinase inhibitors was tested, demonstrating the hPST platforms utility for interrogating teratogenic mechanisms and drug safety prediction. Thus, the hPST assay is a robust predictor of teratogenicity and appears to be an improvement over existing in vitro models.
Page 3 of 34
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Keywords teratogenicity, developmental toxicity, pluripotent stem cell, thalidomide, SOX17, high throughput screening. Introduction The disruption of embryonic development leading to malformation of a developing offspring is a devastating adverse drug response. Currently, due in part to a lack of robust alternative methods, the FDA requires several animal studies to assess the risk of reproductive and embryo-fetal developmental harm prior to the approval of a new drug that would be exposed to women of child bearing potential (Bailey et al., 2009). However, reproductive toxicology studies are often conducted late in the drug development process where unexpected positive results are most costly. Furthermore, the animal studies do not obviate concerns related to teratogens that are uniquely harmful to human fetal development, but not the animal species tested (Shuey and Kim, 2011). Beyond the realm of drug development, recent EU regulations have mandated toxicity testing for high production volume chemicals and eventually lower production volume chemicals. Some estimate that in order to fully comply with these regulations, at least 30,000 compounds would need to be tested at an overwhelming and likely unrealized cost of more than 1 billion euros (Hartung and Rovida, 2009). Therefore, new high-throughput alternatives are needed in a number of fields that can be used to aid in selecting the safest compounds for further development or prioritizing the highest risk chemicals for follow-up investigation (Basketter et al., 2012). A method for assessing a compounds teratogenic risk using mouse embryonic stem (mES) cells was pioneered by the laboratory of Hans Spielman (Seiler and Spielmann, 2011; Spielmann et al., 1997). This method, which was one of the first applications of pluripotent stem cells in toxicology, is referred to as the mouse Embryonic Stem Cell Test, or mEST. The mEST entails the spontaneous embryoid body differentiation for ten days after which beating cardiomyocytes can be observed. The teratogenic risk is assessed by use of a statistical prediction model that compares the concentrations of drug that exhibit 50% inhibition of beating embryoid bodies, 50% cytotoxicity of the mouse ES cells and the concentration inducing cytotoxicity in a mouse differentiated fibroblast line tested in parallel (Seiler and Spielmann, 2011). This original method, while landmark in its application of stem cell differentiation to infer developmental toxicity, has limitations such as the relatively long length of the assay, the labor-intensive methods required for the differentiating the cells, the qualitative nature of scoring beating embryoid bodies, and moderate accuracy of the statistical prediction model (Marx-Stoelting et al., 2009). Since the original publication describing the mEST, many independent labs have sought to improve the in vitro method for assessing teratogenic risk. Most efforts focused on
Page 4 of 34
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
increasing the assay complexity, perhaps based on the assumption that a more complex in vitro system would improve the predictivity of the assay by reflecting additional aspects of the biological complexity of human gestation. For example, attempts to improve the mouse EST have included employing toxicogenomic analysis to the standard cardiomyocyte differentiation protocol (Hewitt et al., 2010; Pennings et al., 2011; van Dartel et al., 2010), transcriptional profiling of 12 genes (Panzica-Kelly et al., 2013), adding additional differentiated cell types such as endothelial cells or bone cells to the assay (Buesen et al., 2009; Festag et al., 2007; zur Nieden et al., 2010). Although some of these methods have increased the predictivity, improvements have often been at the expense of assay throughput. The efforts to develop a human pluripotent stem cell (hPSC) based teratogenicity screen (referred to as the hPST) described herein began with the optimization of a three day, chemically defined, directed differentiation of a monolayer of hPSCs in a 96-well plate format. In contrast to some of the previous studies mentioned above, the hPST employs a seemingly counter-intuitive approach: shortening the stem cell differentiation duration by 3 fold and quantitating protein expression of a single lineage marker, SOX17, as the primary endpoint. In order to gauge utility of the hPST for addressing the needs of both the pharmaceutical and the chemical industries, 71 drug-like compounds with known in vivo effects and 15 environmental toxicants were tested with hPST system. Finally, a small molecule library of 300 kinase inhibitors was tested to assess the suitability of the hPST in a high-throughput screening setting. Together, these data suggest the hPST assay can be a robust predictor of teratogenicity and appears to be an improvement over existing in vitro models.
Page 5 of 34
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
MATERIALS and METHODS. Cell culture. All cell culture was performed at 37oC, 5% CO2. Absence of mycoplasma contamination was routinely confirmed by MycoAlert Detection Kit (#LT07-118, Lonza). Human ES cell lines H9 (from WiCell) and LSJ-1 (obtained from Stanford University) were thawed in mTeSR1 medium (#05850, StemCell Technologies) with 5 M Y-27632 dihydrochloride (#1254, Tocris). 3 million cells were plated in a 100mm cell culture dish (#430293, Corning) precoated with ES cell-qualified Matrigel (#354277, BD Bioscience). Y-27632 was included only during seeding and removed within 24 hours after seeding. mTeSR media was changed every 1 or 2 days. Cells were routinely passed by Dispase (#07923, StemCell Technologies). Cells typically reached near-confluence within 5-6 days. Directed mesendoderm differentiation. For all 96 well plate manipulations, liquid handling was performed by CyBi-Well 96-well Channel Simultaneous Pipettor with 250 DW tips. For differentiation of H9 cells, when cells were about 50% confluent they were dissociated by Accutase (#07920 Stemcell Technologies) and seeded on Matrigel-coated Black/Clear-bottom 96 well plates (#353948, BD Biosciences) in mTeSR1 with 5 M Y-27632 at a density of 10,000 cells/well. Y-27632 was added only during seeding. Media was changed every day. When cells reach ~40% confluency in the 96-well format (usually between 36-72 hours after seeding), the plates were washed with 100 l/well PBS four times (each wash was done by shaking the plate at 650rpm for 3 min by Eppendorf MixMate) and cultured for 24 hours in differentiation media I containing Advanced RPMI Medium 1640 (#12633-012, Invitrogen), 2mM L-Glutamine (#25030-081, Invitrogen), 80ng/ml human Activin A (#338-AC-050, R&D Systems), 20ng/ml human Wnt3a (#5036-WN-010, R&D Systems), 80 units/ml-0.08 mg/ml PenicillinStreptomycin (P0781, Sigma) with testing compound dissolved in a DMSO final concentration of 1%. At 24 hours, cells were washed with PBS two times, and cultured in differentiation media II containing Advanced RPMI Medium 1640, 2mM L-Glutamine, human 80ng/ml Activin A, 0.1% FBS (#10438-026, Invitrogen) and Penicillin-Streptomycin with testing compound. At 72 hours from the seeding (48 hours from addition of differentiation media II), cells were washed with PBS three times at 650 rpm (Eppendorf MixMate), fixed with 4% Formaldehyde Solution (#28908, Thermo) for 15 minutes, washed with PBS once, and then kept in immunofluorescence blocking solution containing PBS with 10% donkey serum (#S30-100ML, Millipore), 2% Sheep serum (#S22-100ml, Millipore) 0.2% Triton X-100 (#T8787, Sigma), 1% BSA (#A9576-50ML, Sigma) for 1-2 days at 4oC.
Page 6 of 34
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
For differentiation of LSJ-1, all procedures were performed in the above described manner with the exceptions that the concentration of Activin A was 40 ng/ml for both 0h and 24h time point, and that Wnt3a was 0.2ng/ml. hPST Assay quality control. Obtaining a robust, consistent differentiation (40-50% of cell population showing SOX17+ nuclei at Day 3) is critical. A suboptimal level of differentiation ( > 70% or < 20% SOX17+ cells) results in technical challenges including higher signal/noise ratio for image analysis and/or variable IC50 values. Differentiation conditions can vary depending on the pluripotent stem cell line, sub-line, passage number, and/or experimental batch of cells. Thus it is critical to optimize and control factors such as seeding density, growth factors concentration, and differentiation length to effectively establish the optimal conditions for each experiment. Cell morphology and confluency was checked every day by microscopy or Cellavista microplate analyzer (Roche Applied Science) during preparation and differentiation phases of the experiment and plates with a growth rate that showed 50% more or less than normal doubling time was not used for further analysis. Normal doubling time was the rate observed when the particular line of cell stock was made. All plates had at least 6 wells of controls treated with 5 M SB-431542 (Fig. 1D) and 2 columns (12 wells) of vehicle control treated with 1% DMSO. All plates included serial dilutions of two reference compounds (usually imatinib and thalidomide). Any plate which does not comply with the following criteria was not used for analysis. (1) Average of SOX17+/DAPI+ (%) in 12 wells of DMSO control is between 25 and 60%, and standard deviation is less than 10%. (2) Imatinib SOX17-IC50 is between 5 and 20 M. (3) Thalidomide SOX17-IC50 is less than 5 M. Once fully optimized and quality controlled frozen stocks were established, failure rate for a 96-well plate was less that 20%. Immunofluorescence. For the hPST, cells were incubated with human SOX17 NL557 affinity purified goat polyclonal antibody (#NL1924R, R&D Systems) diluted 1:20 in blocking solution for 3 hours at 4oC with shaking at 600 rpm for 1 min every hour. The antibody solution was filtered through 0.22 m filter (#SLGP033RS, Millipore) before use to remove precipitate which can interfere with image analysis. After 3 hours, plates were washed twice with blocking solution, and twice with PBS containing 0.1% Triton X-100 (each wash performed at 650rpm for 5 min (Eppendorf MixMate), and then 35 l/well of SlowFade Gold antifade reagent with DAPI (#S36938, Invitrogen) was added and stored at 4oC. For other immunofluorescence assays, the following antibodies were used at the described dilution: T-brachyury, NL557-conjugated, 1:20 (#AF2085R, R&D Systems); OCT4, 1:100
Page 7 of 34
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
(#ab19857, Abcam); NANOG, 1:10 (AF1997, R&D Systems); SOX2, 1:20, NL493conjugated (#NL20181G, R&D Systems); EOMES, 3ug/ml (#AF6166, R&D Systems); FOXA2, 1:400 (#ab40874, abcam); Alexa 488 Donkey Anti-Sheep IgG, 1:500 (#A-11015, Invitrogen); Alexa 488 Donkey Anti-rabbit IgG, 1:500 (#A-21206, Invitrogen); Alexa 488 Donkey Anti-goat IgG, 1:500 (#A-11055, Invitrogen). All primary antibodies were incubated for 2-3 hours at 4oC and secondary antibodies were incubated for 1.5 hours at 4oC.
Microscopy and image data analysis. Microscopy was performed using a Zeiss Axio Observer Z1 fluorescence imaging system with Hamamatsu ORCA-ER digital camera C4742-80. To define the background level, 6 wells on each plate were treated with 5 M SB-431542 which inhibits mesendoderm differentiation by 100% without affecting DAPI+ cell number, and the average of the 6 wells was used as the background level for that particular plate. The adjusted value (Raw Background) was used to determine IC50. High Content platform measurements were performed by Thermo Scientific Cellomics ArrayScan VTI HCS Reader. The number of SOX17+ nuclei was counted with 549 nm channel using 4 fields per well. For DAPI and Alexa488 measurement, 386 nm and 485 nm channel were used respectively. Curve fit analysis and IC50 determinations were performed by GraphPad Prism. RNA isolation and quantitative RT-PCR. RNA was isolated by TaqMan Gene Expression Cells-to-CT Kit (#AM1728, Invitrogen). Real Time quantitative RT-PCR was performed on TaqMan OpenArray RT PCR custom plates (#4456262, Life Technologies) and assessed with OpenArray NT Cycler (Life Technologies, Grand Island, NY) . Ct values of the genes of interest were normalized to the housekeeping gene peptidylprolyl isomerase A (cyclophilin A or PPIA). Compounds. All compounds were stored in a DMSO stock solution, and dissolved in media immediately before each experiment. Compounds with known poor solubility and/or whose cLogP > 5 were not selected for the study. All compounds used for hPST were internally synthesized at Hoffmann-LaRoche except following compounds: 6-Amino-nicotinamide (L06692, AlfaAesar), All-trans Retinoic Acid (554720, Calbiochem), Caffeine (27600, Sigma), CH5424802 (CT-CH542, ChemieTek), Chlorophacinone (45390,Sigma), Clopyralid (36758,Sigma), Crizotinib (4368/10, R & D Systems), Cyproconazol (46068,Sigma), Diniconazole (46049,Sigma), Dinoseb (45453,Sigma), Diquat dibromide (45422,Sigma), Dorsomorphin (P5549, Sigma), Esomeprazole (E7906, Sigma), Fluazinam (34095,Sigma), Flusilazole
Page 8 of 34
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
(45753,Sigma), Hexaconazole (34348,Sigma), Hexazinone (36129,Sigma), Imazamox (34227,Sigma), Imazapyr (37877,Sigma), KH CB19 (4262/1, R & D Systems), Methyldopa (1426002, US Pharmacopeia/USPC), o-Phenylphenol (45529,Sigma), Penicillin (P7794, Sigma), Saccharin (S6047, Sigma), Streptomycin (S9137, Sigma), Thiacloprid (37905,Sigma), TG003 (4336/50, R & D Systems), Triclopyr (32016,Sigma).
Page 9 of 34
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Results Optimizing a three day monolayer differentiation for rapid compound screening
Mesendoderm differentiation occurs at the beginning of gastrulation, one of the first steps of germ layer specification and organogenesis (Murry and Keller, 2008). As in many established directed differentiation methods (Kroon et al., 2008; Murry and Keller, 2008), differentiation is driven by a combination of recombinant growth factors: the Nodal pathway was activated by the addition of Activin A and the Wnt pathway by Wnt3a. Directed differentiation was monitored using immunostaining for well-established
endoderm markers SOX17 and FOXA2, mesoderm markers EOMES and T-brachyury, and pluripotency markers (OCT4, SOX2, and NANOG) (Fig. 1A, B, C, E, Fig. S1, Table S1). On Day 0 before differentiation, ~100% of cells were pluripotent cells and thus positive for OCT4, SOX2, NANOG, while no expression of SOX17, T-brachyury, EOMES were detected. Following the initiation of directed differentiation, pluripotency markers gradually decreased, with about 30% of cells showing SOX2 and OCT4 on day 3, while SOX17, FOXA2, Tbrachyury, and EOMES expression increased. Simultaneous staining experiments revealed that a majority (>90%) of SOX17+ cells at day 3 were also positive for EOMES (Fig. 1C, Table S1) and T-brachyury (Data not shown), suggesting that this cell population is mesendoderm rather than strictly definitive endoderm. SOX17 was selected as the marker of mesendoderm differentiation status for the hPST assay because of the highest signal-tonoise ratio for this analyte, which could be attributed to the antibody specificity to SOX17 as well as the specificity of the SOX17 nuclear translocation event. Thus, the predictive value of SOX17 nuclear translocation for assessing teratogenic risk was estimated by examining the effects of known teratogens on mesendoderm formation, using the differentiation protocol depicted in Figure 2A.
hPST screening assay performance with 71 pharmaceutical compound validation set
For the initial experiment, 71 small molecule pharmaceutical-like compounds were tested using the human ES cell line H9. All 71 compounds had existing in vivo data that interrogated the compounds teratogenic potential. 37 compounds were commercially available and 34 compounds were in-house compounds. The 34 in-house compounds all have been tested in GLP preclinical Segment 2 teratogenicity studies. For these molecules, a compound was considered positive for teratogenic risk if the observed finding in the animal study concluded either: (1) the concentration of compound that demonstrated overt embryo toxicity was lower than the concentration that resulted in maternal toxicity or (2) fetal visceral dysmorphogenesis (beyond minor skeletal defects or delays in ossification) were observed.
Page 10 of 34
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Of the 37 commercially available compounds, 20 were classified based on their FDA labels, 5 from the ECVAM mEST training set classification, and 12 based on in vivo assessments of their teratogenic risk as found in published literature. For compounds where FDA labeling was available, if the FDA pregnancy category indicated on the drug product label was either: C (animals studies demonstrated an adverse effect on the fetus, no conclusive human studies), D (positive evidence of human fetal risk, but the benefit outweighs the risk) or X (positive evidence of human fetal risk, not for use in pregnant women) the compounds were considered positive. Compounds were deemed negative for teratogenic risk if the FDA labeling was either A (animal and well controlled studies in humans failed to demonstrate risk of fetal harm) or B (animals studies failed to demonstrate teratogenic risk, no adequate studies have been completed in pregnant women). A complete list of these compounds along with their FDA pregnancy category labeling or in vivo effects is provided in Table 1 and the more detailed Table S4 that also contains the literature references used. The 71 pharmaceutical compounds with in vivo preclinical data were tested for their ability to disrupt the directed differentiation of human ES cell line H9. Initially, the combination of three markers that delineate mesendoderm, SOX17, EOMES, and Tbrachyury, were examined by immunostaining, followed by quantification via fluorescence microscopy. However it was determined that SOX17 alone provided sufficient predictive value (Table S5, S6). Thus, subsequent experiments relied solely on SOX17 nuclear staining. The interpolated concentration of test compound that resulted in 50% inhibition of the measured lineage marker relative to DMSO control (IC50) was calculated for each test compound (Fig. 2C). In addition, the concentration where a 50% reduction in cell number to control for cytotoxicity and cytostasis was determined using DAPI staining (TC50). The compound-specific IC50 and TC50 values were plotted to enable the assessment of the in vivo/in vitro assay correlation (Fig. 3). Using an IC50 of 30 M as the sole class threshold results in 94% accuracy (97% sensitivity and 92% specificity) for predicting the risk of teratogenicity (Fig. 3, Table 1, 2). Depending on the test compound, the IC50 may reflect either a compound-specific reduction of differentiation as measured by SOX17 protein without a reduction in cell number, such as that observed following thalidomide treatment (Fig. 2B), or a reduction in the number of adherent cells due to either cell death or detachment, such as that observed following diethylstilbestrol treatment (Table 1, Compounds R13 and R22, respectively). Using the number of adherent viable cells alone, as judged by TC-50, as the teratogenicity predictor, results in reduced accuracy (83%) and misclassifies compounds such as thalidomide and retinoic acid, highlighting the importance of measuring a differentiation end point like SOX17 nuclear translocation. To test whether these observations were cell line specific, a second human embryonic stem cell line, LSJ-1
Page 11 of 34
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
from Stanford University, was used to test a subset consisting of 18 compounds from the validation set, resulting in similar concordance (Fig. S3, Table S2).
Molecular analysis of disruptions in signaling pathways following thalidimide and miansarin
treatment
To test the hypothesis that the observed predictivity of the hPST reflected underlying changes in drugspecific effects relevant to the in vivo effects, changes in the major signaling pathways that guide fetal development were examine by qPCR following treatment with two compounds that impair different aspects of fetal development. The potent teratogenic effects of thalidomide resulting in phocomelia (limb shortening and loss) were attributed to inhibitory effects on vasculogenesis (Melchert and List, 2007). Mianserin has been reported to alter neural development in a whole mouse embryo culture model with 15/21 of embryos treated with 10uM for 48hrs exhibiting neural tube defects, forebrain/nasal prominence hypoplasia, defects in maxillary and mandibular arches and eye lens invagination (Lauder et al., 2000). Following the treatment of cells with either 5 M thalidomide or mianserin, (Fig. 4A) the changes in mRNA expression panel of 600 signal transduction genes were determined. Analysis of the largest fold decreases in expression were consistent with drug-specific effects (Fig. 4B). Specifically, thalidomide treatment resulted in decreases in the expression of FZD8 and WNT5B, two gene involved in limb development and VEGFA which is involved in vascular development. In contrast, treatment with mianserin changed genes affecting neural development such as GSC and ID2. These findings support the hypothesis that measuring the disruption of the single lineage marker SOX17 can reflect a broad range of drug-specific effects that alter signaling pathways important for proper embryonic development. Examination of non-pharmaceutical chemicals
Next, the performance of the hPST assay was tested with environmental contaminants including insecticides, herbicides, fungicides, and rodenticides. Due to the lack of discrete standards similar to the FDA pregnancy categories for assessing teratogenic risk of pharmaceuticals, compound with Lowest Effect Level for developmental effects (dLEL) < 50 mg/kg were regarded as positives and dLEL > 200 mg/kg as negatives. Similar arbitrarily determined thresholds were used in previous development toxicity study of pesticides (Kleinstreuer et al., 2011a). To avoid inconsistency across studies using different route of administration, the dLEL was determined from oral dose studies only. By these
Page 12 of 34
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
criteria, 15 test compounds were identified representing diverse chemical structures, functions, and molecular weights (170 - 465Da); 6 compounds with low risk for teratogenicity, and 9 compounds considered potential teratogens (Table 3). Using the threshold of 35 M SOX17-IC50, hPST predicted 13 out of 15 pesticides correctly, for an overall accuracy of 87% (Table 4). Proof-of concept HTS of a 300 compound kinase inhibitor library
To assess the feasibility of using the hPST as high-throughput screening platform and to identify putative pathways whose perturbation may increase the risks of teratogenic effects, a library of ~300 kinase inhibitors was screened (Fig. 5). 302 small chemical kinase inhibitors with targets covering a majority of the human kinome were tested at single concentration of 5 M (Fig. S4). The standard deviation from 20 DMSO controls was 5.1% for DAPI+ cell number (Fig. 5A) and 7.3% for SOX17+ cell number corresponding to Zfactors of .79 and .77, respectively, supporting the robustness of the hPST assay in highthroughput setting (Zhang et al., 1999). Compounds that selectively reduced SOX17 in the absence of cytotoxicity were selected for further examination. As a secondary screen, 10 compounds that that did not reduce DAPI+ cells (ie: were not cytotoxic at the concentration tested) yet decreased SOX17+ cells more than 90% (red column Fig. 5B) were tested in the independent LSJ-1 cell line. Following a broad dose response curve determination (1 -100 M), 5 out of the 10 compounds identified in the primary screen exhibited a clear SOX17 IC50 value below the DAPI TC50 value in the secondary screen, demonstrating qualitatively similar effects between the two independent cell lines (Fig. 5C). Analysis of Kinase target of inhibitors that promote mesendoderm differentiation
Because compounds that increase the efficiency and robustness of directed differentiation of hPSCs towards endoderm-derived tissues such as liver and pancreas could be of value to regenerative medicine applications, the top hits from the kinase library that increased SOX17 expression were also examined further (Borowiak et al., 2009; Zhang et al., 2012; Zhu et al., 2009). Among the 302 compounds tested, 6 compounds (R41 to R46) increased the number of SOX17+ cells by more than 60% (7 times the standard deviation of the DMSO controls) in both H9 and LSJ-1 cells (Table 5). Based on the DiscoveRx KinomeScan in vitro assay, all 6 compounds are predicted to be relatively selective kinase inhibitors, inhibiting between 1 to 15 kinases (data not shown). Analysis of the specific kinase targets that increased the number of SOX17+ cells at Day 3 was performed. Because some kinases were more frequently targeted by library compounds than others (for example,
Page 13 of 34
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
GAK is targeted by 116 compounds while TBK1 is targeted by only one compound), a likelihood of candidate being a true target was estimated by the ratio of the number of compounds that inhibited the kinase observed to the frequency in the library or hypergeometric distribution (Table 6). Phosphoinositide 3-kinase (PI3K), Cdc-like Kinase 4 (CLK4) and Anaplastic lymphoma kinase (ALK) were enriched in the set of compounds that increased SOX17+ cells during the primary screen. Consistent with this result, PI3K inhibitors have been shown to increase endoderm differentiation in a previous study (McLean et al., 2007).
ALK and CLK4 inhibition was further tested with additional specific kinase inhibitors. CLK1-4 are expressed in wide range of cell types including ES cells, embryoid bodies, blastocysts, and endoderm-derived organs, while ALK was detected in embryoid bodies but not in ES cells (data not shown and NCBI EST profile). None of the 8 additional ALK inhibitors tested increased SOX17+ cells significantly (Fig. 6A), suggesting that ALK is unlikely the sole target mediating the increase observed in the primary screen. However, all four CLK4 inhibitors tested, TG003 (Muraki et al., 2004), KH CB19 (Fedorov et al., 2011), and internal compounds R57 and R58 increased SOX17+ cells by at least 50% (7 times SD of DMSO control), which was more than a positive control LY294002 (Fig. 6B). CLK4, appears to be the primary target mediating the increased SOX17+ cells since TG003 and KH CB19 lack CLK3 and CLK2 inhibitor activity respectively (Anastassiadis et al., 2011; Fedorov et al, 2011) and R59, which inhibits CLK1,2,3 but not CLK4, did not increase SOX17+ cells (Fig. 6B, Table S7). Together, these data are consistent with the hypothesis that within the hPST differentiation protocol, the inhibition of CLK4 promotes the differentiation towards SOX17+ cells. Further work would be required to determine if CLK4 inhibition could be an effective means for more efficiently generating endoderm tissues.
Discussion Human cell based assays are needed to improve drug safety. Teratogenic risk assessment is an area where human cell based assays may be of particular value. Another human pluripotent based cell assay was recently described using metabolomics as the discriminating variable (Kleinstreuer et al., 2011b). In this study, H9 human ES cells were treated with compounds for three days in mTESR culture media that is designed to maintain the ES cells in a pluripotent state. With this method, a total of 34 compounds were examined resulting roughly 70-80% prediction of reproductive toxicity. Several other groups have employed transcriptomic analysis of differentiating human ES cells as a means to predict teratogenicity. Mayshar, et al. and Meganathan et al both
Page 14 of 34
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
performed transcriptome profiling of human ES cells spontaneously differentiated in embryoid bodies in the presence of human teratogens (Mayshar et al., 2011; Meganathan et al., 2012). Both groups found treatment specific transcriptional changes, supporting the feasibility of the human ES cell system for detecting human teratogen-specific effects. However, these studies were limited to a small number of compounds and did not identify a single profile capable of distinguishing a broad panel of teratogens. Recently, a European research consortium, ESNATS, aimed at developing human ES cell systems for testing developmental neurotoxicity and reproductive toxicity, reported their comparison of four protocols using the H9 human ES cell line (Krug et al., 2013). One of the protocols was a standard 14 day embryoid body differentiation and the other three protocols were directed differentiations towards neural lineages. For these studies, the ESNATS groups examined the transcriptomes following treatments with either valproic acid or methyl mercury. Although each treatment group gave rise to unique global transcriptional changes, analysis of the transcription factor binding sites revealed a large overlap between the two treatment groups, suggesting common master transcription regulators may play an important role in the observed compound-specific responses. The hPST system described here employed a directed differentiation of human ES cells towards the first stage of organogenesis and examines the expression of a single critical transcription factor that guides early embryonic developmental decisions. The use of SOX17 protein was chosen primarily for pragmatic purposes as the anti-SOX17 antibody exhibited the highest signal-to-noise ratio (5 to 10 fold) and lowest inter-experimental (both plate-to-plate and day-to-day) variance. In our hands, the expression levels of T-brachyury varied greatly among cell populations within a single well and the fluorescence intensity of EOMES and FOXA2 was lower due to lower level of proteins and/or lower avidity of antibody. Similar compound dependent changes in other transcription factors essential to the maintenance of pluripotency (NANOG, OCT4, SOX2) and early germ layer specification (Tbrachyury, EOMES) were observed in the hPST context (data not shown). Therefore, we hypothesize that interrogation of the compound effects during the very first 72 hours of differentiation via single cell quantification of a key regulator of early germ layer specification may be more important than the specific use of SOX17. Furthermore, it may be that examination of protein expression instead of mRNA regulation adds to the sensitivity of the assay since we have observed that the detection of mRNA changes often lags behind changes observed in protein levels. For instance, upon differentiation, loss of OCT4 protein can be observed prior to changes in OCT4 mRNA (data not shown). Given the complexity and duration of human gestation, it may be a surprise that such a short assay would have predictivity for a broad selection of known teratogens. This may be explained by the broad transcriptional competence of undifferentiated pluripotent cells. As
Page 15 of 34
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
differentiation and development proceeds, lineage specification is accompanied by restriction of the transcriptional competence of tissue specific genes (Fig. S5). Thus, examining the very first stages of differentiation allows the interrogation the broadest milieu of signaling pathways. In contrast, an assay based on examining many separate terminally differentiated lineages may compound the cumulative false detection rate and decrease the assays throughput. Based on the findings with the 71 compounds tested, the hPST high predictivity did not rely on knowledge of the in vivo exposure of the drugs in question. For high-throughput safety screens to be effectively employed early in the drug development process, precise knowledge of in vivo potency or in utero exposure levels cannot be a requirement. Clearly, determining the fetal exposures (either total or extrapolated free fraction) in vivo is not a routine or straightforward task. Nevertheless, if true or calculated exposure levels are available, an additional risk assessment evaluation can occur by estimating safety margins for individual compounds in question on a case-by-case basis. The abbreviated duration of the hPST combined with analysis of the single lineage marker increases the theoretical throughput while maintaining sufficient predictive value for ultimate goal of prioritizing compounds and chemicals for further drug development or safety assessments. One potential use for the hPST may be to help prioritize which production volume chemicals or environmental contaminants to prioritize for assessment in definitive reproductive toxicity animal studies. For instance, these compounds could be rapidly screened a single concentration such as 5 M and compounds that showed no cytotoxicity yet selectively decreased SOX17 by greater than 50% versus DMSO controls could be prioritized for further study. Such chemicals may pose greatest risk of selective developmental toxicity in the absence of overt maternal toxicity, similar to thalidomide. Although the quantitation SOX17 protein nuclear localization as the single endpoint of the hPST assay is straightforward, achieving robust and reproducible baseline differentiation conditions was not trivial. This required extensive optimization of growth factor treatments and large master banks of quality controlled cryopreserved cells. This may be due to stochastic factors involved in human ES cell differentiation or inherent passage-topassage and line-to-line variability. Nevertheless, with protocol optimizations, adaptation of this method to other human pluripotent cell lines, including induced pluripotent stem cell lines should be possible. When adapting the assay to the LSJ-1 cell line, we observed that the concentration of WNT3A added needed reduced to 0.1 ng/ml. If LSJ-1 cells were treated with WNT3A concentrations used for H9 differentiation (20 ng/ml), the SOX17 was often expressed in 100% of the cells and the assay no longer provided an adequate level of sensitivity. Thus critical assay components such as concentrations and duration of growth factor treatments to generate mesendoderm must be carefully determined. To assist in the
Page 16 of 34
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
assay quality control, we typically used thalidomide, SB-431542, and imatinib as reference compounds for a monitor of sensitivity. Although 40-60% differentiation was chosen as the starting point for these studies, since the method relies on comparing the ratio of mesendoderm formation in drug treated samples versus control samples, the exact percent mesendoderm cells should not be critical as long as the efficiency is consistent from well to well. In order to best judge the broader applicability of the hPST system described herein, it will be important for other groups to reproduce these findings in independent laboratory settings. In general, human cell based assays can improve safety evaluation. Teratogenicity assessment is an area where human cell based assays have not been implemented in earnest. The data presented herein demonstrate that the hPST can serve as a valuable tool to screen compounds for teratogenic risk, generate novel, testable hypotheses aimed at investigating the molecular mechanisms driving teratogenicity and to identify new possible targets capable of modulating the mesendoderm developmental pathway.
Acknowledgements We would like to thank Claudia McGinnis and Nicole Clemann for their helpful discussions and Thomas Weiser and Thomas Singer for their support during this work.
Page 17 of 34
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Supplementary Data description. Figure S1: Expression of pluripotent and endoderm markers. Figure S2: Example of dense and sparse region observed in differentiation day 3 of H9 cells. Figure S3: Dose-dependent effect of thalidomide on mesendoderm formation in H9 and LSJ-1 cells. Figure S4: Characterization of Kinase Inhibitor Library. Figure S5: Lineage restriction/specification hypothesis Table S1: Population analysis of H9 cells by 3 pluripotent and 4 mesendoderm markers during 3 day differentiation. Table S2: Concordance of the hPST result determined in H9 cells and LSJ-1 cells. Table S3: CAS# and references for 15 environmental toxicants. Table S4: Full table for hPST result of 71 drugs including CAS# and references. Table S5: Addition of EOMES markers does not increase sensitivity. Table S6: Analysis of SOX17+ and EOMES+ cells by double immuno-staining. Table S7: Kinase inhibition profile of CLK4 inhibitors.
Page 18 of 34
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
References
Anastassiadis, T., Deacon, S. W., Devarajan, K., Ma, H. and Peterson, J. R. (2011). Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity. Nat Biotechnol 29(11), 1039-45. 29 Bailey, G. P., Wise, L. D., Buschmann, J., Hurtt, M. and Fisher, J. E. (2009). Pre- and postnatal developmental toxicity study design for pharmaceuticals. Birth Defects Research Part B: 437-445. Developmental and Reproductive Toxicology 86(6), 86 Basketter, D. A., Clewell, H., Kimber, I., Rossi, A., Blaauboer, B., Burrier, R., Daneshian, M., Eskes, C., Goldberg, A., Hasiwa, N., et al. (2012). A roadmap for the development of alternative (nonanimal) methods for systemic toxicity testing - t4 report*. ALTEX 29(1), 3-91. 29 Basma, H., Soto-Gutierrez, A., Yannam, G. R., Liu, L., Ito, R., Yamamoto, T., Ellis, E., Carson, S. D., Sato, S., Chen, Y., et al. (2009). Differentiation and transplantation of human embryonic stem cell-derived hepatocytes. Gastroenterology 136(3), 990-9. 136 Borowiak, M., Maehr, R., Chen, S., Chen, A. E., Tang, W., Fox, J. L., Schreiber, S. L. and Melton, D. A. (2009). Small molecules efficiently direct endodermal differentiation of mouse and human embryonic stem cells. Cell Stem Cell 4(4), 348-58. Buesen, R., Genschow, E., Slawik, B., Visan, A., Spielmann, H., Luch, A. and Seiler, A. (2009). Embryonic stem cell test remastered: comparison between the validated EST and the new 389-400. molecular FACS-EST for assessing developmental toxicity in vitro. Toxicol Sci 108(2), 108 Fedorov, O., Huber, K., Eisenreich, A., Filippakopoulos, P., King, O., Bullock, A. N., Szklarczyk, D., Jensen, L. J., Fabbro, D., Trappe, J., et al. (2011). Specific CLK inhibitors from a novel chemotype for regulation of alternative splicing. Chemistry & biology 18(1), 67-76. 18 Festag, M., Viertel, B., Steinberg, P. and Sehner, C. (2007). An in vitro embryotoxicity assay based on the disturbance of the differentiation of murine embryonic stem cells into endothelial cells. II. Testing of compounds. Toxicology in Vitro 21(8), 1631-1640. 21 Hartung, T. and Rovida, C. (2009). Chemical regulators have overreached. Nature 460(7259), 460 1080-1. Hewitt, M., Ellison, C. M., Enoch, S. J., Madden, J. C. and Cronin, M. T. D. (2010). Integrating (Q)SAR models, expert systems and read-across approaches for the prediction of developmental toxicity. Reproductive Toxicology 30(1), 147-160. 30 Kleinstreuer, N. C., Smith, A. M., West, P. R., Conard, K. R., Fontaine, B. R., Weir-Hauptman, A. M., Palmer, J. A., Knudsen, T. B., Dix, D. J., Donley, E. L., et al. (2011a). Identifying developmental toxicity pathways for a subset of ToxCast chemicals using human embryonic stem cells and metabolomics. Toxicology and applied pharmacology 257(1), 111-21. 257 Kleinstreuer, N. C., Smith, A. M., West, P. R., Conard, K. R., Fontaine, B. R., Weir-Hauptman, A. M., Palmer, J. A., Knudsen, T. B., Dix, D. J., Donley, E. L., et al. (2011b). Identifying developmental toxicity pathways for a subset of ToxCast chemicals using human embryonic stem cells and metabolomics. Toxicol Appl Pharmacol 257(1), 111-21. 257 Kroon, E., Martinson, L. A., Kadoya, K., Bang, A. G., Kelly, O. G., Eliazer, S., Young, H., Richardson, M., Smart, N. G., Cunningham, J., et al. (2008). Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo. Nature 443-52. biotechnology 26(4), 26 Krug, A. K., Kolde, R., Gaspar, J. A., Rempel, E., Balmer, N. V., Meganathan, K., Vojnits, K., Baquie, M., Waldmann, T., Ensenat-Waser, R., et al. (2013). Human embryonic stem cell-derived test systems for developmental neurotoxicity: a transcriptomics approach. Archives of toxicology 87(1), 123-43. 87 Lauder, J. M., Wilkie, M. B., Wu, C. and Singh, S. (2000). Expression of 5-HT(2A), 5-HT(2B) and 5HT(2C) receptors in the mouse embryo. International journal of developmental neuroscience : 653-62. the official journal of the International Society for Developmental Neuroscience 18(7), 18
Page 19 of 34
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Marx-Stoelting, P., Adriaens, E., Ahr, H. J., Bremer, S., Garthoff, B., Gelbke, H. P., Piersma, A., Pellizzer, C., Reuter, U., Rogiers, V., et al. (2009). A review of the implementation of the embryonic stem cell test (EST). The report and recommendations of an ECVAM/ReProTect Workshop. Altern Lab Anim 37(3), 313-28. 37 Mayshar, Y., Yanuka, O. and Benvenisty, N. (2011). Teratogen screening using transcriptome profiling of differentiating human embryonic stem cells. Journal of cellular and molecular 1393-401. medicine 15(6), 15 McLean, A. B., D'Amour, K. A., Jones, K. L., Krishnamoorthy, M., Kulik, M. J., Reynolds, D. M., Sheppard, A. M., Liu, H., Xu, Y., Baetge, E. E., et al. (2007). Activin a efficiently specifies definitive endoderm from human embryonic stem cells only when phosphatidylinositol 3-kinase signaling 29-38. is suppressed. Stem Cells 25(1), 25 Meganathan, K., Jagtap, S., Wagh, V., Winkler, J., Gaspar, J. A., Hildebrand, D., Trusch, M., Lehmann, K., Hescheler, J., Schluter, H., et al. (2012). Identification of thalidomide-specific transcriptomics and proteomics signatures during differentiation of human embryonic stem cells. PloS one 7(8), e44228. Melchert, M. and List, A. (2007). The thalidomide saga. The international journal of biochemistry 1489-99. & cell biology 39(7-8), 39 Muraki, M., Ohkawara, B., Hosoya, T., Onogi, H., Koizumi, J., Koizumi, T., Sumi, K., Yomoda, J., Murray, M. V., Kimura, H., et al. (2004). Manipulation of alternative splicing by a newly 279 24246-54. developed inhibitor of Clks. J Biol Chem 279(23), Murry, C. E. and Keller, G. (2008). Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell 132(4), 661-80. 132 Panzica-Kelly, J. M., Brannen, K. C., Ma, Y., Zhang, C. X., Flint, O. P., Lehman-McKeeman, L. D. and Augustine-Rauch, K. A. (2013). Establishment of a molecular embryonic stem cell developmental toxicity assay. Toxicological sciences : an official journal of the Society of 447-57. Toxicology 131(2), 131 Pennings, J. L. A., van Dartel, D. A. M., Robinson, J. F., Pronk, T. E. and Piersma, A. H. (2011). Gene set assembly for quantitative prediction of developmental toxicity in the embryonic stem cell test. Toxicology 284(1-3), 63-71. 284 Seiler, A. E. and Spielmann, H. (2011). The validated embryonic stem cell test to predict embryotoxicity in vitro. Nat Protoc 6(7), 961-78. Shuey, D. and Kim, J. H. (2011). Overview: developmental toxicology-new directions. Birth Defects Res B Dev Reprod Toxicol. Spielmann, H., Pohl, I., Doring, M. and Liebsch, M. (1997). The embryonic stem cell test (EST), an in vitro embryotoxicity test using two permanent mouse cell lines: 3T3 fibroblasts and embryonic stem cells. In vitro Toxicology 10, 10 119-127. van Dartel, D. A. M., Pennings, J. L. A., de la Fonteyne, L. J. J., van Herwijnen, M. H., van Delft, J. H., van Schooten, F. J. and Piersma, A. H. (2010). Monitoring Developmental Toxicity in the Embryonic Stem Cell Test Using Differential Gene Expression of Differentiation-Related Genes. Toxicological Sciences 116(1), 130-139. 116 Zhang, J. H., Chung, T. D. and Oldenburg, K. R. (1999). A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. Journal of biomolecular screening 4(2), 67-73. Zhang, Y., Li, W., Laurent, T. and Ding, S. (2012). Small molecules, big roles -- the chemical manipulation of stem cell fate and somatic cell reprogramming. J Cell Sci 125(Pt 23), 5609-20. 125 Zhu, S., Wurdak, H., Wang, J., Lyssiotis, C. A., Peters, E. C., Cho, C. Y., Wu, X. and Schultz, P. G. (2009). A small molecule primes embryonic stem cells for differentiation. Cell Stem Cell 4(5), 416-26. zur Nieden, N. I., Davis, L. A. and Rancourt, D. E. (2010). Comparing three novel endpoints for developmental osteotoxicity in the embryonic stem cell test. Toxicology and Applied 91-97. Pharmacology 247(2), 247
Page 20 of 34
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Figure Legends. FIG. 1: Mesendoderm formation in monolayer culture of H9 cells. (A) Immunofluorescence staining shows expression of endoderm marker SOX17 and mesoderm marker EOMES and T-brachyury during differentiation. Pictures of DAPI and SOX17 show the same field. (B) Triple-staining of cells with DAPI, SOX17 and pluripotency marker OCT4 shows that most SOX17-negative cell population are undifferentiated cells. (C) Triple-staining of cells with DAPI, SOX17 and EOMES shows that majority of dense clustered cell population express both SOX17 and EOMES markers while dispersed cells
show neither markers. (D) H9 cells treated with 5 M SB-431542, a known inhibitor of mesendoderm formation shows no SOX17 expression. All scale bars, 100 m. (E) Time course of expression of mesendoderm markers and pluripotent markers. H9 cells were differentiated in the presence of DMSO vehicle control for 3 days and population was analyzed by immunofluorescence staining with 4 markers. . FIG. 2: Dose-dependent effect of thalidomide on mesendoderm formation. (A) Schematic figure showing the timeline of mesendoderm differentiation, compound dosing and immunostaining. (B) H9 cells were differentiated in the presence of different concentration of thalidomide for 3 days and immunofluorescence staining was performed. All scale bars, 100 m. Number of DAPI+ and SOX17+ cells were analyzed and dose dependent curve was drawn (C). All error bars are SD (n = 2)
FIG. 3: Plot of SOX17 and DAPI TC50 values for 71 tested pharmaceutical compounds. The colored boxes on the X-axis delineate the compounds tested. Boxes in red are true positives, blue boxes are true negatives and yellow boxes are incorrectly classified at the 30 M SOX17 IC50 threshold.
FIG. 4: Cellular phenotype and transcriptional profile associated with thalidomide and mianserin treatment. (A) Triple-staining of cells with DAPI, SOX17 and pluripotency marker OCT4 shows cellular phenotype associated with 5 M thalidomide or 15 M mianserin. (B) Effect of thalidomide and mianserin on mRNA level of eight genes, after normalization with DMSO control.
Page 21 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
FIG. 5: Screen of 300 Kinase inhibitor library. H9 cells were differentiated in the presence of library compounds (n = 302) at 5 M according to the standard hPST protocol. (A) SD of DMSO control (n = 20) was 5.1% for DAPI+ cells, and Z-factor was 0.79. Dotted lines indicate 3 x SD of DMSO controls. (B). Frequency histogram of SOX17 expression for compounds that did not exhibit cytotoxicity as judged by a reduction in DAPI per well. SD of DMSO control (n = 20) was 7.3% for SOX17+ cells, and Z-factor was 0.77. Dotted lines indicate 3 x SD of DMSO controls. 10 compounds which decreased SOX17+ cells more than 90% (red column) were further analyzed by secondary dose (1 -100 M) experiment performed in duplicates using LSJ-1 cells (C).
FIG. 6: Effect of ALK and CLK4 inhibitors on mesendoderm differentiation. LSJ-1 cells were differentiated in the presence of compound at 1.5 or 5 M according to the standard hPST protocol. Number of SOX17+ cells were quantitated at differentiation day 3, and average of duplicates were shown. Bars = SD. DMSO vehicle control and LY-294002 serve as negative and positive control respectively. (A) CH5424802, Crizotinib, R51 to R56 are all ALK inhibitors. (B) +, -, w signs in the table show the presence of inhibition, lack of inhibition, and weak inhibition of each kinase respectively.
Page 22 of 34
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Table 1: Summary of IC50, TC50 determined by human pluripotent stem cell test (hPST) and known animal toxicity of 71 pharmaceutical compounds.
hPST Compounds Compound a Source FDA Label
Segment 2 Findings Species c Examined
Test outcome
SOX17 IC50 (M)
DAPI TC50 (M)
Visceral Dysmorpho -genesis?
ET < MTb
Actinomycin D
Positive
<0.01
<0.01
ECVAM FDA Label
All-trans Retinoic Acid
Positive
0.07
33
ECVAM
Cytarabine
Positive
0.08
0.19
ECVAM FDA Label
SB-431542
Positive
0.08
69
Literature
R1
Positive
<0.1
5.0
Roche
Rt
R2
Positive
<0.1
<0.1
Roche
Rt
R3
Positive
<0.1
<0.1
Roche
Rt, Rb
Isotretinoin
Positive
0.2
140
FDA Label
Bromodeoxyuridine
Positive
0.2
3.4
ECVAM
R4
Positive
0.2
0.3
Roche
Rt
Doxorubicin
Positive
0.3
<0.3
FDA Label
Dorsomorphin
Positive
0.3
2.4
Literature
Thalidomide
Positive
0.5
>200
FDA Label
5-Fluorouracil
Positive
0.6
0.7
FDA Label
Dasatinib
Positive
<1
<1
FDA Label
Sorafenib
Positive
<2
<2
FDA Label
Valproic Acid
Positive
2.6
3.3
FDA Label
Sunitinib
Positive
2.8
3.6
FDA Label
Ziprasidone
Positive
2.8
5.1
FDA Label
Rt, Rb
Mianserin
Positive
3.4
60
Literature
Vandetanib
Positive
3.5
4.4
FDA Label
Diethylstilbestrol
Positive
4.4
4.4
FDA Label
Tegaserod
False Positive
4.6
4.5
FDA Label
R5
Positive
<5
5.9
Roche
Rt
R6
Positive
<5
78.0
Roche
Rt
R7
False Positive
5.3
5.6
Roche
Rt
6aminonicotinamide
Positive
7.0
8.0
Literature
Rt
Ritanserin
Positive
7.3
27
Literature
R8
Positive
9.6
9.4
Roche
Y (Rt, C)
Rt, Rb, C
R9
Positive
9.6
9.6
Roche
Y (Rb Only)
Rt, Rb
Gefitinib
Positive
10
10
FDA Label
R10
Positive
10
12
Roche
Y (Rb Only)
Rt, Rb
R11
Positive
10.4
9.7
Roche
Y (Rb Only)
Rt, Rb
Imatinib
Positive
11
22
FDA Label
R12
False Positive
12
23
Roche
Rt, Rb
R13
Positive
12
30
Roche
Y (Rt Only)
Rt, Rb
R14
Positive
26
>200
Roche
Rb
Esomeprazole
Negative
32
101
FDA Label
Page 23 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
R15
Negative
33
33
Roche
Rt, Rb
R16
Negative
33
45
Roche
Rt, Rb
Folate
Negative
39
84
FDA Label
R17
Negative
47
46
Roche
Rt, Rb
R18
False Negative
47
72
Roche
Rt, Rb
R19
Negative
65
>200
Roche
Rt, Rb
R20
Negative
68
55
Roche
Rt
R21
Negative
76
80
Roche
Rt, Rb
R22
Negative
>80
>80
Roche
Rt, Rb, C
R23
Negative
>80
>80
Roche
Rb
R24
Negative
>80
>80
Roche
Rt, Rb
R25
Negative
>80
>80
Roche
Rt, Rb
R26
Negative
>80
>80
Roche
Rt, C
R27
Negative
>80
>80
Roche
Rt, Rb
R28
Negative
>80
>80
Roche
Rt
R29
Negative
>80
>80
Roche
Rt, Rb
R30
Negative
>80
>80
Roche
Rt
Catechin
Negative
97
200.0
Literature
Rt
R31
Negative
124
200.0
Roche
Rt, Rb
Lisuride
Negative
137
122
Literature
Rt
R32
Negative
160
171
Roche
Rt, C
Niacin
Negative
>200
>200
FDA Label
Aspirin
Negative
>200
>200
Literature
Ibuprofen
Negative
>200
>200
Literature
Rt
Aciclovir
Negative
>200
>200
FDA Label
R33
Negative
>200
>200
Roche
Rt
Ketanserin
Negative
>200
>200
Literature
R34
Negative
>200
>200
Roche
Rt, Rb
Streptomycin
Negative
>200
>200
Literature
Methyldopa
Negative
272
462
FDA Label
Saccharin
Negative
>400
>400
ECVAM
Caffeine
Negative
>400
>400
ECVAM
Penicillin
Negative
>400
>400
ECVAM
Note. 71 compounds, 37 commercially available compounds and 34 in-house compounds that had been tested in animal Segment 2 teratogenicity studies were examined. FDA label: A (animal and well controlled studies in humans failed to demonstrate risk of fetal harm); B (animals studies failed to demonstrate teratogenic risk, no adequate studies have been completed in pregnant women). C (animals studies demonstrated an adverse effect on the fetus, no conclusive human studies); D (positive evidence of human fetal risk, but the benefit outweighs the risk); X (positive evidence of human fetal risk, not for use in pregnant women). The full table including references and CAS# is found in Table S4. a ECVAM indicates compound used European Centre for the Validation of Alternative Methods study. b Abbreviation: ET < MT (Embryotoxicity is smaller than maternal toxicity); Y (Yes); N (No) c Abbreviation: M (mouse), Rb (rabbit), Rt (rat), C (cynomolgus monkey), H (human), Z (zebrafish) d C when Recommended Dietary Allowance (RDA) is exceeded
Page 24 of 34
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Table 2: Summary of human pluripotent stem cell test (hPST) of 71 pharmaceutical compounds.
Pharmaceuticals
30 M Cut-off
True
FALSE
Positive
True positive
False positive
Positive predictive value
0.92 3 34 Test outcome Negative False Negative predictive True value negative negative 1 33 0.97
Accuracy Specificity Sensitivity 0.92 0.97
0.94
Page 25 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Table 3: Summary of human pluripotent stem cell test (hPST) result and known animal toxicity of 15 environmental toxicants.
hPST IC50, TC50 (M) Rat ( mg/kg) Rabbit ( mg/kg)
Compound name
Compound class
DAPI
SOX1 7
mLEL
dLEL
mL EL
Rat Phenoty pe
Rabbit Phenotype
hPST classfication
In vivo classification
Concordanc e
dLEL
Chlorophaci none
Rodenticid e
< 1.5
0.1
0.01
0.02
0.02
KD
DF
Pos
Pos
Correct
Fluazinam
Fungicide
< 1.5
< 1.5
250
50
12
SB, KD
IO, DS
Pos
Pos
Correct
Diquat dibromide
Herbicide
4.4
40
IO, KD, SD
IO, SB, LD
Pos
Pos
Correct
Dinoseb
Herbicide
9.1
7.5
10
10
ND
10
IO
BD, DD, SD
Pos
Pos
Correct
Diniconazole
Fungicide
13.4
10.5
20
ND
ND
IO, SB, KD
ND
Pos
Pos
Correct
Hexaconazol e
Fungicide
34.3
31.9
250
2.5
100
50
IO, SB
WD
Pos
Pos
Correct
Flusilazole
Fungicide
> 100
33.5
50
0.4
35
35
KD
WD
Pos
Pos
Correct
Clopyralid
Herbicide
> 100
> 100
250
> 250
250
250
None
BD
Neg
Neg
Correct
Imazamox
Herbicide
> 100
> 100
1000
> 1000
600
> 900
None
None
Neg
Neg
Correct
Imazapyr
Herbicide
> 100
> 100
1000
> 1000
> 400
> 400
None
None
Neg
Neg
Correct
oPhenylpheno l
Fungicide
> 100
> 100
700
> 700
100
> 250
None
None
Neg
Neg
Correct
Triclopyr
Herbicide
> 100
> 100
50
200
ND
ND
IO
ND
Neg
Neg
Correct
Hexazinone
Herbicide
> 100
> 100
400
900
125
> 125
None
None
Neg
Neg
Correct
Thiacloprid
Insecticide
> 100
> 100
50
10
10
10
IO, SB, MB, KD
IO, SB
Neg
Pos
False negative
Cyproconaz ol
Fungicide
> 100
> 100
12
12
50
10
IO, SB, SD, BD
SD
Neg
Pos
False negative
Page 26 of 34
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Note: Compounds
are classified as Pos(itive) when both rat and rabbit dLEL are less than 50 mg/kg, Neg(ative) when they are more than 200 mg/kg. For Triclopyr and Diniconazole, only rat dLEL was used as no rabbit data was available. For all compounds, dLEL obtained from orally administered dose study were used. Compounds are predicted to be Pos by hPST when SOX17 IC50 is less than 35 M. Developmentally toxic phenotype observed at less than 300 mg/kg dose are shown. Phenotype abbreviation: Supernumerary bone (SB), Incomplete ossification (IO), Misshapen bone (MB), Other skeletal defects (SD), Weight decrease (Basma et al.), Kidney defects (KD), Dead fetus (DF), Liver defects (LD), Brain defects (BD), Diaphragmatic defects (DD), Not determined (ND). CAS # and references are included in Table S3.
Page 27 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Table 4: Summary of human pluripotent stem cell test (hPST) of 15 environmental toxicants.
Environmental Toxicants
35 M Cut-off
True
FALSE
Positive
True positive
False positive
Positive predictive value
Test outcome Negative False negative
1.00
True negative
Negative predictive value
0.75
Accuracy Specificiy Sensitivity 1.00 0.78
0.87
Table 5: Effect of 6 compounds on number of SOX17+ cells in H9 and LSJ-1.
R41 R42 R43 R44 R45 R46
Number of SOX17+ cells compared to DMSO control (%) H9 LSJ-1 211 733 201 756 192 689 190 811 179 278 163 767
Page 28 of 34
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Table 6: List of kinase targets which were found enriched in the 6 compound which increased the number of SOX17+ cells. Freq Rank Kinase in Obs2 Obs/Freq 1 library 1 PI3K 0.214 63 283 2 0.136 CLK4 22 3 3 0.118 ALK 17 2 4 0.111 AURKA 9 1 5 0.095 PIM2 21 2 6 0.091 STK4 11 1 7 0.080 AXL 25 2 8 0.071 LTK 14 1 9 0.071 PHKG2 14 1 10 0.067 MAP4K4 15 1 11 0.063 CSNK1D 32 2 12 0.059 CSNK1A1L 17 1 13 0.056 MERTK 18 1 14 0.045 CLK1 44 2 15 0.044 STK3 45 2 16 0.040 VEGFR2 25 1 17 0.038 TNIK 26 1 18 0.036 FLT3 55 2 19 0.034 CLK2 58 2 20 0.034 FLT1 29 1
Note: number of compounds in the entire library which inhibit a particular kinase more than 95% at 10M concentration determined by KINOMEscan assay .
2 1the
Observed number of compound which inhibited a particular kinase more than 95% at 10M in the 6 compounds.
PI3K, the sum of 6 subunits (PIK3C2B, PIK3C2G, PIK3CA , PIK3CB, PIK3CD, PIK3CG) was shown.
3For
Page 29 of 34
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
FIG. 1: Mesendoderm formation in monolayer culture of H9 cells. (A) Immunofluorescence staining shows expression of endoderm marker SOX17 and mesoderm marker EOMES and T-brachyury during differentiation. Pictures of DAPI and SOX17 show the same field. (B) Triplestaining of cells with DAPI, SOX17 and pluripotency marker OCT4 shows that most SOX17-negative cell population are undifferentiated cells. (C) Triple-staining of cells with DAPI, SOX17 and EOMES shows that majority of dense clustered cell population express both SOX17 and EOMES markers while dispersed cells show neither markers. (D) H9 cells treated with 5 M SB-431542, a known inhibitor of mesendoderm formation shows no SOX17 expression. All scale bars, 100 m. (E) Time course of expression of mesendoderm markers and pluripotent markers. H9 cells were differentiated in the presence of DMSO vehicle control for 3 days and population was analyzed by immunofluorescence staining with 4 markers.
179x274mm (300 x 300 DPI)
Page 30 of 34
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
FIG. 2: Dose-dependent effect of thalidomide on mesendoderm formation. (A) Schematic figure showing the timeline of mesendoderm differentiation, compound dosing and immunostaining. (B) H9 cells were differentiated in the presence of different concentration of thalidomide for 3 days and immunofluorescence staining was performed. All scale bars, 100 m. Number of DAPI+ and SOX17+ cells were analyzed and dose dependent curve was drawn (C). All error bars are SD (n = 2)
189x263mm (300 x 300 DPI)
Page 31 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
FIG. 3: Plot of SOX17 and DAPI TC50 values for 71 tested pharmaceutical compounds. The colored boxes on the X-axis delineate the compounds tested. Boxes in red are true positives, blue boxes are true negatives and yellow boxes are incorrectly classified at the 30 M SOX17 IC50 threshold.
194x140mm (300 x 300 DPI)
Page 32 of 34
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
FIG. 4: Cellular phenotype and transcriptional profile associated with thalidomide and mianserin treatment. (A) Triple-staining of cells with DAPI, SOX17 and pluripotency marker OCT4 shows cellular phenotype associated with 5 M thalidomide or 15 M mianserin. (B) Effect of thalidomide and mianserin on mRNA level of eight genes, after normalization with DMSO control. 246x239mm (300 x 300 DPI)
Page 33 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
FIG. 5: Screen of 300 Kinase inhibitor library. H9 cells were differentiated in the presence of library compounds (n = 302) at 5 M according to the standard hPST protocol. (A) SD of DMSO control (n = 20) was 5.1% for DAPI+ cells, and Z-factor was 0.79. Dotted lines indicate 3 x SD of DMSO controls. (B). Frequency histogram of SOX17 expression for compounds that did not exhibit cytotoxicity as judged by a reduction in DAPI per well. SD of DMSO control (n = 20) was 7.3% for SOX17+ cells, and Z-factor was 0.77. Dotted lines indicate 3 x SD of DMSO controls. 10 compounds which decreased SOX17+ cells more than 90% (red column) were further analyzed by secondary dose (1 -100 M) experiment performed in duplicates using LSJ-1 cells (C).
200x167mm (300 x 300 DPI)
Page 34 of 34
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
FIG. 6: Effect of ALK and CLK4 inhibitors on mesendoderm differentiation. LSJ-1 cells were differentiated in the presence of compound at 1.5 or 5 M according to the standard hPST protocol. Number of SOX17+ cells were quantitated at differentiation day 3, and average of duplicates were shown. Bars = SD. DMSO vehicle control and LY-294002 serve as negative and positive control respectively. (A) CH5424802, Crizotinib, R51 to R56 are all ALK inhibitors. (B) +, -, w signs in the table show the presence of inhibition, lack of inhibition, and weak inhibition of each kinase respectively. 256x399mm (300 x 300 DPI)

Toxicol. Sci. 2013 Kameoka Toxsci Kft239

Enviado por

Dados do documento

Direitos autorais

Formatos disponíveis

Compartilhar este documento

Compartilhar ou incorporar documento

Opções de compartilhamento

Você considera este documento útil?

Este conteúdo é inapropriado?

Direitos autorais:

Formatos disponíveis

Toxicol. Sci. 2013 Kameoka Toxsci Kft239

Enviado por

Direitos autorais:

Formatos disponíveis

Toxicological Sciences ToxSci Advance Access published October 23, 2013

A high-throughput screen for teratogens using human pluripotent stem cells

Society of Toxicology Specialty Section Subject Area:

Downloaded from http://toxsci.oxfordjournals.org/ at National University of Singapore on November 30, 2013

Downloaded from http://toxsci.oxfordjournals.org/ at National University of Singapore on November 30, 2013

hPST screening assay performance with 71 pharmaceutical compound validation set

Molecular analysis of disruptions in signaling pathways following thalidimide and miansarin

Downloaded from http://toxsci.oxfordjournals.org/ at National University of Singapore on November 30, 2013

Downloaded from http://toxsci.oxfordjournals.org/ at National University of Singapore on November 30, 2013

Downloaded from http://toxsci.oxfordjournals.org/ at National University of Singapore on November 30, 2013

hPST Compounds Compound a Source FDA Label

Segment 2 Findings Species c Examined

SOX17 IC50 (M)

DAPI TC50 (M)

Visceral Dysmorpho -genesis?

ECVAM FDA Label

All-trans Retinoic Acid

Downloaded from http://toxsci.oxfordjournals.org/ at National University of Singapore on November 30, 2013

ECVAM FDA Label

Downloaded from http://toxsci.oxfordjournals.org/ at National University of Singapore on November 30, 2013

Positive predictive value

Downloaded from http://toxsci.oxfordjournals.org/ at National University of Singapore on November 30, 2013

Accuracy Specificity Sensitivity 0.92 0.97

Downloaded from http://toxsci.oxfordjournals.org/ at National University of Singapore on November 30, 2013

IO, SB, MB, KD

IO, SB, SD, BD

Downloaded from http://toxsci.oxfordjournals.org/ at National University of Singapore on November 30, 2013

Positive predictive value

Test outcome Negative False negative

Negative predictive value

Accuracy Specificiy Sensitivity 1.00 0.78

Table 5: Effect of 6 compounds on number of SOX17+ cells in H9 and LSJ-1.

R41 R42 R43 R44 R45 R46

Downloaded from http://toxsci.oxfordjournals.org/ at National University of Singapore on November 30, 2013

Downloaded from http://toxsci.oxfordjournals.org/ at National University of Singapore on November 30, 2013

179x274mm (300 x 300 DPI)

Downloaded from http://toxsci.oxfordjournals.org/ at National University of Singapore on November 30, 2013

189x263mm (300 x 300 DPI)

Downloaded from http://toxsci.oxfordjournals.org/ at National University of Singapore on November 30, 2013

194x140mm (300 x 300 DPI)

Downloaded from http://toxsci.oxfordjournals.org/ at National University of Singapore on November 30, 2013

Downloaded from http://toxsci.oxfordjournals.org/ at National University of Singapore on November 30, 2013

200x167mm (300 x 300 DPI)

Downloaded from http://toxsci.oxfordjournals.org/ at National University of Singapore on November 30, 2013

Você também pode gostar