original article a novel screening test to predict the

Correspondence: Nobuo Aikawa (E-mail: [email protected])

A novel screening test to predict the developmental toxicity of drugs using human induced pluripotent stem cells

Nobuo Aikawa

Translational Research Unit, R&D Division, Kyowa Kirin Co., Ltd., 1188, Shimotogari, Nagaizumi-cho, Sunto-gun, Shizuoka, 411-8731, Japan

(Received October 16, 2019; Accepted January 22, 2020)

ABSTRACT — In vitro human induced pluripotent stem (iPS) cells testing (iPST) to assess develop-mental toxicity, e.g., the induction of malformation or dysfunction, was developed by modifying a mouse embryonic stem cell test (EST), a promising animal-free approach. The iPST evaluates the potential risks and types of drugs-induced developmental toxicity in humans by assessing three endpoints: the inhibitory effects of the drug on the cardiac differentiation of iPS cells and on the proliferation/survival of iPS cells and human fibroblasts. In the present study, the potential developmental toxicity of drugs was divided into three classes (1: non-developmentally toxic, 2: weakly developmentally toxic and 3: strongly devel-opmentally toxic) according to the EST criteria. In addition, the type of developmental toxicity of drugs was grouped into three types (1: non-effective, 2: embryotoxic [inducing growth retardation/dysfunction]/deadly or 3: teratogenic [inducing malformation]/deadly) by comparing the three endpoints. The present study was intended to validate the clinical predictability of the iPST. The traditionally developmentally toxic drugs of aminopterin, methotrexate, all-trans-retinoic acid, thalidomide, tetracycline, lithium, pheny-toin, 5-fluorouracil, warfarin and valproate were designated as class 2 or 3 according to the EST criteria, and their developmental toxicity was type 3. The non-developmentally toxic drugs of ascorbic acid, sac-charin, isoniazid and penicillin G were designated as class 1, and ascorbic acid, saccharin and isoniazid were grouped as type 1 while penicillin G was type 2 but not teratogenic. These results suggest that the iPST is useful for predicting the human developmental toxicity of drug candidates in a preclinical setting. Key words: Developmental toxicity, Embryotoxicity, Teratogenicity, iPS cell, In vitro

INTRODUCTION

Developmental toxicity caused by drugs, chemi-cals and pesticides is a serious issue that can affect sub-sequent generations. Developmental toxicity is rough-ly divided into embryotoxic (inducing growth retardation or dysfunction), teratogenic (inducing malformation) and death (inducing infertility or stillbirth). Howev-er, these toxicities cannot be evaluated in general tox-icity studies using animals and are instead assessed in reproductive and developmental toxicity studies, which require an extensive number of animals and a long exper-iment term. Therefore, simple assays to achieve simi-lar findings are desired, and many in vitro developmen-tal toxicity assays have been developed, such as the mouse embryonic stem cell test (EST) (Annett et al., 2016; Seiler and Spielmann, 2011; Scholz et al., 1999;

Genschow et al., 2002, 2004), zebrafish test (Truong et al., 2011) and devTOXTM test (Palmer et al., 2013) using human embryonic stem cells or human induced pluripo-tent stem (iPS) cells. We also reported the preliminary method of in vitro iPS cells testing (iPST) (Aikawa et al., 2014) that is a modified version of the EST and a promis-ing animal-free approach.

The EST has been thoroughly studied and is well-un-derstood, and it has been validated by the European Cent-er for the Validation of Alternative Methods (ECVAM). The EST uses two cell lines and three endpoints to predict the developmental toxicity of drugs, chemicals and pes-ticides. The cell lines are mouse embryonic stem (mES) cells and mouse BALB/c 3T3 clone A31 fibroblast (3T3) cells. The three endpoints of the assay are the 50% inhi-bition of cardiac differentiation as the beating generation of the embryonic bodies (EBs), mES cell aggregates and

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Original Article

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the 50% inhibition of the proliferation or survival of mES cells and 3T3 cells for 10 days. The drugs and chemicals are classified into three classes: 1 (non-developmentally toxic), 2 (weakly developmentally toxic) and 3 (strongly developmentally toxic). These classes describe the devel-opmental toxicity potential based on the in vivo effects of drugs and chemicals in animals and/or humans, according to the findings for the linear discriminant functions inte-grating three endpoints.

Thalidomide is a well-known teratogen, which was launched in over 40 countries in 1957 but was with-drawn from the market in most countries by 1962 due to reports it induced human fetal teratogenicity. The ter-atogenic effect of thalidomide differs among species (Vargesson, 2015), and was caused in humans and non-human primates at low doses and in rabbits at high dos-es only but not in hamsters or rodents (Fratta et al., 1965; Schumacher et al., 1968; Teo et al., 2001, 2004). As tha-lidomide has not been tested using the EST, it was sub-jected to the commercial EST kit POCA® Hand1-EST (Suzuki et al., 2011), a modified high-throughput EST. The developmental toxicity was negative, which coincid-ed with the findings in the developmental toxicity study in mice. If human cells were used instead of mouse cells in the EST, the species-specific difference in the drug response would be negated, and the adverse effects of tha-lidomide would have been detected.

The iPST assesses three endpoints, similar to the EST, to predict the developmental toxicity of the drugs using iPS cells instead of mES cells and adult human dermal fibroblasts (fibroblasts) instead of 3T3 cells. One end-point is the inhibition concentration of cardiac differentia-tion for iPS cells, and the others are the cytotoxic concen-trations for iPS cells and fibroblasts. Since a cytotoxicity assay is the general method used for culturing iPS cells and fibroblasts in culture medium containing drugs, there are no technical problems. However, a cardiac differen-tiation assay is considerably difficult to perform because iPS cells, unlike mES cells, do not differentiate sponta-neously into cardiomyocytes. The mES cells are differen-tiated spontaneously into cardiomyocytes after the with-drawal of leukemia inhibitory factor (LIF), which helps maintain the pluripotency of the undifferentiated mES cells, from culture medium (Evans and Kaufman, 1981; Martin, 1981; Wobus et al., 1984). We previously devel-oped a simple protocol to differentiate iPS cells into cardi-omyocytes within a week by the application of biological substances (Aikawa et al, 2015), which made it possible to carry out the cardiac differentiation assay. In a previous study, the teratogenicity effect of thalidomide had already been predicted (Aikawa et al., 2014).

The iPST determined the developmental toxicity in two prediction models. One was the EST prediction mode established in the EST, which involved subdivision into three classes which was based on the in vivo effects in animals and/or humans. The other, as the developmen-tal toxicity prediction model, defined the types of devel-opmental toxicity (e.g., malformation, growth retardation and dysfunction) by comparing each endpoint in the three assays similar to the EST. Embryo and fetal development and growth are affected by the direct effects of drugs as well as by indirect effects on pregnant women, in whom a poor physical condition can adversely influence the sup-ply of nutrition, oxygen, hormones and other molecules to the embryo or fetus. Developmental toxicity is a direct action of drugs on the embryo or fetus without affecting the physiological condition of the pregnant woman. In the iPST, which considers iPS cells as the embryo or fetus and fibroblasts as the pregnant woman, the concentra-tion that results in toxicity to the iPS cells but not to the fibroblasts is considered the level of developmental toxic-ity (Fig. 1). Drugs that are more toxic to the iPS cells than to the fibroblasts have developmental toxicity, and strong toxicity of drugs to the iPS cells differentiation induces teratogenicity or death and strong toxicity to the iPS cells growth/survival induces growth retardation or dysfunc-tion nor death (Fig. 1).

The experimental protocol of the iPST in the present study was a partially modified of a previously reported protocol as follows: the drug treatment period was pro-longed to 6 days (from 4 days) depending on an cardi-ac differentiation period, the ReLeSR™ as a cell dissoci-ation reagent was used instead of dispase to equalize the number of cells seeding, and the author’s original pre-diction criteria, established by assay of the mouse cells, was improved to the classification of the type for devel-opmental toxicity as the developmental toxicity predic-tion model (Aikawa et al., 2014, 2015). In the present study, the iPST was validated internally using various ref-erence drugs. That is, the accuracies of the EST predic-tion model (Annett et al., 2016) and developmental tox-icity prediction model, established using the mouse cells, were verified. First, as a pre-validation examination, the predictability of the developmental toxicity of drugs in humans with the modified protocol was confirmed using thalidomide, a well-known teratogen with high sensitivity for humans; valproate, a well-known teratogen in almost all animal species; and saccharin as a non-teratogen, as it does not affect the pH of the culture medium, unlike ascorbic acid. Next, several reference drugs for which the developmental toxicity has or has not been reported in experimental animals or in epidemiological surveys were

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assessed.

MATERIALS AND METHODS

The present study was conducted according to the pol-icy of the Biological Material Handling Committee of our institute.

Test drugsThe drugs shown in Table 1 were obtained from the

following sources: all-trans retinoic acid (retinoic acid), lithium carbonate (lithium), methotrexate, sodium val-proate (valproate), tetracycline hydrochloride (tetracy-cline), warfarin and thalidomide from FUJIFILM Wako Pure Chemical (Osaka, Japan); aminopterin from Adooq Bioscience (Irvine, CA, USA); 5,5-diphenylhydanto-in (phenytoin), 5-fluorouracil (5-FU) and isoniazid from Sigma-Aldrich Japan (Tokyo, Japan), and ascorbic acid, saccharin and penicillin G potassium salt (Penicillin G)

from Nacalai Tesque (Kyoto, Japan). Ascorbic acid, iso-niazid, saccharin and penicillin G have not been report-ed to show developmental toxicity in humans, where-as all other drugs have some degree of developmental toxicity in humans and experimental animals. All drugs were dissolved in appropriate solvent (dimethyl sulfoxide [DMSO] or medium) and then diluted with the same sol-vent. The final concentration of DMSO was set at ≤ 0.2 vol%, and equal concentrations of DMSO were used in the control and test groups. DMSO at ≤ 0.25 vol% had no effect on the proliferation or viability of iPS or fibroblast cells (unpublished observation).

Cell lines and cell cultureiPS cells

The iPS cells were generated from human bone mar-row mononuclear cells in-house (Kunisato et al., 2011).

The iPS cells were suspended in modified tenneille serum replacer 1 medium (mTeSR™-1; STEMCELL

Fig. 1. Effects of developmental toxic signaling on the developmental stage. In the present study, human induced pluripotent stem cells were used instead of anaplastic embryos, and adult human dermal fibroblasts were used instead of human pregnant women. Developmental toxic signals from pregnant women to embryo/fetus include drugs, radiation, hypoxia, malnutrition and virus. Strong developmental toxic signaling induces death of the embryo in the preimplantation period, death of the embryo or malformation of newborns in the organogenesis period, and death of the fetus or dysfunction of newborns in the fetal growth period. Weak developmental toxic signaling induces death of the embryo or normal growth of the embryo in the preimplantation period, malformation of newborns or normal differentiation of the embryo in the organogenesis period, dysfunction of newborns or normal growth of the fetus in the fetal growth period. An abnormal condition of the pregnant women induces developmental toxic signals inhibits embryo implantation, inducing death of the embryo or growth retarda-tion of the fetus. Thick dotted vertical arrows: strong developmental toxicity signal, thin dotted vertical arrows: weak devel-opmental toxicity signal, hatched horizontal arrows: normal differentiation/growth, shaded arrows: abnormal differentiation/growth, solid arrows: death (infertility or stillbirth), open vertical arrows: normal/weak signal. iPS cells: human induced pluripotent stem cells. Fibroblasts: adult human dermal fibroblasts. (The present illustration is modified from Tuchmann-Duplessis, 1965).

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Developmental toxicity testing using human iPS cells

Technologies, Vancouver, Canada), seeded in a cell cul-ture dish (60 mm × 15 mm) coated with Matrigel® (Corning® International, Tokyo, Japan) and cultured in a 5% CO2 incubator (37°C and humidity-controlled).

FibroblastsHuman dermal fibroblasts-adult, isolated from adult

human skin cryopreserved at primary culture, were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA).

Fibroblasts were cultured using a typical procedure for cell lines, as follows: the cells were suspended in a der-mal fibroblast growth medium (fibroblast medium; DS Pharma Biomedical, Osaka, Japan), seeded in a cell cul-

ture dish (100 mm × 20 mm) coated with collagen type I (Thermo Fisher Scientific, Waltham, MA, USA) and cul-tured in a 5% CO2 incubator (37°C and humidity-control-led).

iPSTThe iPST processes were shown in Fig. 2.

Cytotoxicity assay with human iPS cells and human fibroblastsiPS cellsSubconfluent iPS cell colonies were collected by

ReLeSR™ (STEMCELL Technologies) treatment (few minutes, 37°C). After centrifugation (135 × g, room tem-perature), cell pellets were suspended carefully in fresh

Table 1. List of drugs used in the study.

Drug Class F.W.Test

concentration(µg/mL)

SolventDevelopmental toxicitya)

Clinical Cmax (µg/mL)b)Humans Animals

Aminopterin Antineoplastic 440.41 0.001 - 0.1 DMSOMalformation (e.g. hydrocephalus, skeletal anomalies)

No data 0.132

Methotrexate Antineoplastic 454.44 0.03 - 3 DMSO Malformation (e.g. embryopathy)

Malformation (e.g. missing finger): rat, mouse, rabbit 1.09

All-trans-Retinoic acid

Anti-psoriasis/keratosis 300.44 0.001 - 100 Medium Malformation (e.g. ear

morphological anomalies)

Malformation (e.g. external ear defects): monkey, skeletal abnormality: rat, mouse, rabbit

0.155-0.294

Thalidomide Antineoplastic (Immunomodulant) 285.23 0.3 - 100 Medium Malformation (e.g. no limb)

Malformation (e.g. limb defects): monkey>>rabbit>>ratNo effect: mouse

2.71

Tetracycline hydrochloride Antimicrobial 480.90 0.3 - 30 Medium Malformation (e.g. colored

teeth) Embryotoxicity: rat, mouse 5.00

Lithium carbonate Antimania 73.89 12.5 - 200 Medium Malformation (e.g. cardiovascular anomalies)

Malformation (e.g. cleft palate): mouse, ratNo effect: rabbit, monkey

5.70

Sodium valproate Antiepilepsy/Anticonvulsant 166.19 0.01 - 100,

125 DMSO Malformation (e.g. spina bifida, skeletal anomalies)

Malformation (e.g. cleft lip and palate): mouse, rat, rabbit, monkey

120

5,5-diphenylhydantoin (Phenytoin)

Anticonvulsant 252.27 0.1 - 100* DMSO Malformation (e.g. cleft palate)

Malformation (e.g. cleft palate): mouse, rat 8.97

5-Fluorouracil Antineoplastic 130.08 0.003 - 10 Medium Malformation (e.g. no limb) Malformation (e.g. multi-limb): mouse, rat 0.940

Warfarin Anticoagulant 308.33 0.01 - 100 MediumMalformation (e.g. embryopathy, central nerves anomalies)

Malformation (e.g. nasal hypoplasia, ophthalmologic anomalies): mouse

0.685

L-Ascorbic acid sodium salt Vitamin 198.11 1 - 1000 Medium No data No data 15.0

Isoniazid Antibacterial 137.14 10 - 10000 Medium No dataNo effect: rat, rabbitFetal death: mouse (150 mg/kg, p.o.)

8.00

Saccharin Artificial sweetener 183.18 1 - 3000 Medium No data No effect: mouse, rat, rabbit 0.256Penicillin G potassium salt Antibiotic 372.48 0.3 - 10000 Medium No data No effect: mouse, rat, rabbit 3.44

*: precipitation. DMSO: dimethyl sulfoxide. F.W.: formula weight. Cmax: maximum plasma concentrations of drugs.a) Türck et al., 2000; Scheinhorn and Angelillo, 1977; Loto and Awowole, 2012; Tanba et al., 1969 (Kekkaku-Japanese journal of the

Japanese Society for Tuberculosis); Koren et al., 1998.b) Jaju et al., 1981; Erić and Sabo, 2008; Nahum et al., 2006; Agwuh and MacGowan, 2006.

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mTeSR™-1 at 1 × 105 cells/mL. The cell suspensions were then seeded at 0.1 mL/well in 96-well clear-bot-tom black cell culture plate (Thermo Fisher Scientific) coated with Corning® Matrigel® matrix (Thermo Fisher Scientific) and cultured in a 5% CO2 incubator (37°C and humidity-controlled). After 3 hr, the media of all wells were carefully discarded, and then mTeSR™-1 contain-ing each concentration of drugs or the vehicle was imme-diately added at 0.2 mL/well in triplicate and cultured in a 5% CO2 incubator. Six days later, the cytotoxic effects of the drugs were assessed. The assay medium was replaced with fresh medium of the same composition every day (0.2 mL/well for 5 days, 0.1 mL/well last day).

FibroblastsSubconfluent fibroblasts were collected by the 0.05%

trypsin-EDTA (1%) (Thermo Fisher Scientific) treatment (few minutes, 37°C). After centrifugation (135 × g, room temperature), cell pellets were suspended carefully in fresh fibroblast medium at 1 × 105 cells/mL. The cell sus-pensions were seeded 0.1 mL/well in a Corning® 96-well clear-bottom black cell culture plate (Thermo Fisher Scientific) coated with collagen type 1 and cultured in a 5% CO2 incubator (37°C and humidity-controlled). After 3 hr, the media of all wells was carefully discarded, and

then fibroblast medium containing each concentration of drugs or the vehicle was added at 0.2 mL/well in tripli-cate and cultured in a 5% CO2 incubator. Six days later, the cytotoxicity of drug was assessed. The assay medi-um was replaced with fresh assay medium of the same composition at Day 2, 4 and 5 (0.2 mL/well for 5 days, 0.1 mL/well last day).

Cytotoxicity assayThe viability of cells was assessed using the Cell

meter™ colorimetric cell cytotoxicity assay kit (ATT Bioquest, Sunnyvale, CA, USA). Assay solution (0.02 mL/well) was added to all wells of the 96-well plate with cultured iPS cells or fibroblasts, which were then incubated in a 5% CO2 incubator (37°C and humidi-ty-controlled). Four hours later, the absorbance change at 570 nm and 605 nm was monitored using a microplate reader (SpectraMAX M3; Molecular Devices). The cell viability in each well was determined as the relative per-centage to the vehicle-control wells. Because a high con-centration of ascorbic acid disturbs the absorbance by dis-coloring the culture medium, in the ascorbic acid assay only, the test medium was replaced by no-drug medium (0.1 mL/well) on the day of the assay. After incubation for at least 1 hr in a 5% CO2 incubator, the cytotoxicity of

Fig. 2. Schematic overview of the process of the iPST. The three endpoints of the iPST for assessing the developmental toxicity of drugs are shown here. Two human cell lines were used: iPS cells and adult human dermal fibroblasts. The method of myocardial differentiation of iPS cells was developed for the myocardial differentiation assay of the iPST. The cytotoxicity assay was a widely used in vitro method. iPST: human induced pluripotent stem cell test. iPS cells: human induced pluripo-tent stem cells. Fibroblasts: adult human dermal fibroblasts. EB: embryonic body (aggregated iPS cells).

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drug was assessed.

Cardiac differentiation assay with human iPS cellsSubconfluent iPS cell colonies were collected by dis-

pase (1 mg/mL; STEMCELL Technologies) treatment (37°C, 20 min). After centrifugation (135 × g, 5 min, room temperature), supernatant was discarded, and the cell pellets were suspended by careful pipetting in fresh AggreWell™ EB formation medium (STEMCELL Tech-nologies) supplemented with 10 µmol/L Y-26732 solution (5 mmol/L; FUJIFILM Wako Pure Chemicals). The cell suspension from one culture dish (60 mm × 15 mm) was seeded to 3 wells of an AggreWell™ 800 plate (STEM-CELL Technologies) and then cultured in a 5% CO2 incu-bator. One to 2 days later, the embryonic bodies (EBs) formed from the iPS cells were collected in a conical tube (15 mL) using a reversible cell strainer (37 µm, STEM-CELL Technologies) and reseeded in an ultra-low-attach-ment 6-well culture plate (Sumitomo Bakelite) from one well to another. Culture medium was replaced with Dul-becco’s modified eagle medium: nutrient mixture F-12 + GlutaMax™ (DMEM/F12) supplemented with a serum-free Gibco® B-27® supplement (50X) and a Gibco® MEM non-essential amino acids solution (100X) (NEAA) (B27-DMEM/F12; Thermo Fisher Scientific) (2 mL/well) containing each concentration of drugs, activin-A (100 ng/mL, which is more than 10-fold the ED50; HumanZyme, Chicago, IL, USA), Wnt-3a (100 ng/mL, which is more than 10-fold the ED50; R&D Sys-tems, Minneapolis, MN, USA) and bone morphogenet-ic protein-4 (BMP-4) (100 ng/mL, which is more than 10-fold the ED50; HumanZyme). After one day, medium was replaced with B27-DMEM/F12 containing the same concentrations of drug and noggin (300 ng/mL, which is more than 10-fold the ED50; StemRD, Burlingame, CA, USA) and then cultured for 3 days in a 5% CO2 incuba-tor. Noggin-medium was replaced once with fresh medi-um of the same composition within 3 days. Thereafter, the medium was replaced with DMEM/F12 supplement with 5% Gibco® qualified fetal bovine serum (Thermo Fisher Scientific) and NEAA (Thermo Fisher Scientific) (5% FBS-DMEM/F12) containing the same concentra-tion of drug using a reversible cell strainer (STEMCELL Technologies), and the EBs with medium were transferred to a 96-well cell culture multi-plate (Sumitomo Bakelite) at 24 EBs (1 EB/well) per concentration or vehicle-con-trol.

EBs were observed the occurrence of spontaneous beating under a microscope at least once a day. Two days later, the medium was replaced with the 5% FBS-DMEM/F12 containing no drug. Thereafter, medium was replaced

with the same fresh medium every few days. The occur-rence of spontaneous beating of EBs was observed until the beat ratio exceeded 90% in the vehicle-control group.

Data analysesThe three endpoints were calculated, from which the

potential risks of the developmental toxicity induced by drug was predicted using the EST prediction model shown in Fig. 3 and also the type of developmental toxic-ity was predicted using the developmental toxicity predic-tion model shown in Fig. 3.

The calculation of three endpointsThe endpoint of cardiac differentiation assay was cal-

culated using the probit method in the SAS software pro-gram ver. 9.4 (SAS Institute Japan, Tokyo, Japan) when the cardiac differentiation ratio of vehicle-control exceed-ed 90% and expressed as the 50% differentiation inhibi-tory concentration (ID50) of test drugs. Each endpoint of the cytotoxicity assay on iPS cells and fibroblasts was determined using the logit method in the SAS software program and expressed as the 50% inhibitory concentra-tion (IC50PS for iPS cells and IC50F for fibroblasts) of test drugs.

Classification by the EST prediction modelThe EST prediction model was published as EURL-

ECVAM protocol No. 113: EST (Annett et al., 2016). The linear discriminant functions I, II and III shown in Fig. 3 were calculated by the incorporation of the three end-points (ID50, IC50PS and IC50F), and classified according to the classification criteria shown in Fig. 3 as follows: class 1 (non-developmentally toxic), class 2 (weakly developmentally toxic) and class 3 (strongly developmen-tally toxic), which indicate the potential risk for the drug-induced developmental toxicity.

Classification by the developmental toxicity prediction model

The developmental toxicity prediction model was developed by our lab divided the developmental toxici-ty induced by the test drugs into three types (1: non-effec-tive, 2: embryotoxic [inducing growth retardation or dys-function] or deadly [inducing infertility or stillbirth] or 3: teratogenic [inducing malformation] or deadly [inducing infertility or stillbirth]) (Fig. 3). As already mentioned in introduction section, the developmental toxicity predic-tion model assumed the fibroblasts to be pregnant women and the iPS cells to be the embryo or fetus. In this mod-el, the adverse effects of the drugs on the iPS cells at con-centrations not affecting the fibroblast viability reflect the

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developmental toxicity (Figs. 1 and 3).In the pre-validation examination, the period of car-

diogenesis was calculated from the application of cardi-ac inducers (activin-A, BMP-4 and wnt-3a) until the com-pletion of cardiac differentiation (observed for up to two weeks) to exam whether it can use as parameter for devel-opmental toxicity.

RESULTS

The results of the pre-validation examination with the partially modified protocol and the subsequent internal validation examination were shown.

Pre-validation of iPST protocol modified partiallyThe results of the thalidomide experiment are shown in

Fig. 4 and Table 2. The three endpoints of IC50F, IC50PS and ID50 were > 100, 9.07 and 1.41 µg/mL, respective-ly. The results of the linear discriminant functions were as follows: function I was -5.77, function II was 0.644, and function III was -3.28 (II > I and II > III). According to the EST prediction model, thalidomide was designat-

ed as class 2 drug. In contrast, in the developmental tox-icity prediction model shown in Fig. 3, both the IC50PS and ID50, which indicate surrogate toxicity to an embryo or fetus, were markedly lower than the IC50F, which indi-cates surrogate toxicity to the pregnant woman. Thalid-omide was considered to affect the development of the embryo or fetus at concentrations that were non-toxic in pregnant women, and therefore thalidomide was designat-ed as type 3 drug. Thalidomide was classified as a devel-opmentally toxic/teratogenic drug.

The period of cardiogenesis was 6.7 ± 0.9 days in the control group, while that in the 0.3 µg/mL group was 7.8 ± 1.7 days, that in the 0.6 µg/mL group was 8.6 ± 1.3 days, that in the 1.3 µg/mL group was 8.8 ± 2.0 days, and that in the 2.5 µg/mL group was 9.6 ± 1.8 days. The car-diogenesis period was prolonged with increasing thalido-mide concentrations.

The results of the valproate experiment are shown in Fig. 4 and Table 2. The IC50F, IC50PS and ID50 of val-proate were > 100, 51.1 and 9.63 µg/mL, respectively. The results of the functions were as follows: I was -2.69, II was 2.54, and III was -4.84 (II > I and II > III). Accord-

Fig. 3. Prediction of the developmental toxicity. (a) Three endpoints resulting from the cytotoxicity and myocardial differentiation assays. (b) EST prediction model, which classified the predicted risks of developmental toxicity of drugs by comparing the variables from the linear discriminant functions integrating three endpoints. (c) Developmental toxicity prediction model, which classified the types of developmental toxicity of drugs by a relative comparison of three endpoints. Fibroblasts: adult human dermal fibroblasts. iPS cells: human induced pluripotent stem cells. EST: mouse embryonic stem cell test.

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ing to the EST prediction model, valproate was desig-nated as class 2 drug. In the developmental toxicity pre-diction model, both the IC 50 PS and ID 50 , which indicate surrogate toxicity to an embryo or fetus, were markedly lower than the IC 50 F, which indicates surrogate toxicity to pregnant women. Valproate was considered to aff ect the development of the embryo or fetus at concentrations that were non-toxic in pregnant women; thus, valproate was designated as type 3 drug. Valproate was classifi ed as a developmentally toxic/teratogenic drug.

The period of cardiogenesis was 7.4 ± 1.0 days in the control group, while that in the 1 µg/mL group was 7.3 ± 1.2 days, that in the 3 µg/mL group was 7.1 ± 0.8 days, that in the 10 µg/mL group was 9.0 ± 1.7 days, and that in the 30 µg/mL group was nonexistent. The car-diogenesis period was prolonged by 1 day and more at 10 µg/mL compared with the control group.

The results of the saccharin experiment are shown in Fig. 4 and Table 2 . The IC 50 F, IC 50 PS and ID 50 of saccha-rin were 66.9, 283 and > 1000 µg/mL, respectively. The results of the functions were as follows: I was 77.7, II was 33.8, and III was -28.5. According to the EST pre-diction model, saccharin was designated as class 1 drug. In contrast, in the developmental toxicity prediction mod-el, both the IC 50 PS and ID 50 , which indicate surrogate tox-icity to an embryo or fetus, were higher than the IC 50 F, which indicates surrogate toxicity to pregnant women.

Saccharin did not aff ect the development of the embryo or fetus at concentrations causing adverse reactions in adult or pregnant women; thus, saccharin was designated as type 1 drug. Saccharin was classifi ed as a non-develop-mentally toxic/non-eff ective drug.

The period of cardiogenesis was 7.3 ± 0.1 days in the control group, while that in the 10 µg/mL group was 7.5 ± 0.2 days, that in the 30 µg/mL group was 7.2 ± 0.1 days, that in 100 µg/mL group was 7.5 ± 0.2 days, that in the 300 µg/mL group was 7.6 ± 0.1 days, and that in the 1000 µg/mL group was 8.6 ± 0.2 days. The cardiogenesis peri-od was prolonged by approximately 1 day at 1000 µg/mL compared with the control group.

These fi ndings show that the iPST with partially mod-ifi ed protocol accurately classifi ed the potential risk and type of the developmental toxicity induced by drugs. The period of cardiogenesis was closely related to the ratio of cardiogenesis and was not necessary/additional parameter in the prediction of developmental toxicity.

Validation of the iPST

Table 2 summarizes the endpoints (IC 50 F, IC 50 PS and ID 50 ) by the test drugs in the cytotoxicity assay and the cardiac diff erentiation assay.

Fig. 4 . Pre-verifi cation study of the iPST modifi ed partially from the preliminary protocol. a-(1), b-(1) and c-(1): Results of the iPST. The open triangles (fi broblasts) and open circles (iPS cells) represent the mean ± S.D. of triplicate samples in the cy-totoxicity assay. The closed circles represent the ratio of beating generation in the 24 embryonic bodies per sample concen-tration. The solid lines indicate linear approximation curves. a-(2), b-(2) and c-(2): Days of myocardiogenesis. Each column with a bar represents the mean ± S.D. of days of the myocardiogenesis of embryonic bodies, except for undiff erentiated embryonic bodies. iPST: induced pluripotent stem cell test.

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The classification of the potential risk of developmental toxicity of the drugs in the EST prediction model

Table 2 lists the results of the linear discriminant func-tions. The drugs with the highest values for function III were designated as class 3 according to the EST predic-tion model, including aminopterin, methotrexate and 5-FU (Table 2). The drugs with the highest values for function II were designated as class 2, including retinoic acid, tet-racycline, lithium, phenytoin and warfarin (Table 2). The drugs with the highest values for function I were designated as class 1, including ascorbic acid, isoniazid and penicillin G (Table 2). The test drugs were accurate-ly classified into three classes depending on the potential risks of developmental toxicities in human. It is conclud-ed that the EST prediction model established using mouse cells would be useful for predicting human developmen-tal toxicities of drugs in the iPST using human cells.

The classification of the type of developmental toxicity of the drugs in the developmental toxicity prediction model

The drugs for which both the IC50PS and ID50 were

higher than the IC50F included ascorbic acid and iso-niazid, which were classified as type 1 drugs (Table 2). These drugs may not adversely affect iPS cells (taken to be embryos or fetuses) at concentrations affecting fibrob-lasts (taken to be pregnant women). The drugs for which the ID50 (and IC50PS) were lower than the IC50F includ-ed aminopterin, methotrexate, retinoic acid, tetracycline, lithium, 5-FU and warfarin, which were classified as type 3 drugs according to the criteria dependent on the com-parison of the three endpoints shown in Fig. 3 (Table 2). These drugs may adversely affect iPS cells (taken to be embryos or fetuses) development at concentrations not affecting fibroblasts (taken to be pregnant women). Only penicillin G among the non-teratogenic drugs had a low-er IC50PS than the IC50F, and it was designated as a type 2 drug (Table 2). This drug may have adversely affected iPS cells (taken to be embryos or fetuses) growth at the concentrations not affecting fibroblasts (taken to be adult or pregnant women).

Phenytoin was not classified into a group because all 3 endpoints were the same values (> 30 µg/mL) due to the

Table 2. Three endpoint values, functions values and classification of the developmental toxic potential of test drugs.

DrugTeratogenic

effects in humans

Three endpoints (µg/mL)

Developmental toxicity

prediction model

FunctionEST

prediction model

Prediction by others in vitro assay

IC50F IC50PS ID50 Type I II III Class ESTa) devTOXb) ZETc)

Aminopterin Positive 0.301 0.0524 0.0295 3 -28.1 -13.5 1.21 3 No data Positive No dataMethotrexate Positive 0.835 1.69 0.707 3 -16.2 -6.93 -2.87 3 3 Positive Positive*Retinoic acid Positive 48.8 8.74 6.25 3 -7.06 -0.297 -3.38 2 3 Positive PositiveThalidomide Positive >100$ 9.07 1.41 3 -5.77 0.644 -3.28 2 1# Positive PositiveTetracycline Positive 80.7 10.2 14.2 3 -5.78 0.561 -3.47 2 No data No data No dataLithium Positive 497 94.0 88.3 3 2.78 5.85 -5.56 2 2 Positive No dataValproate Positive >100$ 51.1 9.63 3 -2.69 2.54 -4.84 2 2 Positive PositivePhenytoin Positive >30.0$ >30.0$ >30.0$ No grouping -1.81 1.93 -5.69 2 2 Negative Negative5-FU Positive 1.97 1.73 0.548 3 -17.0 -6.70 -2.08 3 3 Positive PositiveWarfarin Positive 374 534 119 3 5.43 7.42 -7.20 2 2* Negative* PositiveAscorbic acid Negative 192 1430 3000 1 86.4 38.5 -30.9 1 1* Negative NegativeIsoniazid Negative 521 914 1730 1 23.0 14.6 -12.2 1 1 Negative Negative*Saccharin Negative 66.9 283 >1000$ 1 77.7 33.8 -28.5 1 1 Negative NegativePenicillin G Negative 2360 1420 3370 2 17.2 13.3 -9.57 1 1* Negative Negative

#: Result of POCA® Hand1-EST in in-house. *: Different results have been reported. $: The endpoint was not determined because of drug solubility issues or problem with the concentration of dimethyl sulfoxide as the solvent, and the calculation of the linear discriminant functions used 30, 100 or 1000 of the maximum concentration tested. Positive: developmentally toxic/teratogenic. Negative: non-developmentally toxic/non-teratogenic.IC50F: 50% inhibition concentration for the proliferation/survival of human fibroblasts. IC50PS: 50% inhibition concentration for the proliferation/survival of human induced pluripotent stem cells. ID50: 50% inhibition concentration for myocardial differentiation of human induced pluripotent stem cells. EST: mouse embryonic stem cell test. devTOX: human pluripotent stem cell-based developmental toxicity assay. ZET: zebrafish test. 5-FU: 5-fluorouracil.a) Scholz et al., 1999; http://www.stemina.com/toxicology/devtox; Genschow et al., 2004.b) Scheinhorn and Angelillo, 1977; http://www.stemina.com/toxicology/devtox.c) Shaul and Hall, 1977; http://www.stemina.com/toxicology/devtox.

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solubility limit, and therefore comparison was impossi-ble. The type of developmental toxicity induced by drugs except for penicillin G and phenytoin was predicted by the developmental toxicity prediction model. It is sug-gested that the developmental toxicity prediction model may help to predict the types of developmental toxicity of drugs in humans.

DISCUSSION

The classification of the EST prediction model classi-fied the positive control test drugs known to have devel-opmental toxicity (teratogenicity) in humans as devel-opmentally toxic drugs (class 2 or 3) and the negative control test drugs as non-developmentally toxic (class 1). Even though this model was established using mouse cells, the risk of developmental toxicity of test drugs could be accurately predicted using human cells. There-fore, the EST prediction model was verified to be useful in the iPST.

Regarding the classification of the type of toxicity according to the developmental toxicity prediction model, all test drugs except for penicillin G and phenytoin were accurately classified as positive control test drugs for ter-atogenic (malformation) and deadly (type 3) and negative control drugs for non-effective (type 1). Phenytoin, a pos-itive control drug, could not calculate for its three end-points due to its solubility limit, and therefore was not used in the developmental toxicity prediction model. Pen-icillin G, a negative control drug, was classified embry-otoxic (growth retardation and dysfunction) and dead-ly (type 2). In the original EST, penicillin G was deemed a borderline drug between weakly and non-developmen-tally toxic (Scholz et al., 1999) and is considered diffi-cult to classify correctly. The clinical total Cmax of penicil-lin G is 3.44 µg/mL (134.6 µmol/L) (Palmer et al., 2013). The endpoints (50% inhibition concentrations) of the three assays in the present study were between 1420 and 3370 µg/mL, which was > 400-fold the Cmax. At a low-er concentration (e.g., at the 20% inhibition concentra-tion), the cytotoxicity to fibroblasts in the three-endpoint assay was 831 µg/mL, that to iPS cells was 1086 µg/mL and the cardiac differentiation inhibition was 3200 µg/mL (unpublished observation). Even these concentrations were >240-fold the Cmax and the type of toxicity accord-ing to the developmental toxicity prediction model was type 1 (unpublished observation). Therefore, if adminis-tered at a therapeutic concentration, the toxicity of pen-icillin G will be type 1. In fact, penicillin G is frequent-ly administered during pregnancy because it is thought to lack human toxicity and not be teratogenic (Briggs et

al., 2011). Therefore, because the safety margin of peni-cillin G is sufficient, no action in the clinical setting will occur, and penicillin G is unlikely to induce any tera-togenic developmental toxicity. Given the above findings, the developmental toxicity prediction model is expected to accurately predict the developmental toxicity of drugs in the clinical setting by considering the clinical therapeu-tic concentration.

Phenytoin was designated as a class 2 drug, indicat-ing weak developmental toxicity, from 30 μg/mL of sol-ubility limit in the EST prediction model. In the original EST, which used mouse cells, phenytoin was also a class 2 drug (Table 2, EURL-ECVAM protocol No. 113: EST). However, other developmental toxicity assays, such as devTOX (Palmer et al., 2013) and ZET (Truong et al., 2011), incorrectly determined phenytoin to have a nega-tive developmental toxicity (Table 2). Whereas, the orig-inal EST, which are undetected the developmental tox-icity of thalidomide having high sensitivity in humans. The iPST using human cells showed positive findings for phenytoin and thalidomide, making it possible to predict the most accurately the clinical developmental toxicity of drugs.

Among the test drugs classified into classes 2 and 3 according to the EST prediction model, the three end-points of retinoic acid, lithium and warfarin were higher than the clinical therapeutic blood concentrations (Tables 1 and 2). Retinoic acid is an essential factor for embryo and fetal development but impairs development at high exposure (Vandersea et al., 1998). A metabolite of vita-min A (retinoid), retinoic acid is necessary for supporting the life of vertebrates and is usually absorbed from food, as it is not synthesized in the body. An excessive intake of retinoic acid by pregnant women increases the expo-sure of the fetus to retinoic acid above the normal level and increases the teratogenic risk. Many of the teratogen-ic effects of retinoic acid are said to be caused by dietary supplements rather than medicine. Retinoic acid adminis-tered as a medicine is not expected to cause embryotoxic-ity, provided the exposure in pregnant women is proper-ly controlled.

Lithium shows equivalent blood concentrations in pregnant women and embryos/fetuses, and infants with high cord blood levels have a high frequency of compli-cations (Newport et al., 2005). Intake of lithium by preg-nant women increases the risk of congenital heart disease in infants (Diav-Citrin et al., 2014). In the iPST, lithium was shown to be a weakly developmentally toxic drug, whereas the three endpoints in the iPST were more than 10-fold the clinically therapeutic concentration (Tables 1 and 2). On extrapolating the findings of the present

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N. Aikawa

study to humans, a high exposure to lithium is expected to induce developmental toxicity in embryos and fetuses. A retrospective analysis showed that lithium does indeed induce developmentally toxic effects, although at clini-cally therapeutic blood concentrations in pregnant wom-en, it was unlikely to induce such effects. Of note, the use of lithium as a medication during pregnancy is contrain-dicated in Japan, whereas in the West, it is administered with cautions as a medication and is not explicitly con-traindicated.

Warfarin is a vitamin K antagonist and an anticoagulant drug. It reduces the vitamin K redox cycle by inhibiting NAD(P)H-dependent reductase, resulting in the inhibi-tion of the activation of proteins, such as blood coagula-tion factor in the liver and osteocalcin in the bone tissue (Nelsestuen et al., 1974; Kim et al, 2013; Gallieni and Fusaro, 2014). Osteocalcin is a vitamin K-dependent Ca-binding protein involved in bone remodeling (Moser and van der Eerden, 2019). Decreased vitamin K increases the incidence of fractures (Hao et al., 2017). Since warfarin passes through the placenta, it was considered that warfa-rin inhibits the vitamin K redox cycle in the embryo/fetus and subsequently affects bone formation in fetuses. In the present study, the developmentally toxic effects of warfa-rin manifested at a concentration (three endpoints in the iPST) higher than the clinical therapeutic concentration, suggesting that warfarin might not directly induce devel-opmental toxicity at its therapeutic concentration. How-ever, warfarin may affect the embryo/fetus through indi-rect activity, such as its anticoagulant action.

Isoniazid, a negative control drug, was shown to induce fetal hypoplasia (embryotoxicity) when given orally dur-ing pregnancy in rats and rabbits, whereas no teratogene-sis was noted in reproduction studies in humans (Ludford et al., 1973). In the clinical setting, isoniazid administered during pregnancy showed no significant difference in birth rates, birth weight, sex ratios or birth abnormalities compared with placebo (Palmer et al., 2013; Shaul and Hall, 1977). Therefore, isoniazid is considered non-em-bryotoxic and non-teratogenic. In the present study, iso-niazid was classified as a class 1, type 1 drug. The iPST was able to accurately predict the human developmental toxicity of isoniazid. In addition, our assay also accurate-ly predicted the teratogenicity of thalidomide, which has high sensitivity in humans despite its effects being diffi-cult to predict in animals, especially rodents. The iPST can therefore overcome species-specific differences in the developmental toxicity of drugs.

In the iPST, the EST prediction model was considered to predict the general developmental toxicity of drugs in the clinical setting. However, the developmental toxic-

ity prediction model was considered to roughly predict the type of developmental toxicity induced by the drugs. Using both prediction models was thought to allow for a more detailed and accurate risk assessment than using either alone. Incorporating the predicted plasma con-centration of clinical therapeutics may support the accu-rate prediction of the developmental toxicity induced by drugs.

The iPST, as well as the original EST, assessed the car-diac differentiation inducing from the mesoderm formed from iPS cells like embryonic stem cells isolated from blastoderm. The mesoderm is the middle layer of the tril-aminar germ layers formed from blastoderm at a very ear-ly stage of embryonic development. The other two layers are the ectoderm (outer side) and endoderm (inner side). The neural tissue is generally thought to be generated from ectoderm, although a previous report stated that the neural tissue (hindbrain and spinal cord) is generate from axial stem cells, which are the same progenitor cells as seen in the mesoderm, rather than the ectoderm (Takemo-to et al., 2011; Kondoh and Takemoto, 2012). This find-ing suggests that the cardiac differentiation assay in the iPST simultaneously evaluates the neural tissue differen-tiation pathway.

In summary, the iPST using human cells can predict the developmental toxicity induced by drugs in humans. This test may be a better drug screening model than oth-er in vitro assay models for assessing the developmental toxicity induced by drug candidates, as well as chemicals and pesticides, in a preclinical setting. One potential issue associated with the iPST is the potential lot-to-lot varia-tion in iPS cells, which has been shown to have cell-spe-cific differentiation to tissue/organ. The low rate of the cardiac differentiation regarding the induction of iPS cells may not calculate the endpoint ID50 of the cardiac differ-entiation assay in the iPST. Three in-house generated lots were confirmed to have no difference in cardiac differen-tiation, but publicly available commercial lots of iPS cells were not tested. Another potential issue is the difference in embryo/fetus exposure due to the absence of a placen-ta compared to in vivo. Drugs cross the placenta into the fetus by passive diffusion according to the lipid solubil-ity and molecular weight of the drugs and by the active diffusion via placental active transporters. They are then excreted from the placenta by ATP-binding cassette trans-porters (Lankas et al., 1998). In the iPST, because the test drugs are administered to the iPS cells, which are the same as the inner cell mass of a blastocyst at the early-stage of pre-implantation (before placenta development), the absence of placenta might have a negligible effect.

To conduct the cardiac differentiation assay efficient-

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ly, the EB formation process is currently being improved, moving from the AggreWell™800 plate to the PrimeSure-face® plate (96 wells, V-type, low cell binding; Sumitomo Bakelite) with the appropriate edge inside of the V-well. This method will enable testing without moving EBs to another well.

Conflict of interest---- The authors declare that there is no conflict of interest.

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original article a novel screening test to predict the

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