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Summary

Testing for developmental and reproductive toxicology (DART) is a crucial part of the toxicological risk assessment of novel compounds. Embryonic stem cells based assays (like EST test) have a clear potential to fulfil the 3R’s requirements and commonly used for in vitro developmental toxicity tests.

Based on toxicogenomic studies in mammalian stem cells we identified six biomarkers (Bmp4, CK18, FoxA2, Oct4, Sox17 and Vegfr1), to visualise and quantify developmental alterations upon teratogen exposure in the pluripotent stage, in early embryonic lineages and differentiated tissues: heart and liver. Using these biomarker genes, we are generating fluorescently labelled reporter cell lines.

Using mouse embryonic stem cells, we optimised differentiation protocols towards to cardiomyocytes and hepatocytes and confirmed the expression of the selected biomarkers by qPCR. We generated

GFP-CK18 reporter cell lines (liver-specific) and differentiated them towards hepatocytes using a refined 2D and 3D hepatocyte differentiation protocol. We exposed cells during pluripotent stage with two

strong (5-fluorouracil and retinoic acid) and two weak teratogens (thalidomide and diphenylhydantoin) followed by differentiation induction into cardiomyocytes and hepatocytes. Expression of the selected

biomarkers shows alterations upon the teratogenic compound treatment. These data were in line with the observed reduction in the number of beating bodies in the developing cardiomyocytes and the

reduced GFP expression level of the liver-specific CK18 transgenic line.

Development of a stem cell-based reporter assay for in vitro

DART assessment.

Undifferentiated ES cells

Definitive endoderm

Hepatic progenitor cells Meso-endoderm

d3 d21d16d6

Activin A, Chir99021, HGF

ß-Mercaptoethanol DMSO

ß-Mercaptoethanol DMSO

hIPSC Hepatocyte differentiation

A.

B.

2D Hepatocyte differentiation

Undifferentiated mES cells Matured hepatocytes differentiated from mES cells labeled with Ck18 and Alb

d3d0 d21d16d5

Undifferentiated mES cells

LIF ActA. FGF. DEX., Oncostatin

DEX, Oncostatin

Meso-endoderm Definitive endoderm Hepatic progenitor cells

Hepatocytes

3D Cardiomyocyte differentiation

Differentiated mES cells stained with TNNT2

Undifferentiated ES cells

Cardiac mesodermMeso-endoderm Cardiomyocytes

d3d0 d14d5 d7

LIF ActA, Chir99021,

BMP4XAV939

Cardiac progenitor cells

Performance criteria WEC Micromass EST

Predictivity non-embryotoxic 70% 57% 72%

Predictivity weak embryotoxic 76% 71% 70%

Predictivity strong embryotoxic 100% 100% 100%

Precision non-embryotoxic 80% 80% 70%

Precision weak embryotoxic 65% 60% 83%

Precision strong embryotoxic 100% 69% 81%

Accuracy 80% 70% 78%

Overall performance Sufficient Insufficient Sufficient

Early embryonic development

Reporter cell line generation

d4 d7 d10

d14 d17 d21

DMSO control Low exposure Medium exposure High exposure

5’ F

luora

cil

Retin

oic

aci

d

ControlThalid

om

ide

Dip

henyl

hyd

anto

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Effect of teratogens on hepatocyte differentiation

5’Fl

uora

cil

Retin

oic

aci

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0.05nMD

iphenyl

hyd

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inThalid

om

ide

DMSO control Low exposure Medium exposure High exposureSelected Biomarkers

Biomarker gene

Phase in differentiation process

Oct4 Undifferentiated stem cell state

Sox17 Endoderm state

Foxa2 Endoderm state

Bmp4 Mesoderm state

Vegfr1 Mature cardiomyocyte

Ck18 Mature hepatocyte

Overview of the mouse embryonic stem cell line (mES) cardiomyocytes differentiation protocol. mES cells were

differentiated within 14 days into matured cardiomyocytes. Functional cardiomyocytes were identified based on

morphological assessment of contracting cardiomyocytes. High expression of the heart specific structural protein

Troponin T2 (TNNT2 - red) confirms the successful differentiation towards cardiomyocytes at day 14. The cells were

counterstained with nucleic specific marker DAPI (blue) after completing cardiomyocyte differentiation protocol.

Transgenic line generation (A.) Biomarker genes that are selectively expressed at different stages of stem cell differention into mature cardiomyocytes or hepatocytes were selected. The biomarker genes were applied to generate GFP-based reporters using BAC recombination. The liver-specific CK-18 biomarker gene was modified with an N-terminal GFP green fluorescent marker. Successful integration of the GFP reporter into mES cells was confirmed by sequencing. Differentiation of the transgenic lines CK18-GFP towards hepatocytes in 2D cell culture (B). Increased level of GFP signal over time demonstrates the differentiation/maturation process of the hepatocytes. GFP-intensity calculated with Fiji image processing package.

Overview of the mouse embryonic stem cell line (mES) hepatocyte differentiation protocol. mES cells were

differentiated within 21 days into matured hepatocytes. Expression of the liver-specific cytokeratin-18 (green) was

visualised in pluripotent mES cells and differentiated hepatocytes. Successful differentiation of mES cells into functional

hepatocytes was confirmed by Albumin (ALB) immunostaining (red) confirmed the presence of matured hepatocytes.

The cells were counterstained with nucleic specific marker DAPI (blue) after completing hepatocyte differentiation

protocol.

Overview of the hIPSC hepatocyte differentiation protocols (A). hiPSC were differentiated within 21 days into mature hepatocytes. Changes in cell phorphology during stem cell differentiation into hepatocytes are shown t at day 0, 3, 10 and 21 during the differentiation process. Biomarker expression at day 0, 21 of differentiation (B). RNA was collected and expression levels of the biomarker genes was determined by qPCR. The pluripotency marker OCT4 was, significantly decreased after 21 days while the expression of the hepatocyte specific markers ALB and AFP were increased. Student t-test was used for determination of significance. *p<0.05; **p<0.01, *** p<0.001.

d0

Undifferentiated mES cells Adopted from http://madmnemonics.blogspot.nl/2015/06/the-three-primary-germ-layers.html

hIPSC Hepatocyte differentiation upon compound exposure

Teratogenic effect of 5’Fluorouracil and Thalidomide on hIPSC during directed hepatocyte differentiation. hiPSC were exposed to two well-known teratogens, 5-Fluorouracil and Thalidomide for 21 days, in three different concentration.

At day 21 the expression level of the two liver specific biomarkers, α-fetoprotein (AFP) and albumin (ALB) were measured by qPCR. Both compounds showed decreased expression of the biomarkers in a concentration dependent manner. Student t-test was used for determination of significance. *p<0.05; **p<0.01, *** p<0.001.

Hepatocyte

Defective hepatocyte differentiation upon teratogenic compound exposure. Cells were exposed to different teratogenic compounds during mES cell differentiation towards hepatocytes. After 21 days, fluorescence intensity of the hepatocyte specific CK-18-GFP reporter was measured. Presented values are expressed as percentages of control. Student t-test was used for determination of significance. *p<0.05; **p<0.01, *** p<0.001.

Defective cardiomyocyte differentiation upon teratogenic compound exposure. Cells were exposed to teratogenic compounds during mES cell differentiation towards cardiomyocytes. The percentage of beating 3D embryoid bodies, representing functional cardiomyocytes, was determined. Presented values are expressed as percentages of control. Students t-test was used to determine significance. *p<0.05, **p<0.01, ***p<0.001.

A.

B.

Effect of teratogens on cardiomyocyte differentiation

hIPSC cardiomyocyte differentiation

ActA, Chir99021 B27, BMP4 B27, BMP4

Undifferentiated hIPSC cells

Differentiated hIPSC cells towards to cardiomyocytes

Differentiated hIPSC cells co-stained with cardiac specific marker TNNT2.

d3d0 d14d5 d7

Overview of hIPSC cardiomyocytes differentiation protocols. hiPSC were differentiated within 14 days into mature cardiomyocytes. Effective cardiomyocyte differentiation was confirmed by Troponin-T (TNNT2) immunostaining. Biomarker expression at day 0, 21 of differentiation (B). RNA was collected at day 3, 7, 10 and 14 and expression levels of the biomarker genes was determined by qPCR. The pluripotency marker OCT4 was significantly decreased after 14 days, while the expression of the mesodermal state specific marker BMP4 was increased at day 7 and decreased after. Cardiomyocyte specific marker MYH6 showed increase expression significantly increased expression from day 7. Student t-test was used for determination of significance. *p<0.05; **p<0.01, *** p<0.001.

Oct4

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d0 d3 d10 d21

Toxys, Leiden, The Netherlands

P.I. Racz, R.R.M. Ottenheijm, R. Derr, G. Hendriks

Undifferentiated ES cells

Cardiac mesodermMeso-endoderm Cardiomyocytes

Cardiac progenitor cells

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Pluripotent stage specific biomarker expression(OCT4)

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AFPALB

AFPALB

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AFPALB

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ATG STOP

Artificial Chromosome (BAC)

biomarker gene

exon1 exon2intron