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P.I. Racz, R.R.M. Ottenheijm, R. Derr, G. Hendriks · Control Control 25 µ M 25 µ M 50 µ M 50 µ...
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Control Control 25 µM 25µM 50µM 50µM 100µM 100µM 0 1000 2000 3000 *** *** *** ** ** ** Thalidomide Relative expression 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 d21 d16 d6 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 d3 d0 d21 d16 d5 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 mesoderm Meso-endoderm Cardiomyocytes d3 d0 d14 d5 d7 LIF ActA, Chir99021, BMP4 XAV939 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’ Fluoracil Retinoic acid Control Thalidomide Diphenylhydantoin Effect of teratogens on hepatocyte differentiation 5’Fluoracil Retinoic acid 0.0 5n M Diphenylhydantoin Thalidomide DMSO control Low exposure Medium exposure High exposure Selected 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. d3 d0 d14 d5 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 Day3 Day 7 Day 10 Day 14 0.0 0.5 1.0 1.5 2.0 Relative expression ** ** * Bmp4 Day3 Day 7 Day 10 Day 14 0 2 4 6 8 10 Relative expression * * MYH6 Day3 Day 7 Day 10 Day 14 0 50 100 150 200 *** *** Relative expression d0 d3 d10 d21 Toxys, Leiden, The Netherlands P.I. Racz, R.R.M. Ottenheijm, R. Derr, G. Hendriks Undifferentiated ES cells Cardiac mesoderm Meso-endoderm Cardiomyocytes Cardiac progenitor cells A. B. 5'Fluorouracil Control 0.125mM 0.25mM 0.5mM 0.0 0.5 1.0 1.5 2.0 *** *** GFP/DAPI Retinoic acid Control 0.05nM 0.1nM 0.2nM 0.0 0.5 1.0 1.5 * *** GFP/DAPI Diphenylhydantoin Control 25µM 50µM 100µM 0.0 0.2 0.4 0.6 0.8 1.0 GFP/DAPI Thalidomide Control 25µM 50µM 100µM 0.0 0.5 1.0 1.5 GFP/DAPI 5'Fluorouracil Control 0.0625µM 0.125µM 0.25µM 0 50 100 150 ** Beating body Retinoic acid Control 0.05nM 0.1nM 0.2nM 0 50 100 150 Beating body Diphenylhidantoin Control 25µM 50µM 100µM 0 50 100 150 *** *** Beating body Thalidomide Control 0.25mM 0.5 mM 1 mM 0 50 100 150 Beating body Control D21 0.0 0.5 1.0 1.5 *** Pluripotent stage specific biomarker expression (OCT4) Relative expression Control Control 0,125 µM 0,125 µM 0,25 µM 0,25 µM 0,5 µM 0,5 µM 0 2000 4000 6000 *** *** *** ** *** ** 5'Fluorouracil Relative expression AFP ALB AFP ALB Day 0 Day 21 Day 21 0 1000 2000 3000 Hepatocyte specific biomarker expression *** ** Relative expression AFP ALB pgk-Neo FP sa sd G ATG STOP Artificial Chromosome (BAC) biomarker gene exon1 exon2 intron
Transcript of P.I. Racz, R.R.M. Ottenheijm, R. Derr, G. Hendriks · Control Control 25 µ M 25 µ M 50 µ M 50 µ...
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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
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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
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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.
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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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