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The stem-cell test—A novel in vitro assay for teratogenic potential
~
Pergamon
Toxic'. in Vitro Vol. 8, No. 4, pp. 697-701, 1994
0887-2333(94)E0093-9
Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0887-2333/94 $7.00 + 0.00
THE STEM-CELL TEST--A NOVEL I N V I T R O ASSAY FOR TERATOGENIC POTENTIAL D. R. NEWALL and K. E. BEEDLES Genetic and Reproductive Toxicology, Glaxo Group Research Ltd, Ware, Herts. SG12 ODP, UK Abstract--The micromass test for teratogens (Flint and Orton, 1984) has undergone extensive validation for use in reproductive toxicology. A major drawback of this test is its use of primary cells derived from rat embryos. The stem-cell test is a novel assay for teratogenic potential which uses a propagated cell line. Mouse embryonic stem cells (ESC) are maintained in an undifferentiated state in the presence ofleukaemia inhibitory factor (LIF). On removing LIF, the cells differentiate into an embryonic endoderm that is morphologically distinct from ESC. Colonies of ESC are maintained from which cells can be harvested daily, and these cells, when washed free of LIF, form a population of differentiating cells on which the effects of chemicals can be tested. The conditions under which differentiating ESC can be substituted for rat primary embryonic cells in a micromass test protocol have been determined, and the effects of 25 compounds investigated in a blind trial. The stem-cell test predicted the teratogenicity of these compounds with a similar sensitivity and specificity to the micromass test, with the advantage that the test uses a propagated cell line; there is no use of animals.
INTRODUCTION
The micromass test for the assay of teratogenic potential (Flint and Orton, 1984) has undergone extensive development and validation for use in reproductive toxicology. A major drawback of this test, however, is its use of primary cells derived from rat embryos. The test focuses on the effects of compounds on the process of differentiation, and it is the difficulty of producing this event to order in vitro, that has, until recently, militated against the use of a propagated cell line. The D-3 cell line is a line of mouse embryonic stem cells (ESC), routinely used in the manufacture of transgenic mice. These are maintained in an undifferentiated state in the presence of leukaemia inhibitory factor (LIF). On removing the LIF, cells differentiate into an embryonic endoderm that is morphologically distinct from ESC and, importantly, shows an increased area compared with undifferentiated cells. A colony of ESC may thus be maintained from which cells can be harvested daily. These cells can be washed free of LIF and form a population of differentiating cells on which the effects of chemicals can be tested. We have determined the conditions under which differentiating ESC can be substituted for rat primary embyronic cells in a micromass test protocol, and looked at the effects of 25 compounds, in a blind trial, in this novel test system. MATERIALS AND M E T H O D S
A mouse ESC line, D-3, was obtained from the Transgenics Unit, Glaxo G r o u p Research. Cells were Abbreviations: ESC = embryonic stem cells; LIF = leukaemia inhibitory factor; CM =culture medium; LCM = culture medium containing leukaemia inhibitory factor.
maintained in Dulbecco's modified Eagle's medium, supplemented with 15% foetal calf serum, 2 m u L-glutamine, 1 mM sodium pyruvate, 0.1 m u fl-mercaptoethanol, 50 units penicillin/ml, 5 0 # g streptomycin/mi and 103 units LIF/ml. This medium is referred to as LCM, while medium without L I F is referred to as CM. A schematic representation of the test is presented in Fig. 1. Maintenance cultures were washed free of L C M and cells harvested by trypsinization. Cell suspensions were adjusted to a final density of 5 x 104 cells/ml in CM. Aliquots of cell suspension (10#1) were delivered to 96-well plates. Plates were incubated at 37.5°C in a humidified atmosphere containing 5% CO2 for 2 hr to allow cells to settle and attach. After cell attachment, C M containing test material or vehicle/control was added to plates at a volume of 200 #! per well. Plates were returned to the incubator for 7 days. All chemicals were of the purest grade available. The maximum concentration of test material used was 500 #g/ml or the limit of solubility. For each chemical a wide-range trial was performed to determine cytotoxicity, followed by two narrow-range trials. At the end of each incubation period cell survival was assessed using the M T T assay, and the extent of differentiation was determined by fixing and staining cultures with Nile Blue and measuring their area using image analysis. Absorbance of eluted stain in the M T T assay or area of stained cultures, calculated as a percentage of the control value, was plotted against log concentration of test material and IC50 values for cytotoxicity or inhibition of cell differentiation, determined by interpolation. The criterion for identification of teratogens in vitro was specific inhibition of cell differentiation ('two-fold rule'). Test materials were designated as teratogens when the IC50 for cell 697
698
D. R. NEWALL a n d K. E. BEEDLES Wash free of LIF ES C e l l s + L I F
Food plates with CM c o n t a i n i n g v e h i c l e or test material
m.
Cell attachment
D i l u t e to 5 x 104 c e l l s / m l
D e l i v e r 10 t.tl d r o p s to 96well plate
2hr @ 37.5°C
Incubate for 7 days @ 37.5°C
Differentiation-area
EVALUATE Cytotoxicity-MTT
Fig. 1. Schematic r e p r e s e n t a t i o n o f the stem-cell test: ES cells = e m b r y o n i c stem cells; L I F = l e u k a e m i a i n h i b i t o r y factor; C M = culture m e d i u m .
d i f f e r e n t i a t i o n ( D ) w a s l e s s t h a n h a l f t h a t o f t h e ICs0 f o r c y t o t o x i c i t y ( A ) , (i.e. A / D > 2).
o u r i n t e r p r e t a t i o n a f t e r d e c o d i n g a r e g i v e n i n T a b l e 1. T h e r e w e r e f o u r f a l s e n e g a t i v e s ( a c e t a z o l a m i d e , flaminoproprionitrile, caffeine and ethylenethiourea) and three false positives (ascorbic acid, diphenhydramine and furazolidone). The sensitivity (teratogens correctly identified) and specificity (nont e r a t o g e n s c o r r e c t l y i d e n t i f i e d ) w e r e 73 a n d 7 0 % , respectively. The test appeared to identify teratogens requiring activation without an additional metabolizing system.
RESULTS
Control cultures were incubated for 7 days with and without LIF. In the presence of LIF, cultures largely formed into small discrete masses of undifferentiated cells (Plate la), whereas, in the absence of LIF, cells differentiated into a flattened sheet of endoderm-like cells (Plate lb). Despite some spont a n e o u s d i f f e r e n t i a t i o n i n c u l t u r e s w i t h L I F , a 2.5fold increase in area was seen in LIF-free cultures. The results of the blind trial with 25 compounds and
DISCUSSION A c c o r d i n g t o t h e p r e s e n t in vivo c l a s s i f i c a t i o n , t h e sensitivity and specificity of the stem-cell test are not
Table 1. Results of the stem-cell test for 25 coded compounds Stem-cell test
Cytotoxicity [A; IC50 (#g/ml)]
Differentiation [D; ICs0 (#g/ml)]
A/D ratio
Bulsulphan Cyclophosphamide Cytosine arabinoside Retinoic acid
+ + + +
II 143 1.3 x 10 2 7.2 × 10 7
0.9 21 2 x 10 3 1.5 x 10 -7
12.2 6.8 6.5 4.8
Moderate
Diphenylhydantoin Ethylenethiourea Indomethacin
+ +
72 > 500 140
5.8 > 500 52
12.4 - I 2.7
Weak
Acetazolamide Aspirin fl -Aminoproprionitrile Caffeine Dexamethasone Sodium arsenate Methotrexate Procarbazine
+ + + + +
> 500 353 47 78 27 3.6 4.5 x 10 2 > 500
> 500 72 35 68 7.6 0.9 1.6 × 10 2 260
-~ 1 4.9 1.3 1. I 3.6 4.0 2.8 -~2
Non-teratogen
Acetate (sodium) Ascorbic acid Butylated hydroxyanisole Cromolyn (sodium) Diphenhydramine Furazolidone Isoniazid Penicillin G Saccharin Sulfanilamide
+ + + -
> 500 110 14 > 100 20 1.5 > 500 > 500 > 500 > 500
> 500 22 8.3 > 100 2.8 0.3 > 500 > 500 > 500 > 500
~- I 5.0 1.7 -~ 1 7. I 5.0 - 1 -~ 1 ~ 1 ~ 1
Teratogenic status
Compound
Strong
Test materials were designated as teratogens when the IC50 for cell differentiation (D) was less than half of the IC5o for cytotoxicity (A) (i.e. A/D > 2).
Plate I. Mouse
embryonic
stem cells stained with Nile blue (a) after 7 days of incubation after 7 days of incubation without LIF.
699
with LIF, (b)
701
Stem-cell test for teratogenic potential impressive, but are well within the range of values reported for the micromass test, with the advantage that the stem-cell test does not use animals. Furthermore, with the exception of ethylenethiourea, the false positives and negatives seen in this study are common to both test systems. It is interesting to speculate that they are detecting compounds that directly affect differentiation, irrespective of whether this action leads to teratogenicity in vivo, and that the false negatives are compounds that act by way of systems not present in simple cell-based tests. If this is the case, the combining of the stem-cell test with a
more complex in vitro system, such as rat wholeembryo culture, may improve sensitivity and specificity to an acceptable level. At present, the main value of the stem-cell test, as with the micromass test, would seem to be the selection of development candidates within a specific chemical class. REFERENCES
Flint O. P. and Orton T. C. (1984) An in vitro assay for teratogens with cultures of rat embryo midbrain and limb bud cells. Toxicology and Applied Pharmacology 76, 383-395.
Pergamon
Toxic'. in Vitro Vol. 8, No. 4, pp. 697-701, 1994
0887-2333(94)E0093-9
Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0887-2333/94 $7.00 + 0.00
THE STEM-CELL TEST--A NOVEL I N V I T R O ASSAY FOR TERATOGENIC POTENTIAL D. R. NEWALL and K. E. BEEDLES Genetic and Reproductive Toxicology, Glaxo Group Research Ltd, Ware, Herts. SG12 ODP, UK Abstract--The micromass test for teratogens (Flint and Orton, 1984) has undergone extensive validation for use in reproductive toxicology. A major drawback of this test is its use of primary cells derived from rat embryos. The stem-cell test is a novel assay for teratogenic potential which uses a propagated cell line. Mouse embryonic stem cells (ESC) are maintained in an undifferentiated state in the presence ofleukaemia inhibitory factor (LIF). On removing LIF, the cells differentiate into an embryonic endoderm that is morphologically distinct from ESC. Colonies of ESC are maintained from which cells can be harvested daily, and these cells, when washed free of LIF, form a population of differentiating cells on which the effects of chemicals can be tested. The conditions under which differentiating ESC can be substituted for rat primary embryonic cells in a micromass test protocol have been determined, and the effects of 25 compounds investigated in a blind trial. The stem-cell test predicted the teratogenicity of these compounds with a similar sensitivity and specificity to the micromass test, with the advantage that the test uses a propagated cell line; there is no use of animals.
INTRODUCTION
The micromass test for the assay of teratogenic potential (Flint and Orton, 1984) has undergone extensive development and validation for use in reproductive toxicology. A major drawback of this test, however, is its use of primary cells derived from rat embryos. The test focuses on the effects of compounds on the process of differentiation, and it is the difficulty of producing this event to order in vitro, that has, until recently, militated against the use of a propagated cell line. The D-3 cell line is a line of mouse embryonic stem cells (ESC), routinely used in the manufacture of transgenic mice. These are maintained in an undifferentiated state in the presence of leukaemia inhibitory factor (LIF). On removing the LIF, cells differentiate into an embryonic endoderm that is morphologically distinct from ESC and, importantly, shows an increased area compared with undifferentiated cells. A colony of ESC may thus be maintained from which cells can be harvested daily. These cells can be washed free of LIF and form a population of differentiating cells on which the effects of chemicals can be tested. We have determined the conditions under which differentiating ESC can be substituted for rat primary embyronic cells in a micromass test protocol, and looked at the effects of 25 compounds, in a blind trial, in this novel test system. MATERIALS AND M E T H O D S
A mouse ESC line, D-3, was obtained from the Transgenics Unit, Glaxo G r o u p Research. Cells were Abbreviations: ESC = embryonic stem cells; LIF = leukaemia inhibitory factor; CM =culture medium; LCM = culture medium containing leukaemia inhibitory factor.
maintained in Dulbecco's modified Eagle's medium, supplemented with 15% foetal calf serum, 2 m u L-glutamine, 1 mM sodium pyruvate, 0.1 m u fl-mercaptoethanol, 50 units penicillin/ml, 5 0 # g streptomycin/mi and 103 units LIF/ml. This medium is referred to as LCM, while medium without L I F is referred to as CM. A schematic representation of the test is presented in Fig. 1. Maintenance cultures were washed free of L C M and cells harvested by trypsinization. Cell suspensions were adjusted to a final density of 5 x 104 cells/ml in CM. Aliquots of cell suspension (10#1) were delivered to 96-well plates. Plates were incubated at 37.5°C in a humidified atmosphere containing 5% CO2 for 2 hr to allow cells to settle and attach. After cell attachment, C M containing test material or vehicle/control was added to plates at a volume of 200 #! per well. Plates were returned to the incubator for 7 days. All chemicals were of the purest grade available. The maximum concentration of test material used was 500 #g/ml or the limit of solubility. For each chemical a wide-range trial was performed to determine cytotoxicity, followed by two narrow-range trials. At the end of each incubation period cell survival was assessed using the M T T assay, and the extent of differentiation was determined by fixing and staining cultures with Nile Blue and measuring their area using image analysis. Absorbance of eluted stain in the M T T assay or area of stained cultures, calculated as a percentage of the control value, was plotted against log concentration of test material and IC50 values for cytotoxicity or inhibition of cell differentiation, determined by interpolation. The criterion for identification of teratogens in vitro was specific inhibition of cell differentiation ('two-fold rule'). Test materials were designated as teratogens when the IC50 for cell 697
698
D. R. NEWALL a n d K. E. BEEDLES Wash free of LIF ES C e l l s + L I F
Food plates with CM c o n t a i n i n g v e h i c l e or test material
m.
Cell attachment
D i l u t e to 5 x 104 c e l l s / m l
D e l i v e r 10 t.tl d r o p s to 96well plate
2hr @ 37.5°C
Incubate for 7 days @ 37.5°C
Differentiation-area
EVALUATE Cytotoxicity-MTT
Fig. 1. Schematic r e p r e s e n t a t i o n o f the stem-cell test: ES cells = e m b r y o n i c stem cells; L I F = l e u k a e m i a i n h i b i t o r y factor; C M = culture m e d i u m .
d i f f e r e n t i a t i o n ( D ) w a s l e s s t h a n h a l f t h a t o f t h e ICs0 f o r c y t o t o x i c i t y ( A ) , (i.e. A / D > 2).
o u r i n t e r p r e t a t i o n a f t e r d e c o d i n g a r e g i v e n i n T a b l e 1. T h e r e w e r e f o u r f a l s e n e g a t i v e s ( a c e t a z o l a m i d e , flaminoproprionitrile, caffeine and ethylenethiourea) and three false positives (ascorbic acid, diphenhydramine and furazolidone). The sensitivity (teratogens correctly identified) and specificity (nont e r a t o g e n s c o r r e c t l y i d e n t i f i e d ) w e r e 73 a n d 7 0 % , respectively. The test appeared to identify teratogens requiring activation without an additional metabolizing system.
RESULTS
Control cultures were incubated for 7 days with and without LIF. In the presence of LIF, cultures largely formed into small discrete masses of undifferentiated cells (Plate la), whereas, in the absence of LIF, cells differentiated into a flattened sheet of endoderm-like cells (Plate lb). Despite some spont a n e o u s d i f f e r e n t i a t i o n i n c u l t u r e s w i t h L I F , a 2.5fold increase in area was seen in LIF-free cultures. The results of the blind trial with 25 compounds and
DISCUSSION A c c o r d i n g t o t h e p r e s e n t in vivo c l a s s i f i c a t i o n , t h e sensitivity and specificity of the stem-cell test are not
Table 1. Results of the stem-cell test for 25 coded compounds Stem-cell test
Cytotoxicity [A; IC50 (#g/ml)]
Differentiation [D; ICs0 (#g/ml)]
A/D ratio
Bulsulphan Cyclophosphamide Cytosine arabinoside Retinoic acid
+ + + +
II 143 1.3 x 10 2 7.2 × 10 7
0.9 21 2 x 10 3 1.5 x 10 -7
12.2 6.8 6.5 4.8
Moderate
Diphenylhydantoin Ethylenethiourea Indomethacin
+ +
72 > 500 140
5.8 > 500 52
12.4 - I 2.7
Weak
Acetazolamide Aspirin fl -Aminoproprionitrile Caffeine Dexamethasone Sodium arsenate Methotrexate Procarbazine
+ + + + +
> 500 353 47 78 27 3.6 4.5 x 10 2 > 500
> 500 72 35 68 7.6 0.9 1.6 × 10 2 260
-~ 1 4.9 1.3 1. I 3.6 4.0 2.8 -~2
Non-teratogen
Acetate (sodium) Ascorbic acid Butylated hydroxyanisole Cromolyn (sodium) Diphenhydramine Furazolidone Isoniazid Penicillin G Saccharin Sulfanilamide
+ + + -
> 500 110 14 > 100 20 1.5 > 500 > 500 > 500 > 500
> 500 22 8.3 > 100 2.8 0.3 > 500 > 500 > 500 > 500
~- I 5.0 1.7 -~ 1 7. I 5.0 - 1 -~ 1 ~ 1 ~ 1
Teratogenic status
Compound
Strong
Test materials were designated as teratogens when the IC50 for cell differentiation (D) was less than half of the IC5o for cytotoxicity (A) (i.e. A/D > 2).
Plate I. Mouse
embryonic
stem cells stained with Nile blue (a) after 7 days of incubation after 7 days of incubation without LIF.
699
with LIF, (b)
701
Stem-cell test for teratogenic potential impressive, but are well within the range of values reported for the micromass test, with the advantage that the stem-cell test does not use animals. Furthermore, with the exception of ethylenethiourea, the false positives and negatives seen in this study are common to both test systems. It is interesting to speculate that they are detecting compounds that directly affect differentiation, irrespective of whether this action leads to teratogenicity in vivo, and that the false negatives are compounds that act by way of systems not present in simple cell-based tests. If this is the case, the combining of the stem-cell test with a
more complex in vitro system, such as rat wholeembryo culture, may improve sensitivity and specificity to an acceptable level. At present, the main value of the stem-cell test, as with the micromass test, would seem to be the selection of development candidates within a specific chemical class. REFERENCES
Flint O. P. and Orton T. C. (1984) An in vitro assay for teratogens with cultures of rat embryo midbrain and limb bud cells. Toxicology and Applied Pharmacology 76, 383-395.