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Molecular markers in embryonic stem cells
Toxicology in Vitro 15 (2001) 455–461 www.elsevier.com/locate/toxinvit
Molecular markers in embryonic stem cells N.I. zur Nieden*, L.J. Ruf, G. Kempka, H. Hildebrand, H.J. Ahr Bayer AG, Research Toxicology, 42096 Wuppertal, Germany
Abstract Embryonic stem cells are pluripotent cells derived from mouse blastocysts, which have the capacity to differentiate in vitro into a wide variety of cell types. Based on this potential the embryonic stem cell test (EST) has been developed, which represents an assay system for the classification of compounds for their teratogenic potential, based on the morphological evaluation of contracting myocard cells compared to the cytotoxic effects on undifferentiated stem cells and adult 3T3 fibroblasts. To expand the EST, the quantitative expression of the a- and b-myosin heavy chain (MHC) genes under the influence of test compounds was studied employing real-time TaqMan PCR analysis. The molecular evaluation of the MHC genes allows a higher sensitivity for the classification of substances and the transfer of the EST to the molecular level allows to start experimental procedures at day 9 of culture. Thus, the modulated EST holds promise as a new easily quantifiable in vitro screening assay in teratology. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: ES cells; Real-time TaqMan PCR analysis; Teratogenicity
1. Introduction In the field of teratology, several in vitro methods have been introduced to date. As a result, our understanding of the basic molecular and cellular mechanisms of mammalian embryonic development has greatly improved. Among the spectrum of in vitro embryotoxicity tests there are whole embryo and organ culture, micromass assay and the embryonic stem cell test. Embryonic stem cells (ES cells) are pluripotent cells derived from the inner cell mass of mouse blastocysts and have been characterized to differentiate in vitro into a wide variety of cell types representing all three germ layers. Under appropriate culture conditions a part of them differentiates spontaneously into beating myocard cells. The embryonic stem cell test (EST) was developed by Spielmann and colleagues (1997) as an in vitro system representing an assay system for the classification of test chemicals for their teratogenic potential. Three
Abbreviations: ES, embryonic stem; EST, embryonic stem cell test; MHC, myosin heavy chain; 5-FU, 5-fluoruracil; HU, hydroxyurea; PDT, diphenylhydantoin; Sacch, saccharin; Pen G, penicillin G; Thali, thalidomide; Indo, indomethacin; PBS, Dulbecco’s phosphate buffered saline; FCS, fetal calf serum; DMEM, Dulbecco’s modified Eagle’s medium. * Corresponding author. Tel.: +49-202-368074; fax: +49-202364137. E-mail address: [email protected] (N.I. zur Nieden).
different endpoints are evaluated in the EST, which are (a) the morphological evaluation of these contracting myocard cells compared to the general cytotoxic effects of the tested substances on (b) undifferentiated embryonic stem cells and (c) adult 3T3 fibroblasts determined by MTT assay. To improve the EST the quantitative expression of the a- and b-MHC genes under influence of test compounds was studied employing real-time TaqMan PCR analysis. These genes are known to be characteristic for atrial and ventricular cells during early embryonic heart development (Robbins et al., 1990) and are thus promising as marker genes for cardiac development during ES cell differentiation. The effects on MHC expression patterns of some selected compounds with known teratogenic potential representing the three classes non-, weak and strong teratogenic were tested during ES cell differentiation and the results were compared to those acquired with the existing EST. The test compounds used were chosen with reference to the development of the existing EST done by Spielmann et al. (1997).
2. Material and methods 2.1. Cell culture and differentiation Balb/c 3T3 cells were propagated in DMEM (Gibco Life Technologies, Paisley, Scotland) containing 5%
0887-2333/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0887-2333(01)00071-6
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FCS (Greiner, Frickenhausen, Germany), 5% newborn calf serum (Sigma-Aldrich, Steinheim, Germany), 2 mm glutamine (Sigma), 50 U/ml penicillin and 50 mg/ml streptomycin (Sigma), and were passaged every second day. Cells of the mouse ES cell line D3 (American Type Culture Collection, Rockville, MD, USA) were routinely grown as a monolayer in the presence of leukemia inhibitory factor (LIF, 1000 U/ml, Gibco) to maintain their undifferentiated status and passaged every second day. ES cell medium consisted of high glucose DMEM (4.5 g glucose/l, Gibco) supplemented with 20% fetal calf serum (FCS) (Sigma, chosen batches), 50 U/ml penicillin and 50 mg/ml streptomycin, 1% non-essential amino acids (Gibco) and 0.1 mm b-mercaptoethanol (Sigma). Differentiation was carried out in hanging drops (Rudnicki and McBurney, 1987) according to Heuer et al. (1993) with minor modifications. In brief, 20 ml stem cell suspension (3.75104 cells/ml) was placed onto the inner side of the lid of a petri dish filled with phosphate buffered saline (PBS) (Sigma) and then incubated at 37 C with 5% CO2. After culture for 3 days the formed aggregates (embryoid bodies, EBs) were transferred into bacteriological petri dishes. At day 5, EBs were plated separately into 24-well Costar plates. 2.2. Immunofluorescence EBs grown to the desired time point were fixed with ice-cold methanol:acetone (7:3) for 10 min at 20 C and blocked with 10% FCS in PBS for 30 min at room temperature. To identify cardiomyocytes the following primary antibodies were used: the mouse monoclonal antibody anti-myosin cardiac heavy chain a/b MAB 1546 (Chemicon, Temecula, CA, USA) and mouse monoclonal anti-sarcomeric a-actinin (clone EA53, Sigma). The differentiated cells were overlaid with the first antibody (appropriate dilution in PBS, 10% FCS) and incubated at 4 C overnight. The second antibody, a goat anti-mouse IgG F(ab)2 fragment, FITC-conjugated (Pierce, Rockford, IL, USA) was diluted in PBS, 5% FCS containing 0.1 mg/ml Hoechst 33258 (Sigma) to visualize cell nuclei and applied to the cells for 2 h at 4 C. The FITC-conjugated antibodies could be visualized in a Leica fluorescence microscope at an excitation wavelength of 450 nm and an emission wavelength of 490 nm. Corresponding wavelengths for Hoechst 33258 were 270 nm and 380 nm. 2.3. Cytotoxicity measurement The cytotoxic effects of test substances on 3T3 and ES cells were determined in an MTT assay. The mitochondrial dehydrogenase activity was quantified after incubation of substance treated cells with MTT solution (Sigma) for 2 h at 37 C. Absorbance was determined in a spectrophotometer at 570/630 nm.
2.4. RNA isolation and RT-PCR 20 EBs per sample were harvested at various time points in lysis buffer and total RNA was isolated with the RNeasy Midi Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Total RNA was also prepared from different adult mouse tissues using the same kit. Mice (Sv129 strain, male) were obtained from M&B (DN). The amount of RNA was determined using the RiboGreenTM RNA quantitation reagent and kit (Molecular Probes, Leiden, The Netherlands). cDNA was synthesized from 275 ng EB RNA or 250 ng tissue RNA per reaction with 40 U Superscript II, 50 ng random hexamers, 2.5 mm MgCl2, 10 mm DTT and 0.5 mm dNTP in reaction buffer (all Gibco) adding up to a total volume of 25 ml. The reaction mixture was incubated at 25 C for 15 min, at 42 C for 50 min and the cDNA synthesis terminated at 70 C for 15 min. PCR on adult tissues was performed in a Peltier Thermal Cycler (MJ Research, Watertown, MA, USA) with specific primers for a-MHC (50 -GAGCTCACCTACCAGACAGAGGA-30 and 50 -CACCTTCAA0 CTGTAGCTTGTCCAC-3 ), b-MHC (50 -ACCTGTCCAAGTTCCGCAAG-30 and 50 -CTTGTTGACCTGGGACTCGG-30 ) and a/b-MHC (50 -ACCTGTCCAAGTTCCGCAAG-30 and 50 -CTTGTTGACCTGGGACTCGG-30 ). The sequences of the oligonucleotide primers were calculated using the program ‘Primer Express’ from Applied Biosystems (Weiterstadt, Germany) and a BLAST search was performed at NCBI for their specificity. A two-step PCR was carried out with the following cycle conditions: 40 cycles of denaturation at 94 C for 30 s and annealing and elongation for 30 s (59 C for a-MHC, 62 C for b-MHC and a/b-MHC). PCR reactions contained 5 ml of the first strand synthesis reaction, 0.8 mm of each of the primers, 1.5 mm MgCl2, 200 mm dNTP and 2.5 U Taq Polymerase in PCR buffer (all Gibco). Amplified products were visualized by electrophoresis on 3% agarose gels and staining of the gels with ethidium bromide. Gels were photographed under UV with an Imager system (Pharmacia Biotech, Freiburg, Germany). 2.5. Real-time quantitative PCR For quantitative PCR analysis the SYBR1 Green PCR Core Reagents (Applied Biosystems) were used. Direct detection of PCR reaction products was monitored by measuring the increase in fluorescence caused by the binding of SYBR Green to double-stranded DNA in an ABI Prism1 7700 Sequence Detector (Applied Biosystems). Amplification was carried out with the primer set recognizing both isoforms (a/bMHC). Reaction mixtures contained 2.2 ml of the first strand reaction, dNTP Blend (dATP, dCTP and dGTP 0.2 mm each and 0.4 mm dUTP), 3 mm MgCl2, 1.25 U
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AmpliTaq Gold and 0.5 U AmpErase in SYBR PCR buffer. TaqMan PCR began with a Taq Polymerase activating step (95 C for 10 min), all other PCR conditions were as indicated above. Standard curves were constructed with serial dilutions of heart cDNA from adult mice.
3. Results 3.1. Cardiac differentiation of ES cells in vitro To elucidate the appearance of cardiomyocytes within the embryoid bodies, immunofluorescent staining with a-/
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b-MHC and sarcomeric actinin was performed (Plate 1). Cells which were identified as cardiomyocytes by staining of the cytoplasm with a-/b-MHC also showed the characteristic highly organized sarcomeric structures as indicated by indirect immunofluorescence with a-actinin. During optical inspection on day 10 of culture it was estimated that approximately 15–25% of the cells in the embryoid body outgrowths were registered with the antibodies, which corresponds to the amount of cells in the beating areas. Cardiomyocytes in the EB outgrowths began to contract as early as at day 9 and with prolonged time of incubation the number of contracting areas markedly decreased as described (Wobus et al.,
Plate 1. Embryoid bodies at day 12 of culture. ES-cell derived cardiomyocytes characterized by indirect immunofluorescence with (I) a-/b-MHC (III) sarcomeric actinin and anti-mouse IgG FITC. (II) and (IV) Nuclear Blue stain with bis-benzimid. Magnification 320 for (I) and (II) and 200 for (III) and (IV).
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1991) and cardiomyocytes even terminated their spontaneous contractions around day 16. 3.2. Localization of - and -MHC transcripts to the adult mouse heart When conducting TaqMan PCR analyses the amplified products should preferably not exceed a length of 80–100 bp. The primers which were previously used for a- and b-MHC in studies with embryoid bodies (Robbins et al., 1990) amplifying products of 302 bp and 205 bp, respectively, were not applicable. Newly designed primers were tested on diverse adult mouse tissue cDNAs for their capability of detecting the desired amplicon specifically in cardiac tissue. Fig. 1 demonstrates that the new primers were specific for cardiac tissue. This is essential for quantitative analysis of expression patterns since not only cardiomyocytes but also other cell types are present in embryoid bodies.
3.3. Kinetic analysis A kinetic analysis of the investigated gene expression up to day 30 of culture was performed by TaqMan PCR analysis. Starting with day 5 of differentiation there was an increase in the expression of both isoforms of the MHC genes (Fig. 2). The expression curve peaks at day 9 and then declines to a basic level which is reached at day 15 of culture, which was qualitatively in agreement to the observation of beating cells. Thus, the MHC expression analysis gave the conclusion that a reduction in expression induced by teratogenic compounds could be best monitored at day 9 of culture. 3.4. Influence of test compounds As shown in Figs. 3 and 4 and Table 1, six out of seven chemicals affected the expression of the MHC genes at lower concentrations than the concentrations
Fig. 1. (I) Position of primers on the MHC genes. Dotted lines indicate introns. (II)–(IV) Localization of the MHC transcripts to different organs by RT-PCR analysis. (II) a-MHC, 50 ng cDNA/reaction. (III) b-MHC, 25 ng cDNA/reaction. (IV) a-/b-MHC, 25 ng cDNA/reaction. N=no template control, L=liver, K=kidney, Lg=lung, M=muscle, B=bone, C=cartilage, Br=brain, H=heart, Ma=Marker lane, 100 bp ladder (Gibco).
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which cause cytotoxicity. This could be expected since general toxicity as measured by MTT assay is compared to the real-time PCR technique, that quantifies specific toxicity. However, expression analysis is in general far
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more sensitive than differentiation studies monitored by microscopic evaluation, as shown by concentration curves for MHC expression, which are shifted to lower concentrations in comparison to the differentiation of
Fig. 2. Temporal expression pattern of the MHC isoforms in untreated control EBs up to day 30 of culture. (I) a-MHC. (II) a-/b-MHC. (III) bMHC. Values are means, n=3.
Fig. 3. Penicillin G treatment of EBs from day 0 to day 9 of culture. Various endpoints of the EST (MTT assay on ES/D3 cells and on ‘adult’ 3T3 mouse fibroblasts, differentiation assay and MHC transcript) were measured for selected concentrations and normalized to control. The differentiation assay is the microscopical evaluation of beating EBs on day 10 of culture.
Fig. 4. Relationship between IC50 values of the a-/b-MHC expression as measured by quantitative real-time PCR in the TaqMan System and the cytotoxic IC50 values on embryonic D3 cells as determined by MTT-assay.
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Table 1 Results for the tested compounds Teratogenic status in vivo
Substance
Cytotoxicity IC50 D3
Cytotoxicity IC50 3T3
a-/b-MHC expression IC50
Classification MHC expression
Strong
5-Fluoruracil Hydroxyurea Thalidomide Indomethacin Diphenylhydantoin Penicillin G Saccharin
0.012 4.5 810 16 170 870 1000
0.035 1.65 450 45 27 1000 1000
0.0015 0.65 67 55 7.5 650 820
1 1 2 2 2 3 3
Weak
Non-
Classification of compounds by MHC expression was determined using the descriptive statistics existing for the EST by Spielmann et al. (1997). Values stated in mg/ml and are means (n=3).
D3 cells (Fig. 3). In addition, the differentiation assay as well as the expression analyses represent methods for detecting a specific toxicity, which is the potential of the compound to influence embryonic heart development. The microscopic evaluation does not discriminate between EBs where large areas or only single cells beat, whereas in the TaqMan PCR analysis on the molecular level the total MHC cDNA is detected quantitatively. Decrease in expression can thus be revealed more sensitively. A comparison between the cytotoxic IC50 values of undifferentiated D3 cells as determined by the MTT assay and the IC50 values of the MHC expression as quantified by TaqMan PCR analysis (Fig. 4) reveals, that the chemicals tested can be separated into two subclasses. Compounds known to have no teratogenic potential, such as penicillin G (Pen G) and saccharin (Sacch), which are not known to cause any developmental malformations or to harm normal differentiation, influence MHC expression virtually similar as compared to cytotoxicity, since the IC50 values of both endpoints are quite similar. In view of the fact that analyses on the expression level are more sensitive than the evaluation with the MTT test, IC50 values for the MHC expression are slightly lower than the cytotoxic IC50 values. Reduction in expression was caused here by the cytotoxic effect of the substances. Substances belonging to the second subclass, such as 5-fluoruracil (5-FU), hydroxyurea (HU), diphenylhydantoin (PDT) and thalidomide (Thali), which are known to have a teratogenic potential of some kind (strong or moderate teratogen) show significantly different endpoints. The IC50 values determined by MHC expression analysis are considerably smaller than the IC50 values, which were found by MTT assay. Reduction in expression is a specific effect and not caused by the cytotoxicity of the substances. Whereas the strong teratogens 5-FU and HU affect cell processes in general, such as proliferation or DNA synthesis (DeSesso, 1979; Iwama et al., 1983), PDT is a vasoactive teratogen which affects the heart rate of the embryo (Danielsson et al., 1979).
Thalidomide exerts skeletal defects, but also affects normal heart development in man. Only indomethacin (Indo) is shown to be cytotoxic on undifferentiated D3 cells at lower concentrations than the concentration that causes reduction in MHC expression. This might be due to the fact that teratogenic effects of indomethacin appear in vivo as late as in the second and third trimester of the pregnancy. Indomethacin has no impact on the early embryonic development as mirrored by ES cell differentiation up to day 9 and is also a substance, which does not affect the development of the heart but evokes skeletal defects, incomplete virilization or inhibits prostaglandin synthesis (Norton, 1997).
4. Discussion In the course of identifying new and powerful pharmaceutical components, in vitro screening methods are performed prior to test chemicals in vivo on animals or even humans. As disadvantageous consequences on the reproduction of humans account to the worst sideeffects of new pharmaceuticals, promising compounds have to be screened for their teratogenic potential to select suitable candidates for development. For the in vivo testing of chemicals on their teratogenic potential considerable numbers of test animals are used at present. Even the in vitro methods currently used, such as the micromass assay and the whole embryo culture, utilize primarily isolated cells or embryos, implicating that pregnant animals have to be killed. To date, discussions are on their way to find common guidelines for the testing of the teratogenic potential of new industrial chemicals. The present study is based on an unlimited source of cells, which can be—once isolated—kept in permanent culture, thus avoiding the undue consumption of animals. To evade the problem that various other cell types develop within the same embryoid body simultaneously with cardiomyocytes, genes were selected which are
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specifically expressed in cardiomyocytes. For the model system the a-/b-MHC primers were chosen, since the signals acquired in the TaqMan PCR with the primers recognizing both isoforms simultaneously seem to be more sensitive than those acquired with the primer sets recognizing only a single isoform. Modulated expression of the MHC genes under influence of a test compound may provide a new and easily quantifiable endpoint for embryotoxicity testing. The quantitative expression analysis in the TaqMan system has not been developed to replace the MTT assay on embryonic stem cells as well as on ‘adult’ 3T3 fibroblasts, but to determine the decrease in the number of cardiomyocytes differentiating under the influence of a test compound. The chemical concentration that shows a half maximal inhibition of MHC gene expression in embryonic stem cells is a relevant in vitro endpoint for embryotoxic potential. Still, the EST should comprise a method measuring general cytotoxicity (MTT assay) besides another endpoint determining the differentiation processes during ES cell development performed by quantification of the expression of the MHC isoforms representing embryotoxicity. The difference in the concentrations which influence general cytotoxicity and embryotoxicity should embody a relevant in vitro endpoint for the embryotoxic potential of a test substance. The change in the MHC expression under influence of a test compound is an easier quantifiable endpoint in contrast to the microscopical measurement of beating myocard cells. Likewise, the MHC expression can already be evaluated at day 9 of culture compared to the evaluation of beating areas in the embryoid bodies, which is performed at day 10 of culture. In in vitro systems it is always important to take the shortest time possible. The present assay system only estimates cells differentiating into cardiomyocytes, but it is conceivable that teratogens such as indomethacin have an effect in later
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EB development or on other cell types. Other expression profiles might be retrieved when investigating other genes and cells of other types representing different organs than heart. Taken together, the use of real-time PCR with cardiac specific primers may offer an interesting improvement of the ES embryotoxicity test.
References Danielsson, B.R., Azarbayjani, F., Skold, A.C., Webster, W.S., 1997. Initiation of phenytoin teratogenesis: pharmacologically induced embryonic bradycardia and arrhythmia resulting in hypoxia and possible free radical damage at reoxygenation. Teratology 56, 271– 281. DeSesso, J.M., 1979. Cell death and free radicals: a mechanism for hydroxyurea teratogenesis. Medical Hypotheses 5, 937–951. Heuer, J., Bremer, S., Pohl, I., Spielmann, H., 1993. Development of an in vitro embryotoxicity test using murine embryonic stem cell cultures. Toxicology in Vitro 7, 551–556. Iwama, M., Sakamoto, Y., Honda, A., Mori, Y., 1983. Limb deformity induced in chick embryo by hydroxyurea. Journal of Pharmacobiodynamics 6, 836–843. Norton, M.E., 1997. Teratogen update: fetal effects of indomethacin administration during pregnancy. Teratology 56, 282–292. Robbins, J., Gulick, J., Sanchez, A., Howles, P., Doetschmann, T., 1990. Mouse embryonic stem cells express the cardiac myosin heavy chain genes during development in vitro. Journal of Biological Chemistry 265, 11905–11909. Rudnicki, M.A. and McBurney, M.W. (1987). Cell culture methods and induction of differentiation of embryonal carcinoma cell lines. In: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. E.J. Robertson (Ed.) IRL Press, Oxford, pp. 71–112. Spielmann, H., Pohl, I., Do¨ring, B., Liebsch, M., Moldenhauer, F., 1997. The embryonic stem cell test, an in vitro embryotoxicity test using two permanent mouse cell lines: 3T3 fibroblasts and embryonic stem cells. Toxicology in Vitro 10, 119–127. Wobus, A.M., Wallukat, G., Hescheler, J., 1991. Pluripotent mouse embryonic stem cells are able to differentiate into cardiomyocytes expressing chronotropic responses to adrenergic and cholinergic agents and CAE channel blockers. Differentiation 48, 173–182.
Molecular markers in embryonic stem cells N.I. zur Nieden*, L.J. Ruf, G. Kempka, H. Hildebrand, H.J. Ahr Bayer AG, Research Toxicology, 42096 Wuppertal, Germany
Abstract Embryonic stem cells are pluripotent cells derived from mouse blastocysts, which have the capacity to differentiate in vitro into a wide variety of cell types. Based on this potential the embryonic stem cell test (EST) has been developed, which represents an assay system for the classification of compounds for their teratogenic potential, based on the morphological evaluation of contracting myocard cells compared to the cytotoxic effects on undifferentiated stem cells and adult 3T3 fibroblasts. To expand the EST, the quantitative expression of the a- and b-myosin heavy chain (MHC) genes under the influence of test compounds was studied employing real-time TaqMan PCR analysis. The molecular evaluation of the MHC genes allows a higher sensitivity for the classification of substances and the transfer of the EST to the molecular level allows to start experimental procedures at day 9 of culture. Thus, the modulated EST holds promise as a new easily quantifiable in vitro screening assay in teratology. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: ES cells; Real-time TaqMan PCR analysis; Teratogenicity
1. Introduction In the field of teratology, several in vitro methods have been introduced to date. As a result, our understanding of the basic molecular and cellular mechanisms of mammalian embryonic development has greatly improved. Among the spectrum of in vitro embryotoxicity tests there are whole embryo and organ culture, micromass assay and the embryonic stem cell test. Embryonic stem cells (ES cells) are pluripotent cells derived from the inner cell mass of mouse blastocysts and have been characterized to differentiate in vitro into a wide variety of cell types representing all three germ layers. Under appropriate culture conditions a part of them differentiates spontaneously into beating myocard cells. The embryonic stem cell test (EST) was developed by Spielmann and colleagues (1997) as an in vitro system representing an assay system for the classification of test chemicals for their teratogenic potential. Three
Abbreviations: ES, embryonic stem; EST, embryonic stem cell test; MHC, myosin heavy chain; 5-FU, 5-fluoruracil; HU, hydroxyurea; PDT, diphenylhydantoin; Sacch, saccharin; Pen G, penicillin G; Thali, thalidomide; Indo, indomethacin; PBS, Dulbecco’s phosphate buffered saline; FCS, fetal calf serum; DMEM, Dulbecco’s modified Eagle’s medium. * Corresponding author. Tel.: +49-202-368074; fax: +49-202364137. E-mail address: [email protected] (N.I. zur Nieden).
different endpoints are evaluated in the EST, which are (a) the morphological evaluation of these contracting myocard cells compared to the general cytotoxic effects of the tested substances on (b) undifferentiated embryonic stem cells and (c) adult 3T3 fibroblasts determined by MTT assay. To improve the EST the quantitative expression of the a- and b-MHC genes under influence of test compounds was studied employing real-time TaqMan PCR analysis. These genes are known to be characteristic for atrial and ventricular cells during early embryonic heart development (Robbins et al., 1990) and are thus promising as marker genes for cardiac development during ES cell differentiation. The effects on MHC expression patterns of some selected compounds with known teratogenic potential representing the three classes non-, weak and strong teratogenic were tested during ES cell differentiation and the results were compared to those acquired with the existing EST. The test compounds used were chosen with reference to the development of the existing EST done by Spielmann et al. (1997).
2. Material and methods 2.1. Cell culture and differentiation Balb/c 3T3 cells were propagated in DMEM (Gibco Life Technologies, Paisley, Scotland) containing 5%
0887-2333/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0887-2333(01)00071-6
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FCS (Greiner, Frickenhausen, Germany), 5% newborn calf serum (Sigma-Aldrich, Steinheim, Germany), 2 mm glutamine (Sigma), 50 U/ml penicillin and 50 mg/ml streptomycin (Sigma), and were passaged every second day. Cells of the mouse ES cell line D3 (American Type Culture Collection, Rockville, MD, USA) were routinely grown as a monolayer in the presence of leukemia inhibitory factor (LIF, 1000 U/ml, Gibco) to maintain their undifferentiated status and passaged every second day. ES cell medium consisted of high glucose DMEM (4.5 g glucose/l, Gibco) supplemented with 20% fetal calf serum (FCS) (Sigma, chosen batches), 50 U/ml penicillin and 50 mg/ml streptomycin, 1% non-essential amino acids (Gibco) and 0.1 mm b-mercaptoethanol (Sigma). Differentiation was carried out in hanging drops (Rudnicki and McBurney, 1987) according to Heuer et al. (1993) with minor modifications. In brief, 20 ml stem cell suspension (3.75104 cells/ml) was placed onto the inner side of the lid of a petri dish filled with phosphate buffered saline (PBS) (Sigma) and then incubated at 37 C with 5% CO2. After culture for 3 days the formed aggregates (embryoid bodies, EBs) were transferred into bacteriological petri dishes. At day 5, EBs were plated separately into 24-well Costar plates. 2.2. Immunofluorescence EBs grown to the desired time point were fixed with ice-cold methanol:acetone (7:3) for 10 min at 20 C and blocked with 10% FCS in PBS for 30 min at room temperature. To identify cardiomyocytes the following primary antibodies were used: the mouse monoclonal antibody anti-myosin cardiac heavy chain a/b MAB 1546 (Chemicon, Temecula, CA, USA) and mouse monoclonal anti-sarcomeric a-actinin (clone EA53, Sigma). The differentiated cells were overlaid with the first antibody (appropriate dilution in PBS, 10% FCS) and incubated at 4 C overnight. The second antibody, a goat anti-mouse IgG F(ab)2 fragment, FITC-conjugated (Pierce, Rockford, IL, USA) was diluted in PBS, 5% FCS containing 0.1 mg/ml Hoechst 33258 (Sigma) to visualize cell nuclei and applied to the cells for 2 h at 4 C. The FITC-conjugated antibodies could be visualized in a Leica fluorescence microscope at an excitation wavelength of 450 nm and an emission wavelength of 490 nm. Corresponding wavelengths for Hoechst 33258 were 270 nm and 380 nm. 2.3. Cytotoxicity measurement The cytotoxic effects of test substances on 3T3 and ES cells were determined in an MTT assay. The mitochondrial dehydrogenase activity was quantified after incubation of substance treated cells with MTT solution (Sigma) for 2 h at 37 C. Absorbance was determined in a spectrophotometer at 570/630 nm.
2.4. RNA isolation and RT-PCR 20 EBs per sample were harvested at various time points in lysis buffer and total RNA was isolated with the RNeasy Midi Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Total RNA was also prepared from different adult mouse tissues using the same kit. Mice (Sv129 strain, male) were obtained from M&B (DN). The amount of RNA was determined using the RiboGreenTM RNA quantitation reagent and kit (Molecular Probes, Leiden, The Netherlands). cDNA was synthesized from 275 ng EB RNA or 250 ng tissue RNA per reaction with 40 U Superscript II, 50 ng random hexamers, 2.5 mm MgCl2, 10 mm DTT and 0.5 mm dNTP in reaction buffer (all Gibco) adding up to a total volume of 25 ml. The reaction mixture was incubated at 25 C for 15 min, at 42 C for 50 min and the cDNA synthesis terminated at 70 C for 15 min. PCR on adult tissues was performed in a Peltier Thermal Cycler (MJ Research, Watertown, MA, USA) with specific primers for a-MHC (50 -GAGCTCACCTACCAGACAGAGGA-30 and 50 -CACCTTCAA0 CTGTAGCTTGTCCAC-3 ), b-MHC (50 -ACCTGTCCAAGTTCCGCAAG-30 and 50 -CTTGTTGACCTGGGACTCGG-30 ) and a/b-MHC (50 -ACCTGTCCAAGTTCCGCAAG-30 and 50 -CTTGTTGACCTGGGACTCGG-30 ). The sequences of the oligonucleotide primers were calculated using the program ‘Primer Express’ from Applied Biosystems (Weiterstadt, Germany) and a BLAST search was performed at NCBI for their specificity. A two-step PCR was carried out with the following cycle conditions: 40 cycles of denaturation at 94 C for 30 s and annealing and elongation for 30 s (59 C for a-MHC, 62 C for b-MHC and a/b-MHC). PCR reactions contained 5 ml of the first strand synthesis reaction, 0.8 mm of each of the primers, 1.5 mm MgCl2, 200 mm dNTP and 2.5 U Taq Polymerase in PCR buffer (all Gibco). Amplified products were visualized by electrophoresis on 3% agarose gels and staining of the gels with ethidium bromide. Gels were photographed under UV with an Imager system (Pharmacia Biotech, Freiburg, Germany). 2.5. Real-time quantitative PCR For quantitative PCR analysis the SYBR1 Green PCR Core Reagents (Applied Biosystems) were used. Direct detection of PCR reaction products was monitored by measuring the increase in fluorescence caused by the binding of SYBR Green to double-stranded DNA in an ABI Prism1 7700 Sequence Detector (Applied Biosystems). Amplification was carried out with the primer set recognizing both isoforms (a/bMHC). Reaction mixtures contained 2.2 ml of the first strand reaction, dNTP Blend (dATP, dCTP and dGTP 0.2 mm each and 0.4 mm dUTP), 3 mm MgCl2, 1.25 U
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AmpliTaq Gold and 0.5 U AmpErase in SYBR PCR buffer. TaqMan PCR began with a Taq Polymerase activating step (95 C for 10 min), all other PCR conditions were as indicated above. Standard curves were constructed with serial dilutions of heart cDNA from adult mice.
3. Results 3.1. Cardiac differentiation of ES cells in vitro To elucidate the appearance of cardiomyocytes within the embryoid bodies, immunofluorescent staining with a-/
457
b-MHC and sarcomeric actinin was performed (Plate 1). Cells which were identified as cardiomyocytes by staining of the cytoplasm with a-/b-MHC also showed the characteristic highly organized sarcomeric structures as indicated by indirect immunofluorescence with a-actinin. During optical inspection on day 10 of culture it was estimated that approximately 15–25% of the cells in the embryoid body outgrowths were registered with the antibodies, which corresponds to the amount of cells in the beating areas. Cardiomyocytes in the EB outgrowths began to contract as early as at day 9 and with prolonged time of incubation the number of contracting areas markedly decreased as described (Wobus et al.,
Plate 1. Embryoid bodies at day 12 of culture. ES-cell derived cardiomyocytes characterized by indirect immunofluorescence with (I) a-/b-MHC (III) sarcomeric actinin and anti-mouse IgG FITC. (II) and (IV) Nuclear Blue stain with bis-benzimid. Magnification 320 for (I) and (II) and 200 for (III) and (IV).
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1991) and cardiomyocytes even terminated their spontaneous contractions around day 16. 3.2. Localization of - and -MHC transcripts to the adult mouse heart When conducting TaqMan PCR analyses the amplified products should preferably not exceed a length of 80–100 bp. The primers which were previously used for a- and b-MHC in studies with embryoid bodies (Robbins et al., 1990) amplifying products of 302 bp and 205 bp, respectively, were not applicable. Newly designed primers were tested on diverse adult mouse tissue cDNAs for their capability of detecting the desired amplicon specifically in cardiac tissue. Fig. 1 demonstrates that the new primers were specific for cardiac tissue. This is essential for quantitative analysis of expression patterns since not only cardiomyocytes but also other cell types are present in embryoid bodies.
3.3. Kinetic analysis A kinetic analysis of the investigated gene expression up to day 30 of culture was performed by TaqMan PCR analysis. Starting with day 5 of differentiation there was an increase in the expression of both isoforms of the MHC genes (Fig. 2). The expression curve peaks at day 9 and then declines to a basic level which is reached at day 15 of culture, which was qualitatively in agreement to the observation of beating cells. Thus, the MHC expression analysis gave the conclusion that a reduction in expression induced by teratogenic compounds could be best monitored at day 9 of culture. 3.4. Influence of test compounds As shown in Figs. 3 and 4 and Table 1, six out of seven chemicals affected the expression of the MHC genes at lower concentrations than the concentrations
Fig. 1. (I) Position of primers on the MHC genes. Dotted lines indicate introns. (II)–(IV) Localization of the MHC transcripts to different organs by RT-PCR analysis. (II) a-MHC, 50 ng cDNA/reaction. (III) b-MHC, 25 ng cDNA/reaction. (IV) a-/b-MHC, 25 ng cDNA/reaction. N=no template control, L=liver, K=kidney, Lg=lung, M=muscle, B=bone, C=cartilage, Br=brain, H=heart, Ma=Marker lane, 100 bp ladder (Gibco).
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which cause cytotoxicity. This could be expected since general toxicity as measured by MTT assay is compared to the real-time PCR technique, that quantifies specific toxicity. However, expression analysis is in general far
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more sensitive than differentiation studies monitored by microscopic evaluation, as shown by concentration curves for MHC expression, which are shifted to lower concentrations in comparison to the differentiation of
Fig. 2. Temporal expression pattern of the MHC isoforms in untreated control EBs up to day 30 of culture. (I) a-MHC. (II) a-/b-MHC. (III) bMHC. Values are means, n=3.
Fig. 3. Penicillin G treatment of EBs from day 0 to day 9 of culture. Various endpoints of the EST (MTT assay on ES/D3 cells and on ‘adult’ 3T3 mouse fibroblasts, differentiation assay and MHC transcript) were measured for selected concentrations and normalized to control. The differentiation assay is the microscopical evaluation of beating EBs on day 10 of culture.
Fig. 4. Relationship between IC50 values of the a-/b-MHC expression as measured by quantitative real-time PCR in the TaqMan System and the cytotoxic IC50 values on embryonic D3 cells as determined by MTT-assay.
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Table 1 Results for the tested compounds Teratogenic status in vivo
Substance
Cytotoxicity IC50 D3
Cytotoxicity IC50 3T3
a-/b-MHC expression IC50
Classification MHC expression
Strong
5-Fluoruracil Hydroxyurea Thalidomide Indomethacin Diphenylhydantoin Penicillin G Saccharin
0.012 4.5 810 16 170 870 1000
0.035 1.65 450 45 27 1000 1000
0.0015 0.65 67 55 7.5 650 820
1 1 2 2 2 3 3
Weak
Non-
Classification of compounds by MHC expression was determined using the descriptive statistics existing for the EST by Spielmann et al. (1997). Values stated in mg/ml and are means (n=3).
D3 cells (Fig. 3). In addition, the differentiation assay as well as the expression analyses represent methods for detecting a specific toxicity, which is the potential of the compound to influence embryonic heart development. The microscopic evaluation does not discriminate between EBs where large areas or only single cells beat, whereas in the TaqMan PCR analysis on the molecular level the total MHC cDNA is detected quantitatively. Decrease in expression can thus be revealed more sensitively. A comparison between the cytotoxic IC50 values of undifferentiated D3 cells as determined by the MTT assay and the IC50 values of the MHC expression as quantified by TaqMan PCR analysis (Fig. 4) reveals, that the chemicals tested can be separated into two subclasses. Compounds known to have no teratogenic potential, such as penicillin G (Pen G) and saccharin (Sacch), which are not known to cause any developmental malformations or to harm normal differentiation, influence MHC expression virtually similar as compared to cytotoxicity, since the IC50 values of both endpoints are quite similar. In view of the fact that analyses on the expression level are more sensitive than the evaluation with the MTT test, IC50 values for the MHC expression are slightly lower than the cytotoxic IC50 values. Reduction in expression was caused here by the cytotoxic effect of the substances. Substances belonging to the second subclass, such as 5-fluoruracil (5-FU), hydroxyurea (HU), diphenylhydantoin (PDT) and thalidomide (Thali), which are known to have a teratogenic potential of some kind (strong or moderate teratogen) show significantly different endpoints. The IC50 values determined by MHC expression analysis are considerably smaller than the IC50 values, which were found by MTT assay. Reduction in expression is a specific effect and not caused by the cytotoxicity of the substances. Whereas the strong teratogens 5-FU and HU affect cell processes in general, such as proliferation or DNA synthesis (DeSesso, 1979; Iwama et al., 1983), PDT is a vasoactive teratogen which affects the heart rate of the embryo (Danielsson et al., 1979).
Thalidomide exerts skeletal defects, but also affects normal heart development in man. Only indomethacin (Indo) is shown to be cytotoxic on undifferentiated D3 cells at lower concentrations than the concentration that causes reduction in MHC expression. This might be due to the fact that teratogenic effects of indomethacin appear in vivo as late as in the second and third trimester of the pregnancy. Indomethacin has no impact on the early embryonic development as mirrored by ES cell differentiation up to day 9 and is also a substance, which does not affect the development of the heart but evokes skeletal defects, incomplete virilization or inhibits prostaglandin synthesis (Norton, 1997).
4. Discussion In the course of identifying new and powerful pharmaceutical components, in vitro screening methods are performed prior to test chemicals in vivo on animals or even humans. As disadvantageous consequences on the reproduction of humans account to the worst sideeffects of new pharmaceuticals, promising compounds have to be screened for their teratogenic potential to select suitable candidates for development. For the in vivo testing of chemicals on their teratogenic potential considerable numbers of test animals are used at present. Even the in vitro methods currently used, such as the micromass assay and the whole embryo culture, utilize primarily isolated cells or embryos, implicating that pregnant animals have to be killed. To date, discussions are on their way to find common guidelines for the testing of the teratogenic potential of new industrial chemicals. The present study is based on an unlimited source of cells, which can be—once isolated—kept in permanent culture, thus avoiding the undue consumption of animals. To evade the problem that various other cell types develop within the same embryoid body simultaneously with cardiomyocytes, genes were selected which are
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specifically expressed in cardiomyocytes. For the model system the a-/b-MHC primers were chosen, since the signals acquired in the TaqMan PCR with the primers recognizing both isoforms simultaneously seem to be more sensitive than those acquired with the primer sets recognizing only a single isoform. Modulated expression of the MHC genes under influence of a test compound may provide a new and easily quantifiable endpoint for embryotoxicity testing. The quantitative expression analysis in the TaqMan system has not been developed to replace the MTT assay on embryonic stem cells as well as on ‘adult’ 3T3 fibroblasts, but to determine the decrease in the number of cardiomyocytes differentiating under the influence of a test compound. The chemical concentration that shows a half maximal inhibition of MHC gene expression in embryonic stem cells is a relevant in vitro endpoint for embryotoxic potential. Still, the EST should comprise a method measuring general cytotoxicity (MTT assay) besides another endpoint determining the differentiation processes during ES cell development performed by quantification of the expression of the MHC isoforms representing embryotoxicity. The difference in the concentrations which influence general cytotoxicity and embryotoxicity should embody a relevant in vitro endpoint for the embryotoxic potential of a test substance. The change in the MHC expression under influence of a test compound is an easier quantifiable endpoint in contrast to the microscopical measurement of beating myocard cells. Likewise, the MHC expression can already be evaluated at day 9 of culture compared to the evaluation of beating areas in the embryoid bodies, which is performed at day 10 of culture. In in vitro systems it is always important to take the shortest time possible. The present assay system only estimates cells differentiating into cardiomyocytes, but it is conceivable that teratogens such as indomethacin have an effect in later
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EB development or on other cell types. Other expression profiles might be retrieved when investigating other genes and cells of other types representing different organs than heart. Taken together, the use of real-time PCR with cardiac specific primers may offer an interesting improvement of the ES embryotoxicity test.
References Danielsson, B.R., Azarbayjani, F., Skold, A.C., Webster, W.S., 1997. Initiation of phenytoin teratogenesis: pharmacologically induced embryonic bradycardia and arrhythmia resulting in hypoxia and possible free radical damage at reoxygenation. Teratology 56, 271– 281. DeSesso, J.M., 1979. Cell death and free radicals: a mechanism for hydroxyurea teratogenesis. Medical Hypotheses 5, 937–951. Heuer, J., Bremer, S., Pohl, I., Spielmann, H., 1993. Development of an in vitro embryotoxicity test using murine embryonic stem cell cultures. Toxicology in Vitro 7, 551–556. Iwama, M., Sakamoto, Y., Honda, A., Mori, Y., 1983. Limb deformity induced in chick embryo by hydroxyurea. Journal of Pharmacobiodynamics 6, 836–843. Norton, M.E., 1997. Teratogen update: fetal effects of indomethacin administration during pregnancy. Teratology 56, 282–292. Robbins, J., Gulick, J., Sanchez, A., Howles, P., Doetschmann, T., 1990. Mouse embryonic stem cells express the cardiac myosin heavy chain genes during development in vitro. Journal of Biological Chemistry 265, 11905–11909. Rudnicki, M.A. and McBurney, M.W. (1987). Cell culture methods and induction of differentiation of embryonal carcinoma cell lines. In: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. E.J. Robertson (Ed.) IRL Press, Oxford, pp. 71–112. Spielmann, H., Pohl, I., Do¨ring, B., Liebsch, M., Moldenhauer, F., 1997. The embryonic stem cell test, an in vitro embryotoxicity test using two permanent mouse cell lines: 3T3 fibroblasts and embryonic stem cells. Toxicology in Vitro 10, 119–127. Wobus, A.M., Wallukat, G., Hescheler, J., 1991. Pluripotent mouse embryonic stem cells are able to differentiate into cardiomyocytes expressing chronotropic responses to adrenergic and cholinergic agents and CAE channel blockers. Differentiation 48, 173–182.