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Protocol no. 113

EMBRYONIC STEM CELL TEST (EST)

The effect of chemicals on 3T3 cells and on ES cells, a permanent cell line derived from mouse embryonic stem cells, can be used to predict teratogenic potential.

CONTACT

Prof. Dr. med. Horst Spielmann ZEBET BgVV

Diedersdorfer Weg 1

D-12277 Berlin Germany

Tel. (+49)-30-8412-2270 Fax. (+49)-30-8412-2958

RATIONALE

Embryotoxicity testing is either performed in vivo in pregnant animals or in vitro on cultured embryos or embryonic tissues and cells from pregnant animals. Both for in vivo and for ex vivo testing pregnant animals have to be sacrificed. Taking advantage of the potential of embryonic stem (ES) cells to differentiate in culture, a new in vitro embryotoxicity test with permanent cell lines from the mouse is proposed, the Embryonic Stem Cell Test (EST). In the EST two permanent mouse cell lines are used: ES cells, to represent embryonic tissue, and fibroblasts (3T3 cells), to represent adult tissue. The test could be developed only after it was recently found that ES cells can be maintained in the undifferentiated stage in the presence of the cytokine leukemia inhibiting factor (LIF). When released from the undifferentiated stage, ES cells will form embryoid bodies (EB) and differentiate under appropriate conditions into the major embryonic tissues. In addition, ES cells are more sensitive to toxic agents than adult cells (Laschinski, Vogel, and Spielmann, 1991). Therefore, differentiation of ES cells and cytotoxicity of ES cells and 3T3 cells are the endpoints in the ES cell test for predicting the embryotoxic potential of chemicals.

BASIC PROCEDURE

Differentiation of embryonic stem cells The mouse ES cell line D3 is cultured permanently in the presence of LIF, a differentiation inhibition factor. In the absence of LIF, ES cells start to differentiate spontaneously. Several concentrations of the test chemical are added to a stem cell suspension. Supplemented DMEM is placed on the lids of 10-cm petri dishes (Šhanging dropŠ culture according to Wobus and co-workers, 1991). After cultivation for three days, the aggregates are transferred into petri dishes. Two days later EBs are placed into 24-well plates where further development of EBs proceeds into different embryonic tissues (Spielmann et al., 1995; Heuer et al., 1994a,b). Differentiation into contracting myocardial cells occurs spontaneously and is determined by light microscopy after another five days of culture. Cytotoxicity measurement with ES cells and 3T3 cells in the MTT assay Exponentially growing 3T3 cells and ES cells in the absence of LIF are inoculated into 96-well microtiter plates. Two hours after cell seeding, eight concentrations of the test chemical, dissolved in assay medium, are added to each well. After ten days of culture the MTT assay is performed. The absorbance is read on an ELISA reader at 570 nm and at a reference wavelength of 630 nm.

CHEMICALS TESTED

Sixteen test chemicals were selected to evaluate the combined ES cell and 3T3 cell embryotoxicity test system: twelve embryotoxic chemicals recommended by the US Teratology Society (Smith et al., 1983) and four control compounds. All chemicals were provided by Sigma.

Group 1: non-teratogens

Ascorbic acid (sodium salt)
Isoniazid (free base)
Penicillin G (sodium salt)
Saccharin (sodium salt)
Group 2: weak/moderate teratogens
Aspirin (crystalline)
Caffeine (anhydrous)
Dexamethasone (crystalline)
Diphenhydramine (HCl)
Diphenylhydantoin (sodium salt)
Indomethacin
Methotrexate ((+) Amethopterin)

Group 3: strong teratogens

Busulphan
Cytosine arabinoside
5-Fluorouracil (crystalline)
Hydroxyurea
Retinoic acid

REFERENCES

  1. Finney D.G. (1971) Probit Analysis. (3rd Ed.) Cambridge University Press; London. Heuer J., Graeber I. M., Pohl I., and Spielmann H. (1994a) Culture system for the differentiation of murine embryonic stem cells - a new approach to in vitro testing for embryotoxicity and for developmental immunotoxicology. In: European Medicines Research (ed. G.N. Fracchia) IOS Press; Amsterdam, pp. 134-145.
  2. Heuer, J., Graeber I.M., Pohl I., and Spielmann H. (1994b) An in vitro embryotoxicity assay using the differentiation of embryonic mouse stem cells into haematopoietic cells. Toxicol. in Vitro 8: 558-587.
  3. Holzhutter H.G.and Quedenau J. (1995) A novel model-based approach to the mathematical analysis of dose-response-curves. Journal of Biological Systems, in press. Laschinski G., Vogel R., and Spielmann, H. (1991) Cytotoxity test using blastocyst-derived euploid embryonal stem cells: a new approach to in vitro teratogenesis screening. Reproductive Toxicol. 5: 57-64.
  4. Litchfield J.T and Wicoxon F. (1949) A simplified method for evaluating dose-effect experiments J. Pharmacol. Exp. Ther. 96: 99-113. Mosmann T. (1983) Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immun. Meth. 65: 55-63.
  5. Newall, D.R. and Beedles, K.E. (1994) The stem-cell test - A novel in vitro assay for teratogenic potential. Toxic. in Vitro 8: 697-701.
  6. Smith, M.K., Kimmel, G.L., Kochhar, D.M., Shepard, T.H., Spielberg, S.P. and Wilson, J.G. (1983) A selection of candidate compounds for in vitro teratogenesis test validation. Teratogenesis Carcinog Mutagen. 3, 461-480.
  7. Spielmann H., Pohl I., D‡ring B and Moldenhauer F. (1995) In vitro embryotoxicity assay using two permanent cell lines: mouse embryonic stem cells and 3T3 fibroblasts Abstracts of the 23. ETS conference 1995, Dublin: Teratology 51:31A-32A.
  8. Wobus A.M., Wallukat G. and Hescheler J. (1991) Pluripotent mouse embryonic stem cells are able to differentiate into cardiomyocytes expressing chronotropic responses to adrenergic and cholinergic agents and Ca2+ channel blockers. Differentiation 48: 173-182.

IP-113 July 1996