An automated and high-throughput-screening compatible pluripotent stem cell-based test platform for developmental and reproductive toxicity assessment of small molecule compounds
Tóm tắt
The embryonic stem cell test (EST) represents the only validated and accepted in vitro system for the detection and classification of compounds according to their developmental and reproductive teratogenic potency. The widespread implementation of the EST, however, in particular for routine application in pharmaceutical development, has not been achieved so far. Several drawbacks still limit the high-throughput screening of potential drug candidates in this format: The long assay period, the use of non-homogeneous viability assays, the low throughput analysis of marker protein expression and the compatibility of the assay procedures to automation. We have therefore introduced several advancements into the EST workflow: A reduction of the assay period, an introduction of homogeneous viability assays, and a straightforward analysis of marker proteins by flow cytometry and high content imaging to assess the impact of small molecules on differentiation capacity. Most importantly, essential parts of the assay procedure have been adapted to lab automation in 96-well format, thus enabling the interrogation of several compounds in parallel. In addition, extensive investigations were performed to explore the predictive capacity of this next-generation EST, by testing a set of well-known embryotoxicants that encompasses the full range of chemical-inherent embryotoxic potencies possible. Due to these significant improvements, the augmented workflow provides a basis for a sensitive, more rapid, and reproducible high throughput screening compatible platform to predict in vivo developmental toxicity from in vitro data which paves the road towards application in an industrial setting.
Tài liệu tham khảo
Buesen R, Genschow E, Slawik B, Visan A, Spielmann H, Luch A, et al. Embryonic stem cell test remastered: comparison between the validated EST and the new molecular facs-EST for assessing developmental toxicity in vitro. Toxicol Sci: An Official Journal of the Society of Toxicology. 2009;108(2):389–400.
Chen R, Chen J, Cheng S, Qin J, Li W, Zhang L, et al. Assessment of embryotoxicity of compounds in cosmetics by the embryonic stem cell test. Toxicol Mech Methods. 2010;20(3):112–8.
de Jong E, Louisse J, Verwei M, Blaauboer BJ, van de Sandt JJ, Woutersen RA, et al. Relative developmental toxicity of glycol ether alkoxy acid metabolites in the embryonic stem cell test as compared with the in vivo potency of their parent compounds. Toxicol Sci: An Official Journal of the Society of Toxicology. 2009;110(1):117–24.
Di Guglielmo C, Lopez DR, De Lapuente J, Mallafre JM, Suarez MB. Embryotoxicity of cobalt ferrite and gold nanoparticles: a first in vitro approach. Reprod Toxicol. 2010;30(2):271–6.
Doetschman TC, Eistetter H, Katz M, Schmidt W, Kemler R. The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J Embryol Exp Morpholog. 1985;87:27–45.
Eckardt K, Stahlmann R. Use of two validated in vitro tests to assess the embryotoxic potential of mycophenolic acid. Arch Toxicol. 2010;84(1):37–43.
Engle SJ, Puppala D. Integrating human pluripotent stem cells into drug development. Cell Stem Cell. 2013;12(6):669–77.
Genschow E, Spielmann H, Scholz G, Pohl I, Seiler A, Clemann N, et al. Validation of the embryonic stem cell test in the international ecvam validation study on three in vitro embryotoxicity tests. Altern Lab Anim. 2004;32(3):209–44.
Genschow E, Spielmann H, Scholz G, Seiler A, Brown N, Piersma A, et al. The ecvam international validation study on in vitro embryotoxicity tests: results of the definitive phase and evaluation of prediction models. European Centre for the Validation of Alternative Methods. Altern Lab Anim. 2002;30(2):151–76.
Hosoya M, Czysz K. Translational prospects and challenges in human induced pluripotent stem cell research in drug discovery. Cells. 2016;5(4).
Kamelia L, Louisse J, de Haan L, Rietjens I, Boogaard PJ. Prenatal developmental toxicity testing of petroleum substances: application of the mouse embryonic stem cell test (EST) to compare in vitro potencies with potencies observed in vivo. Toxicol in Vitro. 2017;44:303–12.
Kameoka S, Babiarz J, Kolaja K, Chiao E. A high-throughput screen for teratogens using human pluripotent stem cells. Toxicol Sci: An Official Journal of the Society of Toxicology. 2014;137(1):76–90.
Kang HY, Choi YK, Jo NR, Lee JH, Ahn C, Ahn IY, et al. Advanced developmental toxicity test method based on embryoid body's area. Reprod Toxicol. 2017;72:74–85.
Kleinstreuer NC, Smith AM, West PR, Conard KR, Fontaine BR, Weir-Hauptman AM, et al. Identifying developmental toxicity pathways for a subset of toxcast chemicals using human embryonic stem cells and metabolomics. Toxicol Appl Pharmacol. 2011;257(1):111–21.
Kong D, Xing L, Liu R, Jiang J, Wang W, Shang L, et al. Individual and combined developmental toxicity assessment of bisphenol a and genistein using the embryonic stem cell test in vitro. Food Chem Toxicol. 2013;60:497–505.
Le Coz F, Suzuki N, Nagahori H, Omori T, Saito K. Hand1-luc embryonic stem cell test (hand1-luc est): a novel rapid and highly reproducible in vitro test for embryotoxicity by measuring cytotoxicity and differentiation toxicity using engineered mouse es cells. J Toxicol Sci. 2015;40(2):251–61.
Luz AL, Tokar EJ. Pluripotent stem cells in developmental toxicity testing: a review of methodological advances. Toxicol Sci: An Official Journal of the Society of Toxicology. 2018;165(1):31–9.
Martin GR, Wiley LM, Damjanov I. The development of cystic embryoid bodies in vitro from clonal teratocarcinoma stem cells. Dev Biol. 1977;61(2):230–44.
Mueller H, Kassack MU, Wiese M. Comparison of the usefulness of the mtt, atp, and calcein assays to predict the potency of cytotoxic agents in various human cancer cell lines. J Biomol Screen. 2004;9(6):506–15.
Nagahori H, Suzuki N, Le Coz F, Omori T, Saito K. Prediction of in vivo developmental toxicity by combination of hand1-luc embryonic stem cell test and metabolic stability test with clarification of metabolically inapplicable candidates. Toxicol Lett. 2016;259:44–51.
Palmer JA, Smith AM, Egnash LA, Conard KR, West PR, Burrier RE, et al. Establishment and assessment of a new human embryonic stem cell-based biomarker assay for developmental toxicity screening. Birth Defects Res B Dev Reprod Toxicol. 2013;98(4):343–63.
Paquette JA, Kumpf SW, Streck RD, Thomson JJ, Chapin RE, Stedman DB. Assessment of the embryonic stem cell test and application and use in the pharmaceutical industry. Birth Defects Res B Dev Reprod Toxicol. 2008;83(2):104–11.
Park MV, Annema W, Salvati A, Lesniak A, Elsaesser A, Barnes C, et al. In vitro developmental toxicity test detects inhibition of stem cell differentiation by silica nanoparticles. Toxicol Appl Pharmacol. 2009;240(1):108–16.
Peters AK, Steemans M, Hansen E, Mesens N, Verheyen GR, Vanparys P. Evaluation of the embryotoxic potency of compounds in a newly revised high throughput embryonic stem cell test. Toxicol Sci: An Official Journal of the Society of Toxicology. 2008;105(2):342–50.
Petty RD, Sutherland LA, Hunter EM, Cree IA. Comparison of mtt and atp-based assays for the measurement of viable cell number. J Biolumin Chemilumin. 1995;10(1):29–34.
Pouton CW, Haynes JM. Embryonic stem cells as a source of models for drug discovery. Nat Rev Drug Discov. 2007;6(8):605–16.
Riss TL, Moravec RA, Niles AL, Duellman S, Benink HA, Worzella TJ, Minor L. 2004. Cell viability assays. In: Sittampalam GS, Coussens NP, Brimacombe K, Grossman A, Arkin M, Auld D, Austin C, Baell J, Bejcek B, Chung TDY et al., editors. Assay guidance manual. Bethesda (MD).
Rovida C, Hartung T. Re-evaluation of animal numbers and costs for in vivo tests to accomplish reach legislation requirements for chemicals - a report by the transatlantic think tank for toxicology (t(4)). ALTEX. 2009;26(3):187–208.
Scholz G, Genschow E, Pohl I, Bremer S, Paparella M, Raabe H, et al. Prevalidation of the embryonic stem cell test (EST)-a new in vitro embryotoxicity test. Toxicol in Vitro. 1999;13(4–5):675–81.
Seiler A, Visan A, Buesen R, Genschow E, Spielmann H. Improvement of an in vitro stem cell assay for developmental toxicity: the use of molecular endpoints in the embryonic stem cell test. Reprod Toxicol. 2004;18(2):231–40.
Seiler AE, Spielmann H. The validated embryonic stem cell test to predict embryotoxicity in vitro. Nat Protoc. 2011;6(7):961–78.
Shinde V, Sureshkumar P, Sotiriadou I, Hescheler J, Sachinidis A. Human embryonic and induced pluripotent stem cell based toxicity testing models: future applications in new drug discovery. Curr Med Chem. 2016;23(30):3495–509.
Spielmann H, Pohl I, Doring B, Liebsch M, Moldenhauer F. The embryonic stem cell test (EST), an in vitro embryotoxicity test using two permanent mouse cell lines: 3t3 fibroblasts and embryonic stem cells. Dev an Vet. 1997;27:663–9.
Stummann TC, Hareng L, Bremer S. Embryotoxicity hazard assessment of methylmercury and chromium using embryonic stem cells. Toxicology. 2007;242(1–3):130–43.
Stummann TC, Hareng L, Bremer S. Embryotoxicity hazard assessment of cadmium and arsenic compounds using embryonic stem cells. Toxicology. 2008;252(1–3):118–22.
Suzuki N, Ando S, Sumida K, Horie N, Saito K. Analysis of altered gene expression specific to embryotoxic chemical treatment during embryonic stem cell differentiation into myocardiac and neural cells. J Toxicol Sci. 2011a;36(5):569–85.
Suzuki N, Ando S, Yamashita N, Horie N, Saito K. Evaluation of novel high-throughput embryonic stem cell tests with new molecular markers for screening embryotoxic chemicals in vitro. Toxicol Sci: An Official Journal of the Society of Toxicology. 2011b;124(2):460–71.
Suzuki N, Yamashita N, Koseki N, Yamada T, Kimura Y, Aiba S, et al. Assessment of technical protocols for novel embryonic stem cell tests with molecular markers (hand1- and cmya1-ests): a preliminary cross-laboratory performance analysis. J Toxicol Sci. 2012;37(4):845–51.
Tandon R, Brandl B, Baryshnikova N, Landshammer A, Steenpass L, Keminer O, et al. Generation of two human isogenic ipsc lines from fetal dermal fibroblasts. Stem Cell Res. 2018;33:120–4.
West PR, Weir AM, Smith AM, Donley EL, Cezar GG. Predicting human developmental toxicity of pharmaceuticals using human embryonic stem cells and metabolomics. Toxicol Appl Pharmacol. 2010;247(1):18–27.
Zhou R, Cheng W, Feng Y, Wei H, Liang F, Wang Y. Interactions between three typical endocrine-disrupting chemicals (edcs) in binary mixtures exposure on myocardial differentiation of mouse embryonic stem cell. Chemosphere. 2017;178:378–83.