The Human Induced Pluripotent Stem Cell Test as an Alternative Method for Embryotoxicity Testing (original) (raw)
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First steps in establishing a developmental toxicity test method based on human embryonic stem cells
Toxicology in Vitro, 2008
The use of embryonic stem cells is currently the most promising approach to assess developmental toxicity in vitro. In addition, the possibility of using human embryonic stem (hES) cells will increase safety of consumers and patients as false classification of substances due to inter-species variations can be avoided. One validated test based on murine embryonic stem cells, the embryonic stem cell test (EST), consists of following endpoints: IC 50 values of fibroblasts and embryonic stem cells as well as the inhibition of differentiation of mES cells into cardiomyocytes. As a follow up of its successful validation study we established a cytotoxicity assay based on hES cells and human fibroblasts employing two developmental toxicants: 5-fluorouracil (5-FU) and all-trans retinoic acid (RA). The results were compared to historical data from the EST. For 5-FU, no significant differences were obtained between the different cell lines. However, for RA, both test systems produced higher IC 50 values for the fibroblasts than for the stem cells, which is a well-known effect of developmental toxicants. Moreover, the reliability and relevance of several marker genes as possible toxicological endpoints were tested. During early differentiation Oct-4, hTert and Dusp6 showed the most reliable results. Brachyury and GATA-4 were found to be best suited to monitor cardiac differentiation. The late cardiac marker gene TNNT2 demonstrated significant results until day 18. Therefore, these marker genes have the highest potential to serve as endpoints for a developmental toxicity test.
Pluripotent Stem Cells as Tools to Assess Developmental Toxicity: Diversity Instead of Consolidation
Sahu/Handbook of Nanotoxicology, Nanomedicine and Stem Cell Use in Toxicology, 2014
ABSTRACT As pharmaceutical and biotechnology industries develop more potentially toxic compounds, traditional methods of toxicity screening cannot keep up. A conventional approach of screening compounds for their toxic and teratogenic potential heavily relies on multitudes of animal models, which have proven to be timely and expensive and bring forth ethical challenges. These limitations of animal testing have created an eagerness among researchers to develop more predictive in vitro alternative screening assays.The most promising in vitro approach at modernizing toxicity tests is the embryonic stem cell test (EST). The EST assesses toxicity on the naive embryonic cell and also the effect of the putative toxicant on the genesis of a specific tissue endpoint. Importantly, unlike other in vitro models, the EST obviates the need to sacrifice pregnant animals. Although determined suitable for regulatory standards, the classic EST, which is based on mouse cells, is limited in its endpoint analysis and efficient prediction of human response. Those limitations have led to numerous unconsolidated improvements to the assay. This chapter provides an overview of the classic EST as it has been developed in 1997 and illustrates disadvantages of the classic EST as well as the numerous modifications to it.
Use of Murine Embryonic Stem Cells in Embryotoxicity Assays: The Embryonic Stem Cell Test
Embryonic Stem Cell Protocols, 2006
The embryonic stem cell test (EST) takes advantage of the potential of murine embryonic stem (ES) cells to differentiate in culture to test embryotoxicity in vitro. The EST represents a scientifically validated in vitro system for the classification of compounds according to their teratogenic potential based on the morphological analysis of beating cardiomyocytes in embryoid body outgrowths compared to cytotoxic effects on murine ES cells and differentiated 3T3 fibroblasts. Through a number of prevalidation and validation studies, the EST has been demonstrated to be a reliable alternative method for embryotoxicity testing based on the most important mechanisms in embryotoxicity-cytotoxicity and differentiationas well as on differences in sensitivity between differentiated and embryonic tissues. Improvements of the EST protocol using flow cytometry analysis showed that differential expression of sarcomeric myosin heavy chain and α-actinin proteins quantified under the influence of a test compound is a useful marker for detecting potential teratogenicity. The in vitro embryotoxicity test described in this chapter is rapid, simple, and sensitive and can be usefully employed as a component of the risk/hazard assessment process. . Pipetting scheme for the cytotoxicity assay. One test chemical per plate is used (the positive control is placed next to the lowest test concentration).
Toxicology in Vitro, 1999
ÐA project has been started using transgenic embryonic stem cells as a toxicological endpoint in order to register chemical eects on the development of embryonic tissues which are known to be sensitive during their dierentiation. The green¯uorescent protein (GFP) is used as a reporter gene and is linked to a cardiac speci®c promotor. This construct is integrated into the native DNA of undierentiated embryonic stem cells. The expression of GFP was switched on after speci®c activation of the promotor (human-a-actin) which allows a quanti®cation of cardiac cells using the¯uorescence activated cell sorter. Kinetic analysis shows a dierentiation of 25% on cells with activated human-a-actin promotor on day 3, increasing to 86% on day 7, and decreasing again to 35% on day 11. The known animal teratogens retinoic acid and 5-¯uorouracil were chosen and the measurements were compared to the IC 50 values given by other in vitro endpoints in order to investigate the potential of this toxicological endpoint. The results show a higher sensitivity of endpoints which analysed speci®c eects on a selected target tissue. The exposure of embryonic stem cells to chemicals lead to the following IC 50 values: 1.14920.170 ng/ml (cytotoxicity) versus 0.21620.126 ng/ml (GFP expression) after treatment with retinoic acid and 54.225.2 ng/ml (cytotoxicity) versus 26.722 ng/ml (GFP expression) after treatment with 5-¯uorouracil. The data shows the necessity to develop speci®c in vitro methods which take the complexity of embryotoxicology into account.