described, and approved (Genschow et al. 2002; Spielmann et al. 2008) EST as the
in vitro assay for embryotoxicity. Since then, this assay has been utilized for drug
screening (Paquette et al. 2008; Whitlow et al. 2007) to study the toxicity of a panels
of related compounds on development (de Jong et al. 2009) and even for assessing
embryotoxicity of nanomaterials (Di Guglielmo et al. 2010). Establishment of EST
is considered a milestone in the history of stem cell toxicology testing because of its
advantage over in vitro and pre-clinical tests (Genschow et al. 2002).
Even though considered as a very effective assay for toxicology testing, EST, did
have several weaknesses such as for assessing myocardial differentiation, microscopic observation was the sole means for studying the beating areas whereas no
metabolic tests for analysis were not available (Spielmann et al. 2006). Further,
limitation in the EST prediction model was highlighted by another study which was
sponsored by a ECVAM and ReProTect where EST based analysis was able to
classify only 15% of previously untested compounds correctly (Marx-Stoelting
2009).
With the development and technological advancements in the field of biotechnology accompanied with the emergence of new molecular techniques and tools,
modifications in the EST assay were proposed which could overcome the existing
limitations. For evaluating beating cardiomyocytes, the visual microscopic evaluation was replaced by highly sensitive and specific techniques such as fluorescence
activated cell sorting (FACS) and reverse transcription quantitative PCR (RT–
qPCR) providing more quantitative molecular endpoints analysis in terms of gene
and protein expression of myocardial markers (Bigot et al. 1999; Buesen et al. 2009;
Pellizzer et al. 2004b; Riebeling et al. 2011; Seiler et al. 2004; Seiler 2006; Zur
Nieden et al. 2001).
Further, to assess the toxicity of pollutants on various tissue and organs tests were
developed on the basis of the potential of stem cells to differentiate into multiple
lineages including pancreatic, neuronal, osteogenic, and skeletal muscle lineages
(Schmidt et al. 2001; Mori and Hara 2013; Pellizzer et al. 2004a, b; Rolletschek et al.
2004). Based on the new advancements, in 2004 a new EST subtype referred as
molecular multiple-endpoint EST developed, which not only included traditional
cardiomyocyte differentiation as an assay parameter but also incorporated quantitative analysis using RT-qPCR and multilineage stem cell differentiation-based assessment as additions (Zur Nieden et al. 2004).
Above described ESTs used differentiated fibroblasts (3T3 cell line) and ESCs as
assay model. For simplifying and improving the reproducibility of EST based
testing, development of advanced procedures relying exclusively on ESCs were
targeted. High-throughput toxicological analysis techniques such as miRNA and
whole genome profiling using techniques like microarrays and mass spectrometry
were incorporated.
Moreover, the advantage of ESCs over cancerous and other cell types is that they
can be used in the development of toxicity assay. For instance, ESCs have ability to
grow into three-dimensional cell aggregates the so-called organoids. These
organoids or embryoid bodies (EBs) can be considered as miniature organs as
12 Environmental Interaction and Impact on the Life Span of Stem Cells
259
in vitro assay for embryotoxicity. Since then, this assay has been utilized for drug
screening (Paquette et al. 2008; Whitlow et al. 2007) to study the toxicity of a panels
of related compounds on development (de Jong et al. 2009) and even for assessing
embryotoxicity of nanomaterials (Di Guglielmo et al. 2010). Establishment of EST
is considered a milestone in the history of stem cell toxicology testing because of its
advantage over in vitro and pre-clinical tests (Genschow et al. 2002).
Even though considered as a very effective assay for toxicology testing, EST, did
have several weaknesses such as for assessing myocardial differentiation, microscopic observation was the sole means for studying the beating areas whereas no
metabolic tests for analysis were not available (Spielmann et al. 2006). Further,
limitation in the EST prediction model was highlighted by another study which was
sponsored by a ECVAM and ReProTect where EST based analysis was able to
classify only 15% of previously untested compounds correctly (Marx-Stoelting
2009).
With the development and technological advancements in the field of biotechnology accompanied with the emergence of new molecular techniques and tools,
modifications in the EST assay were proposed which could overcome the existing
limitations. For evaluating beating cardiomyocytes, the visual microscopic evaluation was replaced by highly sensitive and specific techniques such as fluorescence
activated cell sorting (FACS) and reverse transcription quantitative PCR (RT–
qPCR) providing more quantitative molecular endpoints analysis in terms of gene
and protein expression of myocardial markers (Bigot et al. 1999; Buesen et al. 2009;
Pellizzer et al. 2004b; Riebeling et al. 2011; Seiler et al. 2004; Seiler 2006; Zur
Nieden et al. 2001).
Further, to assess the toxicity of pollutants on various tissue and organs tests were
developed on the basis of the potential of stem cells to differentiate into multiple
lineages including pancreatic, neuronal, osteogenic, and skeletal muscle lineages
(Schmidt et al. 2001; Mori and Hara 2013; Pellizzer et al. 2004a, b; Rolletschek et al.
2004). Based on the new advancements, in 2004 a new EST subtype referred as
molecular multiple-endpoint EST developed, which not only included traditional
cardiomyocyte differentiation as an assay parameter but also incorporated quantitative analysis using RT-qPCR and multilineage stem cell differentiation-based assessment as additions (Zur Nieden et al. 2004).
Above described ESTs used differentiated fibroblasts (3T3 cell line) and ESCs as
assay model. For simplifying and improving the reproducibility of EST based
testing, development of advanced procedures relying exclusively on ESCs were
targeted. High-throughput toxicological analysis techniques such as miRNA and
whole genome profiling using techniques like microarrays and mass spectrometry
were incorporated.
Moreover, the advantage of ESCs over cancerous and other cell types is that they
can be used in the development of toxicity assay. For instance, ESCs have ability to
grow into three-dimensional cell aggregates the so-called organoids. These
organoids or embryoid bodies (EBs) can be considered as miniature organs as
12 Environmental Interaction and Impact on the Life Span of Stem Cells
259