76
The embryonic stem cells, derived from the blastocyst of different species, grow
in vitro as a cohesive monolayer that retains the multipotency of the original cells in
the presence of feeder cells and factors that contrast the differentiation. In their
absence, the embryonic stem cells start to differentiate and grow as three-dimensional
clusters, the embryoid bodies, in which the differentiation spontaneously begins.
Modified methods allowed the controlled production of embryoid bodies with
similar fate, and their development into precursors and specialized tissues, or miniorgans (Dahlmann et al. 2013; Ferguson and Subramanian 2018; Kurosawa 2007;
Outten et al. 2012; Xie et al. 2017). These cultures are especially interesting to study
the tracking and interplay between cells and nanoparticles in a 3-D space.
A few studies focused on testing the toxicity of nanoparticles on embryoid bodies. Both silver and golden nanoparticles displayed some toxicity, which was strong
for the functionalized nanoparticles, the silver citrate and the golden thiolate
(Begum et al. 2016; Sathuluri et al. 2011; Senut et al. 2016). Chondroitin sulfate
nanoparticles and mesoporous silica were instead highly biocompatible and could
promote safe and sustained delivery of growth and differentiation factors in culture,
reducing the necessity of frequent inoculation (Garcia-Bennett et al. 2014; Lim
et al. 2011). The magnetic nanoparticles, such as superparamagnetic iron oxides,
seemed to be also safe; they entered the lysosomal compartment of the cells and
gave a significant magnetic signal. In addition, they accelerated the formation of
embryoid bodies by attraction of the embryonic stem cells in a variable magnetic
field and could induce the mesodermal cardiac differentiation through magnetically
induced mechanical distortion (Du et al. 2017; Nejadnik et al. 2012; Parsa
et al. 2015).
Special interest developed on the neurotoxicity of nanoparticles on the embryonic stem cell-derived and embryoid bodies with neural differentiation. All the
nanoparticles tested, such as polyethylene, iron oxide, double-layered magnesium
and aluminum hydroxides, and silver, were neurotoxic. The neural differentiation
and the associated gene expression were reduced. In some cases, death, apoptosis,
and oxidative stress appeared around 100 μg/ml (Hoelting et al. 2013; Oh et al.
2016; Rostami et al. 2015; Wu et al. 2015).
In conclusion, the embryonic toxicity of nanoparticles, evaluated with the embryonic stem cell test method and its variants, is generally moderate, the only exception
being functionalized nanoparticles and certain metal or metal oxides.
3.5.4 Zebrafish Embryo Test
The Zebrafish Embryo Test is a variant of the Fish Embryo Test, planned to test the
environmental pollution and including also the medaka and the fathead minnow as
model species. The zebrafish embryonic test is highly standardized and validated to
test the embryotoxicity; its predictivity is demonstrated, and the guidelines are
available as published papers or online documents (Beekhuijzen et al. 2015; Brannen
et al. 2016; Ducharme et al. 2013; Dumitrescu et al. 2019; Hamm et al. 2019; Haque
A. G. Cattaneo
The embryonic stem cells, derived from the blastocyst of different species, grow
in vitro as a cohesive monolayer that retains the multipotency of the original cells in
the presence of feeder cells and factors that contrast the differentiation. In their
absence, the embryonic stem cells start to differentiate and grow as three-dimensional
clusters, the embryoid bodies, in which the differentiation spontaneously begins.
Modified methods allowed the controlled production of embryoid bodies with
similar fate, and their development into precursors and specialized tissues, or miniorgans (Dahlmann et al. 2013; Ferguson and Subramanian 2018; Kurosawa 2007;
Outten et al. 2012; Xie et al. 2017). These cultures are especially interesting to study
the tracking and interplay between cells and nanoparticles in a 3-D space.
A few studies focused on testing the toxicity of nanoparticles on embryoid bodies. Both silver and golden nanoparticles displayed some toxicity, which was strong
for the functionalized nanoparticles, the silver citrate and the golden thiolate
(Begum et al. 2016; Sathuluri et al. 2011; Senut et al. 2016). Chondroitin sulfate
nanoparticles and mesoporous silica were instead highly biocompatible and could
promote safe and sustained delivery of growth and differentiation factors in culture,
reducing the necessity of frequent inoculation (Garcia-Bennett et al. 2014; Lim
et al. 2011). The magnetic nanoparticles, such as superparamagnetic iron oxides,
seemed to be also safe; they entered the lysosomal compartment of the cells and
gave a significant magnetic signal. In addition, they accelerated the formation of
embryoid bodies by attraction of the embryonic stem cells in a variable magnetic
field and could induce the mesodermal cardiac differentiation through magnetically
induced mechanical distortion (Du et al. 2017; Nejadnik et al. 2012; Parsa
et al. 2015).
Special interest developed on the neurotoxicity of nanoparticles on the embryonic stem cell-derived and embryoid bodies with neural differentiation. All the
nanoparticles tested, such as polyethylene, iron oxide, double-layered magnesium
and aluminum hydroxides, and silver, were neurotoxic. The neural differentiation
and the associated gene expression were reduced. In some cases, death, apoptosis,
and oxidative stress appeared around 100 μg/ml (Hoelting et al. 2013; Oh et al.
2016; Rostami et al. 2015; Wu et al. 2015).
In conclusion, the embryonic toxicity of nanoparticles, evaluated with the embryonic stem cell test method and its variants, is generally moderate, the only exception
being functionalized nanoparticles and certain metal or metal oxides.
3.5.4 Zebrafish Embryo Test
The Zebrafish Embryo Test is a variant of the Fish Embryo Test, planned to test the
environmental pollution and including also the medaka and the fathead minnow as
model species. The zebrafish embryonic test is highly standardized and validated to
test the embryotoxicity; its predictivity is demonstrated, and the guidelines are
available as published papers or online documents (Beekhuijzen et al. 2015; Brannen
et al. 2016; Ducharme et al. 2013; Dumitrescu et al. 2019; Hamm et al. 2019; Haque
A. G. Cattaneo