An abbreviated protocol for multilineage neural differentiation of murine embryonic stem cells and its perturbation by methyl mercury
Introduction
Chemical risk assessment is currently still highly dependent on globally harmonized experimental animal studies. Reproductive toxicity testing proposed within the European chemical safety legislation (REACH) has been estimated to require ∼60% of the total animals needed for toxicity testing [1]. Therefore, this area of toxicity testing is a high priority area for designing alternative assays to reduce experimental animal use. An established in vitro test system for developmental toxicity is the embryonic stem cell test (EST) [2], in which the developmental toxic effect of compounds on stem cell differentiation towards beating cardiomyocytes is assessed. Despite the promise of this in vitro methodology, a recent validation study suggests a number of in vivo developmental toxic compounds are misclassified as negative in the EST, one of which being methyl mercury chloride [2]. Misclassifications may be partly due to the prediction model used, in which both proliferation and differentiation parameters play a role. However, these compounds may have been misclassified, because they may not affect mesodermal-derived cardiomyocyte differentiation in vivo, but primarily affect alternative differentiation routes, such as the ectodermal or the endodermal routes [2]. Additional alternative in vitro embryonic stem cell differentiation assays complementary to the EST may improve the prediction for such compounds.
Several neural differentiation protocols for mouse embryonic stem (ES) cells have been described since the mid 1990s, most of which were developed for mechanistic studies of embryonic cell differentiation [3], [4]. These methods use a variety of factors to reach a similar extent of neural differentiation. They include retinoic acid (RA) [3], [5], serum deprivation [4], hormones and growth factors and supporting matrices for a range of different neural end points [6]. Most methods make use of three-dimensional embryoid body (EB) formation [3], [4], while some methods use a two-dimensional monolayer culture [7], [8], or a combination of these two [5]. For the rapid prediction of neurodevelopmental toxicity, it is urgently needed to develop a short duration, high-throughput model, which sufficiently mimics the in vivo differentiation of ES cells into neuronal-type cells. In the present study we have designed a testing model based on methods described by Okabe et al. [4] and Bibel et al. [5] using EB formation and stimulation of neural differentiation using RA and serum deprivation. These models appear to approach the in vivo situation in terms of their neural differentiation pattern. However, we have reduced the length of a combination of these two protocols to enable increased throughput of compound testing. Furthermore, multilineage (ectodermal, mesodermal and endodermal) differentiation of the combined protocols was studied.
In vivo studies have shown that the development of the brain is a process sensitive towards developmental toxic challenges [9], [10], [11]. During neural system development of the embryo, stem cells differentiate into many types of neurons, glial cells and neuronal epithelial cells [12]. In this highly tuned process, interaction between specific cell types is essential for proper differentiation and the establishment of optimal ratios of cell types in the brain in time and space. It is known from in vivo studies that neurodevelopmental toxicants, such as methyl mercury [13] and ethanol [14], can influence these ratios and disrupt the developing brain. Therefore, improvement of assessing the putative effects of neurodevelopmental toxicants is expected by monitoring over time the varying quantities of specific neural cell types using a battery of differentiation markers.
In this study, we used methyl mercury chloride (MeHgCl) to evaluate the responsiveness of our assay. During the past century, catastrophic methyl mercury poisonings in Japan and Iraq have shown its neurodevelopmental toxicity [13], [15]. Today, methyl mercury is widely used as a model neurodevelopmental toxic compound [16], [17], [18].
In the present study we have successfully designed a 13-day differentiation protocol, in which multiple lineages of neural and other brain-associated cells are formed. In addition, we developed a screening method using a group of differentiation markers which may be used for predicting neurodevelopmental toxicity. Finally, the model was shown to have differential sensitivity to a developmental neurotoxicant, MeHgCl. This work represents the first steps towards an assay for assessing developmental neurotoxicity in vitro
Section snippets
Culture media
Complete medium (CM) contained Dulbecco's modified eagle's medium (DMEM) (Gibco BRL, Gaithersburg, MD, USA) supplemented with 20% fetal bovine serum (Hyclone, Logan, UT, USA), 1% nonessential amino acids (Gibco BRL, Gaithersburg, MD, USA), 1% penicillin/streptomycin (Gibco BRL, Gaithersburg, MD, USA), 2 mM l-glutamine (Gibco BRL, Gaithersburg, MD, USA) and 0.1 mM β-mercapto ethanol (Sigma–Aldrich, Zwijndrecht, The Netherlands). Low serum medium (LS), had the same composition as CM except that the
Abbreviation of the neural differentiation protocol
In order to enhance the throughput of the neural differentiation assay, shorter versions of the original combined 20 days neural differentiation protocols (long protocol) were designed and tested. Initial morphological observations showed that neural differentiation was present from day 7 onwards. Therefore the ITS phase was abbreviated to study its effect on the course of neural differentiation. With this in mind, we developed two new protocols, in which the ITS phase was reduced from the
Abbreviation of the protocol
The first aim of this study was to design a more efficient embryonic stem cell neural differentiation assay. To abbreviate the published 20-day protocols, the ITS phase was reduced from 7 days in the long protocol to 4 days or 1 day in the medium and short protocols, respectively. Morphological observations (Fig. 2) and immunocytochemical staining of key markers for pluripotency, neural precursor cells and mature neural cells (Fig. 3) showed similar profiles when comparing all three protocols.
Conflict of interest
None.
Acknowledgement
This research was supported by grant MFA 6809 from The Netherlands Foundation for Technological Sciences (STW).
References (47)
- et al.
Retinoic acid promotes neural and represses mesodermal gene expression in mouse embryonic stem cells in culture
Biochem Biophys Res Commun
(1996) - et al.
Development of neuronal precursor cells and functional postmitotic neurons from embryonic stem cells in vitro
Mech Dev
(1996) - et al.
Defined conditions for neural commitment and differentiation
Methods Enzymol
(2003) - et al.
Dose–response analysis of infants prenatally exposed to methyl mercury: an application of a single compartment model to single-strand hair analysis
Environ Res
(1989) - et al.
Embryotoxicity hazard assessment of methylmercury and chromium using embryonic stem cells
Toxicology
(2007) - et al.
Differentiation of pluripotent embryonic stem cells into the neuronal lineage in vitro gives rise to mature inhibitory and excitatory neurons
Mech Dev
(1995) - et al.
Molecular and functional analysis of a novel neuronal vesicular glutamate transporter
J Biol Chem
(2001) - et al.
Two genes encode distinct glutamate decarboxylases
Neuron
(1991) - et al.
The method of mouse embryoid body establishment affects structure and developmental gene expression
Tissue Cell
(2009) - et al.
The role of GABA in the early neuronal development
Int Rev Neurobiol
(2005)
Disentangling cellular proliferation and differentiation in the embryonic stem cell test, and its impact on the experimental protocol
Reprod Toxicol
Hazard assessment of methylmercury toxicity to neuronal induction in embryogenesis using human embryonic stem cells
Toxicology
Alternative approaches can reduce the use of test animals under REACH
Eur Comm Rep
Validation of the embryonic stem cell test in the international ECVAM validation study on three in vitro embryotoxicity tests
Altern Lab Anim
Differentiation of mouse embryonic stem cells into a defined neuronal lineage
Nat Neurosci
Neural commitment of embryonic stem cells: molecules, pathways and potential for cell therapy
J Pathol
High-throughput screening-compatible single-step protocol to differentiate embryonic stem cells in neurons
Stem Cells Dev
OECD guidelines for the testing of chemicals/section 4: health effects test no. 426: developmental neurotoxicity study
Qualitative and quantitative comparability of human and animal developmental neurotoxicity
Environmental factors associated with a spectrum of neurodevelopmental deficits
Ment Retard Dev Disabil Res Rev
Embryonic stem cells: prospects for developmental biology and cell therapy
Physiol Rev
Mercury exposure and child development outcomes
Pediatrics
Numbers of neurons and glia in mature rat somatosensory cortex: effects of prenatal exposure to ethanol
J Comp Neurol
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2020, Food and Chemical ToxicologyCitation Excerpt :This is also true for a variant protocol, inducing osteogenic differentiation (de Jong et al., 2014). Pluripotency of embryonic stem cells provides the potential to differentiate into cell types from all embryonic layers (Stummann and Bremer, 2012), with differentiation into neuronal cells as a presently much explored model for developmental neurotoxicity (de Leeuw et al., 2019; Theunissen et al., 2010). It remains to be established whether these particular differentiation directions will all be responsive to a specific compound at all, or if so, with a similar sensitivity.