An abbreviated protocol for multilineage neural differentiation of murine embryonic stem cells and its perturbation by methyl mercury

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Abstract

Alternative assays are highly desirable to reduce the extensive experimental animal use in developmental toxicity testing. In the present study, we developed an improved test system for assessing neurodevelopmental toxicity using differentiating embryonic stem cells. We advanced previously established methods by merging, modifying and abbreviating the original 20-day protocol into a more efficient 13-day neural differentiation protocol. Using morphological observation, immunocytochemistry, gene expression and flow cytometry, it was shown predominantly multiple lineages of neuroectodermal cells were formed in our protocol and to a lower extent, endodermal and mesodermal differentiated cell types. This abbreviated protocol should lead to an advanced screening method using morphology in combination with selected differentiation markers aimed at predicting neurodevelopmental toxicity. Finally, the assay was shown to express differential sensitivity to a model developmental neurotoxicant, 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).

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