Neuroendocrine Cell Culture Derived From Embryonic Stem Cells as a Model System to Investigate Developmental Neurotoxicity of Endocrine Disruptors

Mohammed El Majdoubi, Ph.D. Associate Professor of Neuroscience

Department of Natural Sciences and Mathematics, Dominican University of California

DOI: https://doi.org/10.5897/PP2018/0010

Copyright © 2018 Author(s) retain the copyright of this article.

This article is published under the terms of the Creative Commons Attribution License 4.0.

Neuroendocrine cells are a set of specialized neurons located mainly in the hypothalamic areas of the brain. Rather than forming synaptic connections with other neurons, neuroendocrine cells release their product, neuro-hormones directly into the blood to act on their endocrine targets all over the body. Neuroendocrine cells are important because they control vital functions such as growth, reproduction, metabolism, energy balance and stress responses (Swanson et al., 1987).

Unfortunately, neuroendocrine cells are the major neuronal target of endocrine disruptors which can produce adverse developmental, reproductive, neurological and behavioral effects in both humans and wildlife (Gore, 2008). A wide range of both natural and man-made substances are thought to cause endocrine disruption by mimicking or blocking normal functioning of body hormones, including pharmaceuticals, pesticides and plasticizers. Endocrine disruptors may be found in many everyday products such as plastic bottles, metal food cans, detergents, flame retardants, food, toys, cosmetics, and pesticides. Many of these products have been found to cause life-threatening illnesses in laboratory animals. Research suggests that endocrine disruptors may pose the greatest risk during prenatal and early postnatal development when neuronal systems are forming (McLachlan 2001).

Neuroendocrine cells integrate and control several homeostatic processes that are required for our survival. Disruption of the development of neuroendocrine systems (ie, developmental neurotoxicity) thus has the potential of perturbing important physiological processes with lifelong consequences. However, because of the limitations of animal-based models and traditional cell culture models of neuronal development, the mechanisms of this developmental neurotoxicity are poorly understood.

This major public health concern emphasizes the need to establish effective cell culture models of developing neuroendocrine cells, which would allow the investigation of the mechanisms of endocrine disruptions directly on their primary cell targets in an accessible and controlled environment. Neuroendocrine cell cultures derived from stem cells recapitulate important developmental processes such as proliferation and differentiation. The wide range of manipulations possible in cultures of developing neuroendocrine cells allow for experiments that are difficult or impossible in embryos, primary hypothalamic neuronal cultures or transformed neuroendocrine cell lines.

Embryonic stem cells are undifferentiated cells that can replicate indefinitely and turn into neurons following exposure to retinoic acid, a metabolite of vitamin A (Gottlieb and Huettner, 1999). In recent years, stem cell-derived neuronal models have become available and may offer distinct advantages over traditional neuronal model systems for investigating neurotoxic effects of environmental pollutants. As part of this collective effort, my laboratory has developed an efficient and easily reproducible protocol to direct the differentiation of over 30% of pluripotent mouse embryonic stem cells into neuroendocrine cells in vitro using a combination of retinoic acid and forskolin, an active compound found in the roots of the Indian coleus which is a tropical plant related to mint. (El Majdoubi, 2011).

Using this model we recently assessed the neurotoxicity of four well studied heavy metal compounds found in the environment: mercury, cadmium, lead, and manganese. Undifferentiated embryonic stem cells were generally more sensitive to higher physiological doses of all four compounds which inhibited cell proliferation and induced cell death. In contrast, lower physiological doses of these compounds did not impact the growth of embryonic stem cells but did interfere with their neuronal and neuroendocrine differentiation (El Majdoubi et al., 2016). These results indicate that our neuroendocrine cell culture derived from stem cells could, therefore, become a useful alternative model system for investigating neurotoxicity of environmental pollutants such as heaving metals and commercial compounds at the cellular level. We are currently using this model to characterize developmental neurotoxicity of endocrine disruptors such as polybrominated diphenyl ethers (PBDE) and bisphenol A (BPA) which are used as flame retardants and in polycarbonate plastics, respectively.

Through this project, we hope to increase knowledge about the effects of endocrine disruption by providing a cell culture model that will help elucidate the molecular mechanisms of endocrine disrupting chemicals on various differentiation stages of neuroendocrine cells under the microscope. The use of cultured cells has many advantages over the use of an intact animal or an isolated preparation as an experimental approach. Cultured cells provide a more controlled and easily manipulated cellular environment than that achievable in an intact animal. There are fewer potential problems with access to the cells of interest and the distribution of pharmacological agents. Because cells in culture continue to grow and divide, unlike isolated experimental preparations, they can be used to study long-term questions about differentiation, development, and the regulation of gene expression. If our neuroendocrine cell culture model turns out to be suitable for assessing the mechanisms of endocrine disruption, then the need for laboratory animal testing would be reduced and possibly replaced with a high throughput screening system. This would greatly facilitate classification and governmental regulation of endocrine disrupting chemicals.

REFERENCES

El Majdoubi, M (2011). Stem cell-derived in vitro models for investigating the effects of endocrine disruptors on developing neurons and neuroendocrine cells. Journal of Toxicology Environmental Health B Critical Review 14:292-299.

El Majdoubi M, McDonald C, Morrow T, Zagzoog M (2016). Effects of Heavy Metals on Proliferation and Neuronal Differentiation of Mouse Embryonic Stem Cells. Cytotherapy, 18 (Suppl): S76

Gore AC. (2008) Developmental programming and endocrine disruptor effects on reproductive neuroendocrine systems. Frontiers in Neuroendocrinology 29: 358-374.

Gottlieb DI, Huettner JE. (1999). An in vitro pathway from embryonic stem cells to neurons and glia. Cells Tissues Organs 165:165-172.

McLachlan J. (2001) Environmental signalling: what embryos and evolution teach us about endocrine disrupting chemicals. Endocrine Reviews 22:319-341.

Swanson LW. (1987). The Hypothalamus. In: Hokfelt T, Bjorklund A, Swanson LW, (eds.), Handbook of chemical neuroanatomy, Vol 5. Amsterdam: Elsevier. pp 1-124.