Progress toward the development of in vitro model system for chemical-induced developmental neurotoxicity: potential applicability of stem cells.

Abstract

committed toward the neuronal lineage and are very similar to the fetal neural stem cells. In our so far published papers, we have successfully attempted to establish stem cell-based in vitro model systems for the study of the various aspects of DNT and have proven that human umbilical cord blood-derived stem cells (hUCBSCs) when differentiated into neurons serve as one of the classical tools for the same. These were employed to assess the developmental neurotoxicity potential of Monocrotophos (MCP), a known developmental neurotoxin. Differentiating cells were given MCP exposure at different stages of maturation, i.e., day 2, 4 and 8, and significant changes in the markers of differentiation, proliferation, injury and receptors were studied. Our group reported that there was a significant upregulation of the different MAPKs, apoptosis and neurogenesis markers and a down regulation in the various cell proliferation markers during the neuronal differentiation process. Expression analysis was done both at the transcriptional (RT-PCR) as well as translational levels (immunocytochemistry and Western blotting). As differentiation proceeded, there was a significant quantitative reduction in the expression of stems markers, i.e., CD133, MYC, NANOG, SHH, KLF4, SOX2, POU5F1 and FRAP1, whereas the differentiation marker expression was significantly increased except in case of NGF, CHRM2 and NR42A. The highest expression for differentiation markers, i.e., NFM, MBP, NFH, NCM, BDNF and STAT4, was seen on day 8. These results support that hUCBSCs successfully differentiated into the neuronal lineage. Post MCP exposure there was again a significant alteration in the various markers of apoptosis, oxidative stress, metabolism, etc. It was concurred from the results that MCP exposure leads to ROS generation, oxidative stress, depleted glutathione levels and even promoted apoptosis. Receptor studies further demonstrated that the Developmental neurotoxicity (DNT) has been the prime focus of our research group since it is one of the most crucial and significant parts of the neuroscience research arena. The restricted availability of live human fetal brain tissue and major ethical issues makes the field further challenging. So far, it has been best represented via the in vivo model systems; however, the inaccurate extrapolation of data to humans restricts its scope and applications. Therefore, there is a dire need for samples (cells and tissues) of human origin. Based on the OECD guidelines 426, rodent data are present for about 200 compounds, but there is a scarcity of data available for humans. The developing brain is highly vulnerable to even minute doses of substances which are otherwise harmless for a fully developed mature brain. This is primarily because of the poorly developed blood–brain barrier, low oxygen levels and high mitotic rates. The entire process of brain development is quite complex, and in order to generate DNT models in vitro, cells and tissues of human origin are needed. Stem cells with their inherent potential for pluripotency and unique ability for unlimited proliferation serve as one of the best tools. Stem cells can be made to differentiate into the neuronal lineages and can thereby sufficiently mimic cells of the developing brain. Human umbilical cord blood was employed for the isolation of stem cells as it is not only a rich source of stem cells, but also a waste material, therefore does not pose any major ethical dubious. CD34+ hematopoietic stem cells (HSCs)/non-hematopoietic stem cells (non-HSCs) derived from cord blood are greatly