Tyroxine Hydroxylase-Positive Neuronal Cell Population is Increased by Temporal Dioxin Exposure at Early Stage of Differentiation from Human Embryonic Stem Cells.

Sailendra Sarma,Seiichiroh Ohsako,Reiko Nagano

doi:10.3390/ijms20112687

Abstract

Background: The neurological effects of short-term dioxin exposure during the fetal period is an important health risk in humans. Here, we investigated the effects of dioxin on neural differentiation using human embryonic stem cells (hESCs) to evaluate human susceptibility to dioxin. Methods: Using an enzymatic bulk passage, neural differentiation from human ESCs was carried out. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was added to various stages of culture. The expression levels of the neuronal markers microtubule-associated protein 2 (MAP2) and thyroxine hydroxylase (TH) were measured by RT-qPCR and image analysis of immunostaining. Results: Although early-stage neuronal cells are quite resistant to TCDD, the numbers of neural rosettes and increases in mRNA expression levels and the number of cells positive for MAP2 and TH were significant by temporal exposure at embryoid body stage (Day9-exposure group). In contrast, the TCDD exposures against ESCs (Day0-exposure group) and differentiated neural cells (Day35-exposure group) were not affected at all. The increment was similarly observed by continuous exposure of TCDD from Day9 through Day60. Conclusions: These results indicated that dioxin exposure during the early stage of differentiation from hESCs increases the contents of neuronal cells, especially TH-positive neuronal cells. Regulations of aryl hydrocarbon receptor (AHR) signaling in an early stage of embryogenesis should be investigated extensively to understand the mechanism underlying the increase in neuronal cell populations and to apply the knowledge to regenerative medicine.

Highlights

Dioxin and related substances are ubiquitous environmental pollutants causing a wide variety of pathological alterations that affect human health owing to a diverse range of toxic effects [1]
During continuous culture after the withdrawal of TCDD, neural rosettes, which are ring-like structures indicating the sprouting neuronal progenitors, were observed seven days after changing neural proliferation medium (NPM) (Figure 2A)
RT-qPCR analysis on Day60 showed that the copy number of thyroxine hydroxylase (TH) mRNA significantly increased in the 1 nM TCDD-exposed group (Figure 6D). These results indicate that continuous exposure to TCDD stimulates the increase in neuronal cell population during developmental processes

Summary

Introduction

Dioxin and related substances are ubiquitous environmental pollutants causing a wide variety of pathological alterations that affect human health owing to a diverse range of toxic effects [1]. Ahr-mediated signaling is suspected to target the central nervous system during the early developmental stage in laboratory mouse models, as shown by numerous descriptive reports on, for example, abnormal behavior, recognition disorder, or abnormal neuronal cell migration associated with TCDD exposure [9,10,11,12,13]. Due to the large difference in toxicity, in vitro studies by using human stem cells as models are important for determining TCDD toxicity against central nervous system development and neuronal cell differentiation during an early stage of the fetus. Regulations of aryl hydrocarbon receptor (AHR) signaling in an early stage of embryogenesis should be investigated extensively to understand the mechanism underlying the increase in neuronal cell populations and to apply the knowledge to regenerative medicine

Methods

Results

Discussion

Conclusion