Early Transcriptomic Changes upon Thalidomide Exposure Influence the Later Neuronal Development in Human Embryonic Stem Cell-Derived Spheres.

Mami Kikegawa,Hiromi Nishikawa,Tomohiro Ito,Xian-Yang Qin,Hiroko Nansai,Hideko Sone

doi:10.3390/ijms21155564

Abstract

Stress in early life has been linked with the development of late-life neurological disorders. Early developmental age is potentially sensitive to several environmental chemicals such as alcohol, drugs, food contaminants, or air pollutants. The recent advances using three-dimensional neural sphere cultures derived from pluripotent stem cells have provided insights into the etiology of neurological diseases and new therapeutic strategies for assessing chemical safety. In this study, we investigated the neurodevelopmental effects of exposure to thalidomide (TMD); 2,2′,4,4′-tetrabromodiphenyl ether; bisphenol A; and 4-hydroxy-2,2′,3,4′,5,5′,6-heptachlorobiphenyl using a human embryonic stem cell (hESC)-derived sphere model. We exposed each chemical to the spheres and conducted a combinational analysis of global gene expression profiling using microarray at the early stage and morphological examination of neural differentiation at the later stage to understand the molecular events underlying the development of hESC-derived spheres. Among the four chemicals, TMD exposure especially influenced the differentiation of spheres into neuronal cells. Transcriptomic analysis and functional annotation identified specific genes that are TMD-induced and associated with ERK and synaptic signaling pathways. Computational network analysis predicted that TMD induced the expression of DNA-binding protein inhibitor ID2, which plays an important role in neuronal development. These findings provide direct evidence that early transcriptomic changes during differentiation of hESCs upon exposure to TMD influence neuronal development in the later stages.

Highlights

There has been worldwide concern over the increasing number of patients with depression and children with developmental disorders [1,2]
Our results showed that TMD significantly increased the total cell numbers, and the presence of microtubule-associated protein 2 (MAP2)-positive and tyrosine hydroxylase (TH)-positive neuronal cells in a dose-dependent manner (Figure 1B and Figure S1A)
The recent technology using three-dimensional neuronal sphere models derived from pluripotent stem cells has provided new insights into the etiology of neurological diseases and new therapeutic strategies for assessing chemical safety

Summary

Introduction

There has been worldwide concern over the increasing number of patients with depression and children with developmental disorders [1,2]. Environmental factors including exposure to chemicals such as pesticides and air pollutants during the developmental age could play a role in the development of neurodevelopmental diseases [4,5]. We previously developed in vitro models using hESCs for studying the neurodevelopmental toxicities caused by various environmental pollutants [11,12,13]. To understand the molecular events underlying the neurodevelopmental effects of environmental chemicals including drugs, endocrine disruptors, and flame retardants, we have studied the effects of TMD and three environmental pollutants including BDE-47, BPA, and 4OH-PCB187 on global gene expression during neurosphere formation and during the following differentiation into neuronal cells

Results and Discussion

Immunocytochemistry and Image Analysis

Microarray Gene Expression Profiling

Knowledge-Based Pathway Analysis and Network Analysis

Conclusions