TET2-mediated DNA hydroxymethylation of TGFB1 is related to selective intrauterine growth restriction in monochorionic twin pregnancies.

INTRODUCTION: Selective intrauterine growth restriction (sIUGR), which specifically occurs in monochorionic (MC) twins, usually has a poor prognosis and the underlying mechanisms are not well understood. It is an ideal model for exploring epigenetic-modified mechanisms for fetal development in MCDA twins due to eliminating the interference of different heritable backgrounds and intrauterine environments among individuals. METHODS: The levels of ten-eleven translocation 2 (TET2) and its upstream and downstream targets miR-29b-3p and transforming growth factor beta 1 (TGFB1) were determined using RT‒qPCR, western blotting, and immunohistochemistry. Using TET2 overexpression and knockdown methods, we investigated the role of TET2 in trophoblast functions. The regulatory relationships among TET2, miR-29b-3p, and TGFB1 were explored by cell migration assay, invasion assay, apoptotic ratio assays, Western blot, hMeDIP-qPCR and dual-luciferase assay. RESULTS: A consistent upregulation of TET2 and TGFB1 was observed in the smaller placental shares compared to the larger placental shares in sIUGR. Gain-of-function studies of TET2 in trophoblasts showed decreased cell invasion and increased apoptosis, whereas loss-of-function studies of TET2 rescued this effect. Mechanistic studies revealed that miR-29b-3p and TGFB1 were the upstream factor and downstream target of TET2, respectively. Furthermore, miR-29b-3p/TET2/TGFB1-smad was identified as a unique axis that regulates trophoblast invasion, migration, and apoptosis in a DNA hydroxymethylation-dependent manner. DISCUSSION: We elucidated the functional roles of TET2 and DNA hydroxymethylation in trophoblasts and identified a novel DNA regulatory mechanism, providing a basis for further exploration of DNA epigenetic regulatory patterns in sIUGR.

Chemical-based primary human hepatocyte monolayer culture for the study of drug metabolism and hepatotoxicity: Comparison with the spheroid model.

Traditionally cultured monolayers of primary human hepatocytes (PHHs) deteriorate within days and thereby become unsuitable for drug-related studies. PHH spheroids (3D PHHs) maintain liver functions for weeks, but are considerably more demanding. Recently, a chemical-based approach (5C PHHs) succeeded in long-term culture of hepatocyte monolayers, but it remains unclear whether the drug-related functions are preserved. To clarify this, we compared the 5C and 3D PHHs in terms of gene expression analysis, proteomic analysis, functionality (basal and induced activities of representative CYP450 enzymes and urea and albumin secretions), survival in culture, and sensitivity to representative drugs. In all comparisons, which spanned culture durations of up to 4 weeks, the 5C PHHs performed at least as well as the 3D PHHs. Hence, the novel 5C PHH monolayer format combines the convenience of the traditional monolayer format with the functionality and maintainability of the spheroid format. Our results suggest that 5C PHH monolayers can be used more conveniently and efficiently for high-throughput drug screening, preclinical drug safety evaluations, and mechanistic studies.

Basal-type lumenogenesis in extraembryonic endoderm stem cells models the early visceral endoderm.

Cultured rat primitive extraembryonic endoderm (pXEN) cells easily form free-floating multicellular vesicles de novo, exemplifying a poorly studied type of morphogenesis. Here, we reveal the underlying mechanism and the identity of the vesicles. We resolve the morphogenesis into vacuolization, vesiculation and maturation, and define the molecular characteristics and requirements of each step. Vacuolization is fueled by macropinocytosis and occurs by default if not blocked by high cell density or matrix proteins. Fine-tuned cell-cell contact then forms nascent three-cell vesicles with vacuole-derived lumina. In maturation, the vesicles complete epithelialization, expand via mitosis and continued fluid uptake, and differentiate further. The mature vesicles consist of a simple squamous epithelium with an apical-outside/basal-inside polarity that we trace back to the single cell stage. The polarity and gene expression pattern of the vesicles are similar to those of the early visceral endoderm. pXEN cells provide a useful in vitro model for study of matrix-independent, basal-type lumenogenesis and the physiology of the visceral endoderm.This article has an associated First Person interview with the first author of the paper.

Transcriptome analysis shows ambiguous phenotypes of murine primitive endoderm-related stem cell lines.

Primitive endoderm (PrE)-related cell lines (XEN, pXEN and nEnd cells) show key features of the PrE. By transcriptome analysis, we show: (a) Compared to embryonic stem cells, PrE-related cell lines are less in vivo like, although early nEnd cells are most similar to the PrE. (b) These cell lines show post-PrE features of parietal (XEN and pXEN cells) or visceral (nEnd cells) endoderm, likely driven by Tgf-ß and Wnt/Activin signaling, respectively. (c) pXEN and nEnd cell lines additionally show pre-PrE features. Hence, neither pXEN nor nEnd cell cultures represent a distinct in vivo entity. Rather, their properties are compatible with mixed and hybrid phenotypes. Our findings indicate that pre-PrE, PrE and early post-PrE phenotypes result from different niches, which need to be better understood to derive cell lines that distinctly represent the early stages of the extraembryonic endoderm.

Isolation of primitive mouse extraembryonic endoderm (pXEN) stem cell lines.

Mouse blastocysts contain the committed precursors of the extraembryonic endoderm (ExEn), which express the key transcription factor Oct4, depend on LIF/LIF-like factor-driven Jak/Stat signaling, and initially exhibit lineage plasticity. Previously described rat blastocyst-derived ExEn precursor-like cell lines (XENP cells/HypoSCs) also show these features, but equivalent mouse blastocyst-derived cell lines are lacking. We now present mouse blastocyst-derived cell lines, named primitive XEN (pXEN) cells, which share these and additional characteristics with the XENP cells/HypoSCs, but not with previously known mouse blastocyst-derived XEN cell lines. Otherwise, pXEN cells are highly similar to XEN cells by morphology, lineage-intrinsic differentiation potential, and multi-gene expression profile, although the pXEN cell profile correlates better with the blastocyst stage. Finally, we show that pXEN cells easily convert into XEN-like cells but not vice versa. The findings indicate that (i) pXEN cells are more representative than XEN cells of the blastocyst stage; (ii) mouse pXEN, rather than XEN, cells are homologs of rat XENP cells/HypoSCs, which we propose to call rat pXEN cells.

Stable expression of Y-box protein 1 gene in early development of the abalone Haliotis diversicolor.

Abalone animals are import models for the study of the early development of marine invertebrates. However, systematical evaluations of internal control genes (ICG) have seldomly been performed. In this study, ten candidate genes were cloned and surveyed for their stability throughout the early developmental period of H. diversicolor using qPCR. In a period from fertilized egg to postlarva, three genes, Y-box protein 1 (YB1), ornithine decarboxylase antizyme 1 (OAZ1) and eukaryotic translation initiation factor 5A (EIF5A), were found to be the most stable and could be used as ICGs. It is suggested that using two genes jointly, such as YB1 and OAZ1, could be sufficiently reliable to normalize the temporal dynamics of other genes. Normalized by YB1/OAZ1, some rough features of early development of a small abalone were characterized. This is the first report of the temporal dynamics of metabolic activities and overall mRNA abundance of abalone animals in early stages. It is also the first time the multi-functional gene YB1 has been described as an internal control for early developmental biology studies. Phylogeny and function of YB1 are further discussed.