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.

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.

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.

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‐β 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.

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 molecular characteristics and requirements of each step. Vacuolization is triggered by reducing both matrix proteins and cell-cell contact, and driven by macropinocytosis. Fine-tuned cell-cell contact then forms nascent 3-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 the early visceral endoderm. pXEN cells provide a useful in vitro model for studying matrix-independent, basal-type lumenogenesis and the physiology of the visceral endoderm.