A Novel Monoclonal Antibody to Human Laminin alpha 5 Chain Strongly Inhibits Integrin-Mediated Cell Adhesion and Migration on Laminins 511 and 521

Peer reviewe

MUC20 expression marks the receptive phase of the human endometrium

Research question: How does mucin MUC20 expression change during the menstrual cycle in different cell types of human endometrium? Design: Study involved examination of MUC20 expression in two previously published RNA-seq datasets in whole endometrial tissue (n = 10), sorted endometrial epithelial (n = 44) or stromal (n = 42) cell samples. RNA-Seq results were validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in whole tissue (n = 10), sorted epithelial (n = 17) and stromal (n = 17) cell samples. MUC20 protein localization and expression were analysed in human endometrium by immunohistochemical analysis of intact endometrial tissue (n = 6) and also Western blot of cultured stromal and epithelial cells (n = 2). Results: MUC20 is differentially expressed in the endometrium between the pre-receptive and receptive phases. We show that MUC20 is predominantly expressed by epithelial cells of the receptive endometrium, both at the mRNA (RNA-Seq, P = 0.005; qRT-PCR, P = 0.039) and protein levels (Western blot; immunohistochemistry, P = 0.029). Conclusion: Our results indicate MUC20 as a novel marker of mid-secretory endometrial biology. We propose a model of MUC20 function in the hepatocyte growth factor (HGF)-activated mesenchymal-epithelial transition (MET) receptor signalling specifically in the receptive phase. Further investigations should reveal the precise function of MUC20 in human endometrium and the possible connection between MUC20 and HGF-activated MET receptor signalling. MUC20 could potentially be included in the list of endometrial receptivity markers after further clinical validation.Peer reviewe

Changes in Laminin Expression Pattern during Early Differentiation of Human Embryonic Stem Cells

<div><p>Laminin isoforms laminin-511 and -521 are expressed by human embryonic stem cells (hESC) and can be used as a growth matrix to culture these cells under pluripotent conditions. However, the expression of these laminins during the induction of hESC differentiation has not been studied in detail. Furthermore, the data regarding the expression pattern of laminin chains in differentiating hESC is scarce. In the current study we aimed to fill this gap and investigated the potential changes in laminin expression during early hESC differentiation induced by retinoic acid (RA). We found that laminin-511 but not -521 accumulates in the committed cells during early steps of hESC differentiation. We also performed a comprehensive analysis of the laminin chain repertoire and found that pluripotent hESC express a more diverse range of laminin chains than shown previously. In particular, we provide the evidence that in addition to α1, α5, β1, β2 and γ1 chains, hESC express α2, α3, β3, γ2 and γ3 chain proteins and mRNA. Additionally, we found that a variant of laminin α3 chain—145 kDa—accumulated in RA-treated hESC showing that these cells produce prevalently specifically modified version of α3 chain in early phase of differentiation.</p></div

The expression of laminin α5, β1, β2 and γ1 chains in differentiating RA-treated hESC.

<p>(A) Immunofluorescence analysis of laminin (LM) chains α5, β1, β2 and γ1 and OCT4 in RA-treated hESC. Laminin chains (red) and OCT4 (green) were detected with appropriate antibodies. Cell nuclei were labeled with DAPI (blue). Scale bar: 100 μm. (B) Multilayer confocal microscopy was used to visualize the LM β1 chain (red) localization and OCT4 (green) expression in RA-treated hESC. Cell nuclei were labeled with DAPI (blue). Scale bar: 20 μm. (C) Immunoprecipitation of laminin-511 and -521 from RA-treated hESC. The protein complexes were immunoprecipitated using laminin α5 chain-specific antibody. The laminin α5, β1, β2 and γ1 chains were detected by Western blot analysis using corresponding antibodies.</p

Optimizing bone morphogenic protein 4-mediated human embryonic stem cell differentiation into trophoblast-like cells using fibroblast growth factor 2 and transforming growth factor-beta/activin/nodal signalling inhibition

Several studies have demonstrated that human embryonic stem cells [hESC] can be differentiated into trophoblast-like cells if exposed to bone morphogenic protein 4 [BMP4] and/or inhibitors of fibroblast growth factor 2 [FGF2] and the transforming growth factor beta [TGF-beta]/activin/nodal signalling pathways. The goal of this study was to investigate how the inhibitors of these pathways improve the efficiency of hESC differentiation when compared with basic BMP4 treatment. RNA sequencing was used to analyse the effects of all possible inhibitor combinations on the differentiation of hESC into trophoblast-like cells over 12 days. Genes differentially expressed compared with untreated cells were identified at seven time points. Additionally, expression of total human chorionic gonadotrophin [HCG] and its hyperglycosylated form [HCG-H] were determined by immunoassay from cell culture media. We showed that FGF2 inhibition with BMP4 activation up-regulates syncytiotrophoblast-specific genes [CGA, CGB and LGALS16], induces several molecular pathways involved in embryo implantation and triggers HCG-H production. In contrast, inhibition of the TGF-beta/activin/nodal pathway decreases the ability of hESC to form trophoblast-like cells. Information about the conditions needed for hESC differentiation toward trophoblast-like cells helps us to find an optimal model for studying the early development of human trophoblasts in normal and in complicated pregnancy. (C) 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.Peer reviewe

RA induces hESC differentiation and concomitant upregulation of HAND1 and GATA-4 expression.

<p>(A) Immunofluorescence analysis of OCT4 (green) in RA-treated hESC. Cell nuclei were labeled with DAPI (blue). Scale bar: 100 μm. (B) Flow cytometric analysis of OCT4, SSEA-3, NANOG, SOX-2, GATA-4 and HAND1 expression in RA-treated hESC on day 3 and 5. Untreated hESC (grown in mTeSR1) harvested at the identical time-points were used as controls. Average Fold Change based on Median Fluorescence Intensity (MFI) values was calculated in relation to corresponding control (mTeSR1) samples. Statistical significance with P-values less than 0.05 are labeled with “*” (C) Flow cytometric analysis of RA-treated and control (mTeSR1) hESC cells co-stained with antibodies recognizing OCT4 and GATA-4 or HAND1. Overlays of RA-treated (red) and control (mTeSR1, blue) hESC populations are presented. (D) Flow cytometric analysis of RA-treated and control (mTeSR1) hESC co-stained with GATA-4 and HAND1-specific antibodies. The percentages of cell populations in each quadrant are indicated on the density plots.</p

hESC express a diverse range of laminin chains.

<p>(A) Western blot analysis of indicated laminin (LM) chains in hESC. The lysates of JEG-3 (α1 chain), A431 (α2, α3, β1-β3, γ1- γ3), A549 and JAR (α5) cells and human platelets (α4) were used as controls. See the text for a detailed explanation. (B) Flow cytometric analysis of laminin (LM) chains α1, α2, α3 and α5 in hESC on day 3 and 5 of RA treatment. Untreated hESC (mTeSR1) were used as controls. Average Fold Change based on Median Fluorescence Intensity (MFI) values was calculated in relation to corresponding control (mTeSR1) samples. Statistical significance with P-values less than 0.05 are labeled with “*”.</p