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

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

Laminin α5 chain expression increases in differentiating hESC.

<p>Flow cytometric analysis of RA-treated (red population) and control (mTeSR1, blue population) hESC stained with antibodies recognizing laminin (LM) α5 chain and SSEA-3 (A) or HAND1 (B).</p

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

Melanocytes in the Skin – Comparative Whole Transcriptome Analysis of Main Skin Cell Types

<div><p>Melanocytes possess several functions besides a role in pigment synthesis, but detailed characteristics of the cells are still unclear. We used whole transcriptome sequencing (RNA-Seq) to assess differential gene expression of cultivated normal human melanocytes with respect to keratinocytes, fibroblasts and whole skin. The present results reveal cultivated melanocytes as highly proliferative cells with possible stem cell-like properties. The enhanced readiness to regenerate makes melanocytes the most vulnerable cells in the skin and explains their high risk of developing into malignant melanoma.</p></div

Uniquely expressed genes in MC.

<p>The list of genes, expressed in MC, but not in KC and FB.</p><p>Uniquely expressed genes in MC.</p

The number of differentially expressed genes in each study group - melanocyte (MC), keratinocyte (KC), fibroblast (FB) and whole skin (Skin).

<p>Red triangles – upregulated genes, blue triangles – downregulated genes.</p

The number of detected and uniquely expressed genes.

<p>The number of detected and uniquely expressed genes.</p

Comparative pathway analysis of MC and KC, FB and whole skin.

<p>Red plots indicate pathways, which were prominently expressed in MC. Blue plots mark pathways, which were downregulated in MC (A, B, C) and concomitantly upregulated in the whole skin (A), KC (B) and FB (C), respectively. The word sizes in wordclouds are defined by the p-values.</p