Rapidly Self-Renewing Human Multipotent Marrow Stromal Cells (hMSC) Express Sialyl Lewis X and Actively Adhere to Arterial Endothelium in a Chick Embryo Model System

There have been conflicting observations regarding the receptors utilized by human multipotent mesenchymal bone marrow stromal cells (hMSC) to adhere to endothelial cells (EC). To address the discrepancies, we performed experiments with cells prepared with a standardized, low-density protocol preserving a sub-population of small cells that are rapidly self-renewing.Sialyl Lewis X (SLeX) and α4 integrin expression were determined by flow cytometry. Fucosyltransferase expression was determined by quantitative realtime RT-PCR. Cell adhesion assays were carried out with a panel of endothelial cells from arteries, veins and the microvasculature in vitro. In vivo experiments were performed to determine single cell interactions in the chick embryo chorioallantoic membrane (CAM). The CAM is a well-characterized respiratory organ allowing for time-lapse image acquisition of large numbers of cells treated with blocking antibodies against adhesion molecules expressed on hMSC.hMSC expressed α4 integrin, SLeX and fucosyltransferase 4 and adhered to human EC from arteries, veins and the microvasculature under static conditions in vitro. In vivo, hMSC rolled on and adhered to arterioles in the chick embryo CAM, whereas control melanoma cells embolized. Inhibition of α4 integrin and/or SLeX with blocking antibodies reduced rolling and adhesion in arterioles and increased embolism of hMSC.The results demonstrated that rapidly self-renewing hMSC were retained in the CAM because they rolled on and adhered to respiratory arteriolar EC in an α4 integrin- and SLeX-dependent manner. It is therefore important to select cells based on their cell adhesion receptor profile as well as size depending on the intended target of the cell and the injection route

Rapidly Self-Renewing Human Multipotent Marrow Stromal Cells (hMSC) Express Sialyl Lewis X and Actively Adhere to Arterial Endothelium in a Chick Embryo Model System

There have been conflicting observations regarding the receptors utilized by human multipotent mesenchymal bone marrow stromal cells (hMSC) to adhere to endothelial cells (EC). To address the discrepancies, we performed experiments with cells prepared with a standardized, low-density protocol preserving a sub-population of small cells that are rapidly self-renewing.Sialyl Lewis X (SLeX) and α4 integrin expression were determined by flow cytometry. Fucosyltransferase expression was determined by quantitative realtime RT-PCR. Cell adhesion assays were carried out with a panel of endothelial cells from arteries, veins and the microvasculature in vitro. In vivo experiments were performed to determine single cell interactions in the chick embryo chorioallantoic membrane (CAM). The CAM is a well-characterized respiratory organ allowing for time-lapse image acquisition of large numbers of cells treated with blocking antibodies against adhesion molecules expressed on hMSC.hMSC expressed α4 integrin, SLeX and fucosyltransferase 4 and adhered to human EC from arteries, veins and the microvasculature under static conditions in vitro. In vivo, hMSC rolled on and adhered to arterioles in the chick embryo CAM, whereas control melanoma cells embolized. Inhibition of α4 integrin and/or SLeX with blocking antibodies reduced rolling and adhesion in arterioles and increased embolism of hMSC.The results demonstrated that rapidly self-renewing hMSC were retained in the CAM because they rolled on and adhered to respiratory arteriolar EC in an α4 integrin- and SLeX-dependent manner. It is therefore important to select cells based on their cell adhesion receptor profile as well as size depending on the intended target of the cell and the injection route

A Forward-Genetic Screen and Dynamic Analysis of Lambda Phage Host-Dependencies Reveals an Extensive Interaction Network and a New Anti-Viral Strategy

Latently infecting viruses are an important class of virus that plays a key role in viral evolution and human health. Here we report a genome-scale forward-genetics screen for host-dependencies of the latently-infecting bacteriophage lambda. This screen identified 57 Escherichia coli (E. coli) genes—over half of which have not been previously associated with infection—that when knocked out inhibited lambda phage's ability to replicate. Our results demonstrate a highly integrated network between lambda and its host, in striking contrast to the results from a similar screen using the lytic-only infecting T7 virus. We then measured the growth of E. coli under normal and infected conditions, using wild-type and knockout strains deficient in one of the identified host genes, and found that genes from the same pathway often exhibited similar growth dynamics. This observation, combined with further computational and experimental analysis, led us to identify a previously unannotated gene, yneJ, as a novel regulator of lamB gene expression. A surprising result of this work was the identification of two highly conserved pathways involved in tRNA thiolation—one pathway is required for efficient lambda replication, while the other has anti-viral properties inhibiting lambda replication. Based on our data, it appears that 2-thiouridine modification of tRNAGlu, tRNAGln, and tRNALys is particularly important for the efficient production of infectious lambda phage particles

3-Dimensional images of cells in rhodamine-labeled vesicles of chick embryo CAM.

<p>Orthologous projections of z-stacked photomicrographs of the CAM at 200× magnification. Crosshairs indicate cell of interest. <b>A.</b> B16F1 melanoma cells primarily embolized in the overlying capillary plexus (arrowhead) and at the ends of tapering arterioles. <b>B</b>. An hMSC, retaining its shape, adhered in a large vessel (dashed lines) lying beneath the capillary plexus.</p

Real time assay of cells in vessels of the chick embryo CAM.

<p><b>A.</b> Schematic for injecting cells or beads into a large vein of the CAM and capturing images for 3 to 10 minutes at either 40× or 100× magnification. <b>B. (upper panel).</b> Green B16F1 melanoma cells were primarily embolized in the capillary bed and had distorted morphology (∧). <b>(lower panel).</b> Green hMSC retained a regular morphology and were found both within arteries (†) and within the capillary beds (#). Images taken 10 minutes after injection of the cells. Arrows indicate direction of blood flow. Magnification 100×.</p

Distribution of hMSC to arteries/arterioles, veins and capillaries/end arterioles in the CAM.

<p><b>A.</b> Distribution of hMSC compared to lymphocytes and effects of pre-treatment with anti-SLeX and/or anti-α4 integrin (n = 5). <b>B</b>. Distribution in arteries of hMSC from 5 preparations from 5 different donors of marrow repeated 5 times.</p

Comparison of single-cell NF-κB activation dynamics for three different LPS preparations.

<p>Intensity represents the relative nuclear localization of p65-dsRed fusion protein, calculated as mean nuclear intensity divided by initial mean cytoplasmic intensity. The concentration for all three preparations was 0.5 µg/mL. The black line shows the average time course for all cells; the light blue traces are ten randomly selected individual cells. The number of active cells (N), as well as the maximum peak amplitude (Peak Amp), and time elapsed until the maximum amplitude is reached (Time to Peak) are also shown. The maximum intensity is indicated by a dot and the two dashed lines indicate how Peak Amp and Time to Peak are determined. The duration of the first peak (Peak Width) is also shown. This value is determined by drawing a horizontal line at the intensity that is halfway between the minimum and maximum peak value. The region above the line and shaded in green denotes the time during which the p65-dsRed nuclear intensity is more than half of the maximum p65-dsRed nuclear intensity. Below each plot, corresponding representative microscope images are shown for the first 200 minutes after stimulation, as labeled.</p

Blocking paracrine signaling by TNF across all concentrations and preparations.

<p>The average time course for N number of active cells is plotted for cells stimulated in the absence (blue trace, top value of n) and presence (orange trace, bottom value) of sTNFRII, which competes to bind TNF. Concentrations are indicated at left, and the preparation at top.</p

The potency window for each of the LPS preparations.

<p>The fraction of active cells is plotted as a function of concentration for values spanning nine orders of magnitude, as shown.</p

Activation dynamics for each of the LPS preparations and several concentrations and summary statistical comparisons.

<p>(A) As in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053222#pone-0053222-g001" target="_blank">Figure 1</a>, the average and ten randomly selected traces of active cells are shown, as well as Time to Peak, Peak Amp and N. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053222#pone-0053222-g001" target="_blank">Figure 1</a> legend for more details. Concentrations are indicated at left, and the preparation at top. The very lowest and highest concentrations are not shown here (but appear in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053222#pone-0053222-g004" target="_blank">Figure 4</a>), because virtually no cells were found to be active in the first case, and the traces are essentially identical to their nearest neighbor in the second. (B) Time-to-peak values for each LPS preparation are shown with standard deviations, across each concentration. LPS preparations are indicated with different colors as labeled in D. (C) Peak amplitude values for each LPS preparation are shown with standard deviations across each concentration. (D) The correspondence between time-to-peak and peak amplitude is shown for each LPS preparation, including all concentrations.</p