Effect of overexpression of HCMV US2 protein on MSC in a complement-mediated cytotoxicity assay.

<p>(A) The results depict representative data from a flow cytometry-based complement-mediated cytotoxicity assay from at least 3 independent experiments. The assay was performed in the presence of human IgM and rabbit serum complement (experimental) or in the presence of human IgM and absence of the rabbit serum complement (control). MSC stained with calcein were counted as viable, cells stained with Ethidium homodimer-1 were counted as dead, and cells doubly positive were counted as having membrane damage. Doubly negative cells were excluded from the analysis. A live/dead control (top) was used in order to set up the quadrant gates for alive, dead, and membrane-damaged populations and to set parameters for compensation between the 2 fluorescent channels. The experimental assay provided the percentage of dead cells under experimental conditions. The control assay provided the percentage of spontaneously dead cells. The same conditions were applied for non-transduced MSC and MSC-US2. (B) The percentage of cytotoxicity for each cell line was calculated as follows: (percent of dead cells under experimental conditions – percent of spontaneously dead cells)/(100 – spontaneously dead)*100. The percentage of experimental or spontaneously dead cells was determined by calculating the percentage of cells that were positive for Ethidium homodimer-1, and negative for calcein. The results depict the mean ± SEM of three independent experiments. * indicates p<0.05.</p

Up-regulation of CD46 surface expression on MSC by HCMV US proteins.

<p>(A) MSC, MSC-E, MSC-US2, MSC-US3, MSC-US6, and MSC-US11 were analyzed by flow cytometry for CD46 expression. Top- Percentage of CD46 positive cells for each MSC population. Bottom- MFI ratio for CD46 expression on each MSC population. MFI ratio  =  (Median Fluorescence Intensity for CD46/Median Fluorescence Intensity for isotype control). The results represent the mean ± SEM from at least three independent experiments. * indicates p<0.05 when comparing MSC-US cells with non-transduced MSC. 0(B) Each panel depicts representative data of at least three independent experiments. Black filled histograms correspond to different MSC cell populations stained with antibody against CD46 and unfilled histograms are the staining with the corresponding isotype control.</p

Up-regulation of CD59 surface expression on MSC by HCMV US proteins.

<p>(A) MSC, MSC-E, MSC-US2, MSC-US3, MSC-US6, and MSC-US11 were analyzed by flow cytometry for expression of CD59. Each panel depicts representative data of at least three independent experiments. Black filled histograms correspond to different MSC cell populations stained with antibody against CD59, and unfilled histograms are the corresponding isotype control staining. The MFI ratio for each MSC cell line was obtained by performing the following calculation: MFI ratio  =  (Median Fluorescence Intensity for CD59/Median Fluorescence Intensity for isotype control). (B) MFI ratio for each MSC population is shown. The results represent the mean ± SEM from at least three independent experiments. * indicates p<0.05 when comparing MSC-US cells with non-transduced MSC.</p

Up-regulation of CD55 surface expression on MSC by HCMV US proteins.

<p>(A) MSC, MSC-E, MSC-US2, MSC-US3, MSC-US6, and MSC-US11 were analyzed for CD55 surface expression by flow cytometry. Top- Percentage of CD55 positive cells for each MSC population. Bottom- MFI ratio for CD55 expression on different MSC populations. MFI ratio  =  Median Fluorescence Intensity for CD55/Median Fluorescence Intensity for isotype control. The results represent the mean ± SEM from at least three independent experiments. * indicates p<0.05 when comparing MSC-US cells with non-transduced MSC. (B) Each panel depicts representative data of at least three independent experiments. Black filled histograms correspond to each MSC cell population stained with antibody against CD55 and unfilled histograms correspond to isotype staining.</p

PCR primers for testing US-transduced and untransduced MSC.

a<p>F, Forward Primer; R, Reverse Primer.</p

Expression of HCMV US proteins by MSC decreases PBMNC proliferation and correlates with the levels of HLA-I.

<p>(A) Each of the transduced and untransduced MSCs were used as stimulators and were co-cultured with responders human PBMNC. After five days, DNA synthesis was assayed with the BrdU cell proliferation colorimetric ELISA. Data represents mean ± SEM of four independent experiments. In each experiment the specific stimulator-responder co-culture was performed in triplicate (* indicates p<0.01 and were considered statistically significant compared to MSC-E levels). (B) Furthermore a direct correlation was found between the levels of HLA-I MFI and human PBMNC proliferation.</p

Expression of HCMV US proteins protects MSC against NK cell lysis.

<p>Stimulators, MSC-E, MSC-US2, MSC-US3, MSC-US6, MSC-US11 and untransduced MSC were co-cultured independently with different concentrations of NK-92MI cells at 20∶1, 10∶1, 5∶1 and 1∶1 effector∶target ratios for 4 hrs. Release of lactate dehydrogenase was measured after cell lysis by ELISA. The percent specific lysis was calculated for each cell population and effector∶target ratio. The percentage of spontaneous lysis for all of the MSC tested in cytotoxicity assays ranged from 0.3–9.09%. Data represents the mean ± SEM of 6 independent experiments for each ratio between NK92MI cells and each transduced and untransduced cell line. (* indicates statistically significant difference between % specific lysis of US transduced MSC and MSC-E).</p

Quantification of Human MSC engraftment in fetal sheep liver.

<p>(A) Standard curve for human GAPDH amplification. Different Ct values were obtained by amplification of GAPDH at different percentages of human MSC/(human MSC+sheep MSC cells) concentrations. qPCR reaction for each concentration was repeated three times. (B) Percentage of total human cells engrafted in fetal liver of sheep that were injected with different MSC. The results are mean ± SEM of twelve replicates of qPCR reactions for MSC-E and MSC-US6 transplanted animals or six replicates of qPCR reactions for MSC-US11 transplanted animals. (<i>C</i>) Representative immunofluorescence staining and confocal microscopy of NPT-II and albumin staining of liver sections from sheep receiving MSC-US6 cell line. Images were captured using a <i>Fluoview 1000</i> confocal microscope with a 40× objective. The white arrow marks the area of the inset. Scale bars: 50 µm main picture; 12.5 µm on inset <i>(D) (top)</i> on the left, quantification of total NPT-II positive cells in fetal liver of sheep that were injected with the different MSC cell lines. On the top right, quantification of dual NPT-II and albumin positive cells in fetal liver of animals that were injected with the different MSC cell lines. On the bottom left, quantification of dual NPT-II and alpha-fetoprotein positive cells in fetal liver of sections from animals that were injected with the different MSC. On the bottom right, quantification of dual NPT-II and Hepar-I positive cells in fetal liver of sheep that were injected with the different MSC cell lines. The results are means ± SEM of at least 90,000 cells that were counted for NPT-II staining within the fetal liver sections from sheep that were injected with the different MSC. * p<0.05.</p

US transduced MSC possess the same differentiative capacities as untransduced MSC.

<p>(A) Adipocytic differentiation of transduced and untransduced MSC. Lipid vacuoles were observed by red staining and nuclei by blue staining. (B) Osteogenic differentiation of transduced and untransduced MSC. Red staining indicated alkaline phosphatase activity, blue staining the nuclei, and black staining the calcium deposits. All images were captured with an Olympus IX-71 microscope at 40× original magnification.</p

Generation of US-transduced MSC.

<p>(A) HCMV US2, US3, US6 and US11 cDNA sequences were cloned into the pMSCVneo plasmid between EcoRI and XhoI or Sal-I. US genes were driven by the MSCV LTR promoter while the Neomycin resistance marker gene (NeoR) was under the control of an internal PGK promoter. (B) Total RNA was extracted from each of the transduced and untransduced MSC populations and, after reverse transcription, cDNAs were obtained and amplified using specific primers for each of the US genes, NeoR and B-Actin. (C) Light microscope image at 10× original magnification of the different MSC populations showing similar morphology during cell culture. Images were captured with an Olympus IX-71 microscope.</p