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

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

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

Clearance from the circulation of hMSC, melanoma cells and 10 µm inert beads.

<p>Inflexible inert 10 µM beads and B16F1 are cleared from circulation faster than hMSC. <b>A.</b> Values for cellular flux calculated as the average number of cells or 10 µm beads counted within vessels each minute in the CAM at 100× magnification. B. Values expressed as percentage flux were calculated as cells or beads in one minute as % of total observed in 10 minutes (n≥6).</p

Low passage hMSC express α4 integrin and SLeX.

<p>hMSC derived from four preparations from four different donors were assayed for expression of α4 integrin and SLeX by flow cytometry. Passage 1 cells were plated overnight to recover adherent viable cells and then re-plated at 100 cells/cm². The cells were harvested when 70 to 80% confluent.</p

Purified rhTSG-6 suppressed LPS-induced inflammation in mice.

<p>(A) Schematic for the experiment. (B, C) rhTSG-6 suppressed LPS-induced levels of mRNA for IL-6 and IFNγ in spleen. Statistical calculation was performed by a one-way ANOVA followed by the Tukey multiple comparison post hoc test. Data are presented as mean ± S.E.M. Symbol: RQ, relative quantity.</p

Synthesis of rhTSG-6 by culture of rhTSG-6/CHO-S cells in OCDPF medium and in a bioreactor that controlled pH.

<p>(A) Expansion of cells and oxygen saturation. (B) Yield of rhTSG-6 and pH of medium. (C) Photomicrographs demonstrating that rhTSG-6/CHO-S cells are largely single-cell suspensions. Scale bars = 100 μm and 50 μm (insert) (D) Yield of monomeric rhTSG-6. Western blot of medium with antibodies to hTSG-6. Values in (A) and (B) are means of 3 replicates.</p

CHO cell line stably transfected to synthesize rhTSG-6 forms aggregates resulting in a decrease of protein production.

<p>Cells were incubated in CD CHO basal media and in spinner culture. (A i) Diagram of expression plasmid of TSG-6 under control of human elongation factor promoter 1α and insertion of sequences for Myc and His tags at the C-terminus. (A ii) Expression of rhTSG-6 (antibody clone A38.1.20) and the His tag by immunocytochemistry. Scale bar = 10 μm. (A iii) Expression by Western blotting. (B) Number of control CHO cells (CHO-S) and rhTSG-6 synthesizing cells (rhTSG-6/CHO-S). Data are presented as mean ± S.D. (C) rhTSG-6 content and pH of medium from cultures of rhTSG-6/CHO-S cells. Data are presented as mean ± S.D. (D) Western blots with antibodies to the His-tag of medium from cultures of rhTSG-6/CHO-S cells. Monomeric rhTSG-6 was detected after day 3 but not after day 4, apparently because of aggregation. Abbreviations: IN, input sample; FT, flow through. (E) Aggregates of rhTSG-6/CHO-S cells after 4 days of culture. Scale bars = 500 μm and 250 μm (insert). (F) Immunolabeling with rhTSG-6 antibody (clone A38.1.20), HABP (biotin-conjugated HA binding proteins), and DAPI staining of aggregates. Scale bars = 100 μm and 50 μm (insert).</p

Addition of heparin to medium improved yield of rhTSG-6 in spinner cultures.

<p>(A) Effect on number of rhTSG-6/CHO-S cells. (B) Effect on yield of rhTSG-6. (C) Effect on yields of protein aggregates/complexes (H-rhTSG-6) and monomeric rhTSG-6 (L-rhTSG-6). Western blots with antibodies to hTSG-6 on medium from day 4 cultures of rhTSG-6/CHO-S cells. (E) Effects on pH of medium. Values are mean ± S.D. of 3 replicates of each sample in (A), (B), and (D).</p

Purification of rhTSG-6 from 5 or 6 liters cultured media of the bioreactor.

<p>(A) Schematic for the purification steps. (B) Assay by SDS-PAGE of rhTSG-6 eluted from the Q-Sepharose FF column. Symbols: IN, input sample; CB, Coomassie Blue staining of gel. (C) Endotoxin content of conditioned culture medium, eluate from the His-tag column and Q-Sepharose FF column. Values are mean ± S.D. of 3 replicates. (D) Deglycosylation of the purified rhTSG-6. Arrows indicate deglycosylated forms rhTSG-6.</p