Demographic features and platelet count of subjects enrolled in this study.

<p>ND: not determined.</p>*<p>Data were derived from 16 healthy donors.</p

Positive frequencies of circulating anti-GPIIb/IIIa and anti-GPIb antibody-producing B cells, and their combination in patients with primary ITP, various thrombocytopenic conditions, and healthy controls.

<p>SLE, liver cirrhosis, and post-HSCT are conditions potentially causing secondary ITP, whereas aplastic anemia and MDS are non-ITP disease controls.</p

Clinical findings in patients with primary ITP, stratified by the presence or absence of circulating anti-GPIb or anti-GPIIb/IIIa antibody-producing B cells.

<p>Clinical findings in patients with primary ITP, stratified by the presence or absence of circulating anti-GPIb or anti-GPIIb/IIIa antibody-producing B cells.</p

Anti-GPIIb/IIIa and anti-GPIb antibody-producing B cells in the circulation of patients with various thrombocytopenic conditions and healthy controls.

<p>Cut-off values for anti-GPIIb/IIIa and anti-GPIb antibody-producing B cells were 2.0 and 2.4 per 10⁵ PBMCs, respectively. Bars indicate the mean, and asterisks indicate statistical significance (<i>P</i><0.05).</p

SDF-1 is required for generating MOMCs.

<p>CD14⁺ monocytes were cultured on fibronectin in the presence of serial concentrations of SDF-1. (<b>A</b>) The generation of spindle-shaped adherent cells, expressed as a proportion (%) of those generated from culturing monocytes alone. The results shown are the mean and SD of 10 independent experiments. (<b>B</b>) Morphology of adherent cells obtained in a culture with 100 ng/ml SDF-1. Bars: 200 µm. (<b>C</b>) Cell-surface CD34 expressed on adherent cells obtained in a culture with 100 ng/ml SDF-1, as analyzed by flow cytometry. Closed histograms indicate CD34 expression; open histograms represent staining with isotype-matched control mAb. (<b>D</b>) Multidifferentiation potential of adherent cells obtained in a culture with 100 ng/ml SDF-1. Cells treated for osteogenic, chondrogenic, adipogenic, and endothelial induction for 1 week were analyzed by immunohistochemical staining for Cbfa1, Sox-9, or PPARγ (red) in combination with CD45 (green), or for eNOS or Tie-2 (red) in combination with DAPI (blue), and were observed under a fluorescence microscope. Representative results of 3 independent experiments are shown. Bars: 50 µm. (<b>E</b>) AMD 3100, a CXCR4 antagonist, suppressed the generation of MOMCs. Circulating monocytes were cultured with platelet-conditioned medium on fibronectin in the presence of 0, 1, or 5 ng/ml AMD 3100. Results are expressed as a proportion (%) of the number of spindle-shaped adherent cells obtained in the culture of monocytes alone. *<i>P</i><0.05, compared with culturing without AMD 3100.</p

Soluble factor(s) released from activated platelets are required for MOMC generation.

<p>CD14⁺ monocytes were cultured alone or in combination with platelets or platelet-conditioned medium on fibronectin. (<b>A</b>) Morphology of adherent cells on culture day 7. Bars: 200 µm. (<b>B</b>) Spindle-shaped adherent cells generated in the indicated culture, expressed as a proportion (%) of those generated in a culture of monocytes alone. Results show the mean and SD of three independent experiments. (<b>C</b>) Scatter plots and surface expression of CD34 on adherent cells, analyzed by flow cytometry. CD34 expression is shown by a closed histogram; open histograms represent staining with isotype-matched control mAb. (<b>D</b>) The morphology and CD34 expression of adherent cells obtained from cultures with platelet-conditioned medium prepared by stimulating platelets with thrombin or ADP. Bars: 200 µm. Cell-surface CD34 expression, analyzed by flow cytometry, is shown by closed histograms.</p

Platelets are required for the generation of MOMCs.

<p>CD14⁺ monocytes were cultured on fibronectin with platelets or platelet-depleted CD14⁻ PBMCs, or on fibronectin alone. (<b>A</b>) Morphology of adherent cells on culture day 7. Bars: 200 µm. (<b>B</b>) The total number of cells and the number of spindle-shaped adherent cells generated are expressed in proportion (%) to those generated by monocyte culture alone. The results shown are the mean and SD of three independent experiments. (<b>C</b>) Cell-surface CD14 and CD34 expression on the cultured adherent cells, as analyzed by flow cytometry. (<b>D</b>) The multidifferentiation potential of adherent cells obtained by culturing monocytes and platelets. Cells treated for osteogenic, chondrogenic, adipogenic, and endothelial induction for 1 week were analyzed by immunohistochemical staining for Cbfa1, Sox-9, or PPARγ (red) in combination with CD45 (green), or for eNOS or Tie-2 (red) in combination with DAPI (blue), and were observed under a fluorescence microscope. Representative results of three independent experiments are shown. Bars: 50 µm.</p

Screening of MOMC differentiation factor(s): 16 candidate cytokines, growth factors, and chemokines.

<p>CD14⁺ monocytes were cultured on fibronectin with or without serial concentrations of various soluble factors released by activated platelets and having a MW <30 kDa. The generation of spindle-shaped adherent cells was expressed as a proportion (%) of those generated by culturing monocytes alone. Results show the mean and SD of five independent experiments. *<i>P</i><0.05, compared with the culture without exogenous factors. IL, interleukin; EGF, epidermal growth factor; bFGF, basic fibroblast growth factor; TGFβ, transforming growth factor-β; PDGF, platelet-derived growth factor; GROα, growth-related oncogene-α; ENA78, epithelial cell-derived neutrophil-activating peptide 78; TARC, thymus and activation-regulated chemokine; SDF-1, stromal cell-derived factor-1; PF4, platelet factor 4; RANTES, regulated upon activation, normal T cell expressed and secreted; MIP-1α, macrophage inflammatory protein-1α; MCP-3, monocyte chemotactic protein-3; and NAP2, neutrophil-activating peptide 2.</p

The MOMC generation activity resides in the MW <30-kDa fraction of platelet-conditioned medium.

<p>CD14⁺ monocytes were cultured on fibronectin with or without unfractionated or fractionated platelet-conditioned medium prepared by stimulating platelets with thrombin. (<b>A</b>) Adherent cell morphology on culture day 7. Bars: 200 µm. (<b>B</b>) The generation of spindle-shaped adherent cells in the indicated cultures, expressed as a proportion (%) of those generated by culturing monocytes alone. Results show the mean and SD of three independent experiments.</p

MOMC precursors are enriched in CD14⁺CXCR4^high cells.

<p>(<b>A</b>) Dot-plot analysis of expression of CD14 and CXCR4 in CD14⁺ monocytes derived from 2 independent donors. Peripheral blood samples were anticoagulated with heparin or sodium citrate immediately after blood sampling. (<b>B</b>) Dot-plot analysis of CXCR4 expression in CD14⁺ cells, gated by CXCR4^high and CXCR4^low cells for fluorescent-activated cell sorting. Cell-surface CXCR4 on CXCR4^high and CXCR4^low cells is shown as histograms. (<b>C</b>) Morphology of adherent cells obtained from CD14⁺CD11a⁺, CD14⁺CXCR4^high, or CD14⁺CXCR4^low cells cultured on fibronectin with platelet-conditioned medium. Bars: 200 µm. (<b>D</b>) The generation of spindle-shaped adherent cells, expressed as a proportion (%) of those generated by culturing CD14⁺CD11a⁺ cells. Results show the mean and SD of three independent experiments. (<b>F</b>) Multidifferentiation potential of cells obtained by culturing CD14⁺CXCR4^high and CD14⁺CXCR4^low cells. Cells treated for osteogenic, chondrogenic, adipogenic, and endothelial induction for 1 week were analyzed by immunohistochemical staining for Cbfa1, Sox-9, or PPARγ (red) in combination with CD45 (green), or for eNOS or Tie-2 (red) in combination with DAPI (blue), and were observed under a fluorescence microscope. Representative results of 3 independent experiments are shown. Bars: 50 µm.</p