Platelet-derived stromal cell-derived factor-1 is required for the transformation of circulating monocytes into multipotential cells.

BACKGROUND: We previously described a primitive cell population derived from human circulating CD14(+) monocytes, named monocyte-derived multipotential cells (MOMCs), which are capable of differentiating into mesenchymal and endothelial lineages. To generate MOMCs in vitro, monocytes are required to bind to fibronectin and be exposed to soluble factor(s) derived from circulating CD14(-) cells. The present study was conducted to identify factors that induce MOMC differentiation. METHODS: We cultured CD14(+) monocytes on fibronectin in the presence or absence of platelets, CD14(-) peripheral blood mononuclear cells, platelet-conditioned medium, or candidate MOMC differentiation factors. The transformation of monocytes into MOMCs was assessed by the presence of spindle-shaped adherent cells, CD34 expression, and the potential to differentiate in vitro into mesenchymal and endothelial lineages. RESULTS: The presence of platelets or platelet-conditioned medium was required to generate MOMCs from monocytes. A screening of candidate platelet-derived soluble factors identified stromal cell-derived factor (SDF)-1 as a requirement for generating MOMCs. Blocking an interaction between SDF-1 and its receptor CXCR4 inhibited MOMC generation, further confirming SDF-1's critical role in this process. Finally, circulating MOMC precursors were found to reside in the CD14(+)CXCR4(high) cell population. CONCLUSION: The interaction of SDF-1 with CXCR4 is essential for the transformation of circulating monocytes into MOMCs

Fibronectin Binding Is Required for Acquisition of Mesenchymal/Endothelial Differentiation Potential in Human Circulating Monocytes

We previously reported monocyte-derived multipotential cells (MOMCs), which include progenitors capable of differentiating into a variety of mesenchymal cells and endothelial cells. In vitro generation of MOMCs from circulating CD14+ monocytes requires their binding to extracellular matrix (ECM) protein and exposure to soluble factor(s) derived from circulating CD14- cells. Here, we investigated the molecular factors involved in MOMC generation by examining the binding of monocytes to ECM proteins. We found that MOMCs were obtained on the fibronectin, but not on type I collagen, laminin, or poly-L-lysine. MOMC generation was followed by changes in the expression profiles of transcription factors and was completely inhibited by either anti-α5 integrin antibody or a synthetic peptide that competed with the RGD domain for the β1-integrin binding site. These results indicate that acquisition of the multidifferentiation potential by circulating monocytes depends on their binding to the RGD domain of fibronectin via cell-surface α5β1 integrin

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

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

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

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

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