Identification of Molecular Factors Required for Transdifferentiation of Human Circulating Monocytes into Multipotential Cells.

Recently, we have identified a primitive cell population termed monocyte-derived multipotential cells (MOMCs), which have a fibroblast-like morphology and a unique phenotype positive for CD14, CD45, CD34, and type I collagen. MOMCs are derived from in vitro culture of circulating CD14⁺ monocytes, and contain progenitors capable of differentiating into a variety of mesenchymal cells, neuron, and endothelium. Thus, human MOMCs are attractive candidates for an autologous transplantable cell source for therapeutic tissue regeneration. In vitro induction of MOMCs from circulating CD14⁺ monocytes requires their binding to matrix proteins such as fibronectin, and exposure to soluble factors derived from peripheral blood CD14⁻ cells, but detailed processes remain unknown. Here, we investigated molecular factors involved in MOMC induction using human peripheral blood monocyte cultures. Induction of MOMCs was defined as having all of features, including fibroblastic morphology, CD34⁺ phenotype, and potentials of differentiating into multiple cell lineages. First, we set up MOMC cultures on fibronectin, type I collagen, laminin, or poly-L-lysine, and found that MOMCs were efficiently obtained exclusively in the presence of fibronectin. Since fibronectin is a ligand for β1 integrins, we examined effects of blockade of these interactions on MOMC generation. Induction of MOMCs was completely inhibited by anti-α₅-integrin monoclonal antibody, but not by anti-α₄-integrin antibody. In addition, a synthetic peptide that competed with the RGD domain of fibronectin suppressed generation of MOMC, but a CS-1 domain peptide had no effect, indicating that binding of α₅β₁ integrin to the RGD domain of fibronectin is required for monocytes to acquire a multipotential property. Next, highly enriched monocytes were cultured on fibronectin with various peripheral blood cell subsets to identify cells producing soluble factors required for MOMC generation. As a result, MOMCs were obtained exclusively in the presence of platelets. MOMC induction was also observed when conditioned medium prepared by stimulating platelets with various agonists, such as thrombin and ADP, was used instead of whole platelets. When platelet-derived condition medium was fractionated based on the molecular weight (MW), MOMC induction activity was retained in the fraction of MW < 30,000. Then, we selected a list of 16 candidate factors that are released from activated platelets and < 30,000 in MW, and screened them using two different strategies: evaluation of MOMC induction efficiency in monocyte cultures by adding individual candidate factors; and FACS sorting of circulating monocytes into cells with high and low expressions of receptor for individual candidate factors, followed by evaluation of MOMC induction. As a result, SDF-1was selected as a sole factor with capacity to promote generation of MOMCs. Moreover, MOMC generation was significantly more prominent in cultures with monocytes with high expression of CXCR4, a receptor for SDF-1, than in cultures of monocytes with low CXCR4 expression. In summary, circulating monocytes are able to transdifferentiate into multipotential cells through the fibronectin-α₅β₁ integrin and SDF-1-CXCR4 interactions. This information is helpful in establishing an optimal MOMC culture condition used in cell transplantation for tissue regeneration.