Identification of a Novel Stromal Cell Membrane Protein that Binds to Megakaryocytes.

In bone marrow, hematopoiesis-supporting cells or stromal cells govern the growth, survival and differentiation of hematopoietic stem cells. We previously reported the method to produce the functional platelets from murine embryonic stem (ES) cells in vitro, using the coculture system with OP9 stromal cells. Also in this system, the cell contact between ES-derived cells and stromal cells is essential for megakaryocyte differentiation, and even after growth factors including TPO were supplied, ES-derived megakaryocytes cannot be maintained without stromal cells. In this study, we searched membrane proteins that were expressed in the stromal cells and were involved in the cell contact with megakaryocytes. We employed the retrovirus-based signal sequence trap (SST) method to identify such membrane proteins from murine stromal cells. 117 signal sequence-containing clones were identified, and the clones were further screened by the binding capacity to megakaryocytic cell lines. We finally identified a novel protein that contains two Ig domains and belongs to the transmembrane-type immunoglobulin superfamily. RT-PCR analysis showed that this protein is expressed in bone marrow stromal cell lines including OP9, and vascular endothelial cell lines, whereas no expression was detected in any hematopoietic cell lines. We next created a cDNA construct in which the extracellular domain of the protein was linked to IgG Fc domain, and the construct was transfected into 293 cells. The fusion protein was purified from the culture supernatant. By flow cytometer, the purified recombinant protein bound to megakaryocytic cell lines and CD41-positive bone marrow megakaryocytes. The binding to murine Sca-1-positive and human CD34-positive immature cells was also detected. The binding was dependent on calcium, but was not inhibited by RGD peptide. The effect on in vitro differentiation system of ES cells was further tested. The addition of the fusion protein showed no influences on the maintenance of undifferentiated state of ES cells. However, after the differentiation induction, the protein facilitated morphological change of the ES cells. Further analysis will contribute to understanding of the mechanisms by which the stromal cell contact regulates megakaryocyte differentiation and platelet production.