BM FRC-like cells positively regulate megakaryocyte expansion via the CLEC-2/PDPN interaction. (A) Scheme of coculture of megakaryocyte progenitors within BM stromal layer containing BM FRC-like cells. (B) Representative ICC image of coculture of CLEC-2 WT megakaryocytes (top) or cKO megakaryocytes (bottom) with BM stromal cells. BM FRC-like cells were detected as PDPN⁺ cells (green). Megakaryocyte lineages were detected as CD41⁺ cells (red). (C-D) Expansion level of CLEC-2 cKO megakaryocytes was reduced as compared with that of CLEC-2 WT megakaryocytes in coculture with BM FRC-like cells. (E-H) Treatment with a PDPN blocking antibody (anti-PDPN, clone: 8F11, 15 μg/mL) suppressed megakaryocyte expansion of CLEC-2 WT (E-F), but not cKO cells (G-H) in coculture with BM FRC-like cells. Representative ICC images of CD41⁺ megakaryocytes in coculture with BM FRC-like cells (C,E,G), and quantitative absolute numbers of CD41⁺ megakaryocytes in fields, n = 4 per group (D,F,H). Scale bar, 100 μm for panels B, C, E, and G. The error bars represent SEMs. *P < .05, **P < .01. MK, megakaryocyte; N.S., a nonsignificant difference.