CD41⁺CMP possesses robust megakaryocyte-specific lineage potential in vitro. (A-B) Colony assays with conventional semisolid culture system (MethoCult supplemented with cytokines; n = 6). (A) Representative overall view of colonies derived from single BM-derived CD41⁺ CMPs and MEPs after 14 days on 35-mm dishes. CD41⁺ CMPs did not give rise to colonies under this culture condition (left). Images were created by using the image-stitching function of BZ-X700 (Keyence, Osaka, Japan) at low magnification (×4). (B) Single-cell colony assays. CFUs derived from 100 single HSPCs in Methocult (left) or in serum-free liquid culture (right) were enumerated (means ± standard deviations [SDs]; n = 6). CD41⁺ CMPs barely produced colonies in both assays. (C-D) Megakaryocyte lineage potential was evaluated using the specific culture system with collagen-based medium and cytokines, which is highly optimized for CFU-Meg (Megacult-C; n = 6). (C) Representative overall views of colonies derived from CD41⁺ CMPs and MEPs after 14 days using 4× (upper; scale bars, 250 µm) and 20× (lower; scale bars, 50 µm) objectives (BZ-X700). CFU-Megs derived from MEPs (pink arrow) were found far less frequently, and the size of each CFU-Meg was much smaller compared with those from CD41⁺ CMPs. Staining was performed according to the manufacturer's instructions. (D) Bar graphs represent numbers of burst-forming unit (BFU)/CFU-Meg colonies derived from 1000 cells. CD41⁺ CMPs gave rise exclusively to megakaryocyte colonies without any nonmegakaryocyte colonies. Both the number and size of megakaryocyte colonies produced from CD41⁺ CMPs far exceeded those of MEPs (means ± SDs; n = 6; megakaryocyte). (E) Cytospin preparation after 7-day culture of CD41⁺ CMPs in liquid medium (May-Giemsa staining). Progenies of CD41⁺ CMPs possessed distinct morphological features of megakaryocytes, such as polyploidy and bleb formation. The composite image from 3 representative views were shown. Images were captured by a BH2 microscope (Olympus, Tokyo, Japan) and Digital Sight DS-5M (Nikon, Tokyo, Japan) with 60× objective lens. E, erythroid; GEMM, mixed.