GFP-expressing megakaryocytes and platelets derived from ES cells. In panels A and B, megakaryocytes and platelets expressing GFP were produced from ES cells transfected with pCX-EGFP. (A) Paired photographs of phase-contrast microscopy (left column) and fluorescence microscopy (right column) are shown (original magnification, × 400). Proplatelet-bearing megakaryocytes are strongly GFP⁺ up to the top of the proplatelets (top and middle rows, day 14). GFP⁺ platelets are observed on or under the stromal layer, although platelets are hardly recognized under the phase-contrast microscopy (bottom row, day 15). (B) Platelets in the culture supernatants (day 15) were stained with anti-CD41 antibody followed by PE-conjugated secondary antibody, and 2-color flow cytometric analysis (PE and GFP) was performed. Platelets derived from GFP-transfected ES cells are both GFP⁺ and anti-CD41⁺ (right panel). Results of the platelets stained with a control antibody (middle), and of the platelets derived from untransfected ES cells (left) are shown. (C) Platelets on day 15 derived from ES cells transfected with GFP linked to megakaryocyte-specific PF4 promoter (pBK-PF4-GFP) are also GFP⁺ by flow cytometry (gray histogram). Open histogram represents the fluorescence of platelets derived from untransfected ES cells.