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. 2008 Aug 4;205(8):1917–1927. doi: 10.1084/jem.20071482

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© 2008 Nishikii et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jem.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 1. — c-Kit⁺αIIb⁺ cell selection from day 6 EB determines the megakaryocytic lineage. (A) Protocol used in this study. (B) EB formation or OP9 co-culture was used for ESC differentiation from day 0 through day 6 in vitro. On the indicated days of culture, expression of Flk-1, integrin αIIb subunit (CD41), or CD31 was examined in both groups of cultured cells. The graph shows mean ± SEM from three independent experiments. (C) GPIbα and GFP expression in GPIbα-ESCs is regulated by the human GPIbα promoter. GFP is detectable in cultured cells that exhibit megakaryopoiesis (day 9) but not in progenitors (day 6). In the bottom box, cells with surface-expressed GFP also express human GPIbα. The cells on day 9 were fixed and stained to mark human GPIbα to confirm the colocalization with GFP expression at the membrane of the cells. Bars, 10 μm. (D) Representative dot plots for the expression of individual antigens on 10,000 living cells from day 6 EB. c-Kit⁺αIIb⁺ cells corresponded with Flk-1⁺ cells (not depicted) but not with VE-cadherin⁺ cells. And >96% of c-Kit⁺αIIb⁺ cells are found in the CD9⁺Sca-1⁻ fraction. (E) EB cells were sorted as shown in D, and 20,000 cells per well of a 6-well plate were seeded onto OP9 stroma cells in the presence of TPO. The left graph shows the number of generated GFP⁺ cells (mean ± SEM) per well of a 6-well plate on day 12 of culture. Representative images from day 12 culture dishes (phase or fluorescence microscopy [insets: bar, 50 μm] of four different fraction-derived cells at day 6; i, c-Kit⁺/αIIb⁺; ii, c-Kit⁻/αIIb⁺; iii, c-Kit⁺/αIIb⁻; iv, c-Kit⁻/αIIb⁻ [phase: bar, 50 μm]) and of Wright-Giemsa–stained cytospin preparation on day 12 derived from c-Kit⁺/αIIb⁺ fraction of day 6 were also shown. Bar, 50 μm. Results indicate that c-Kit⁺/αIIb⁺ fraction of day 6 (i) selectively yields megakaryocytes on day 12. Similar results have been obtained from three independent experiments. (F) Expression of genes related to the megakaryocytic lineage as determined by RT-PCR of cells from day 0, 6, or 8 cultures as indicated. (G) Wright-Giemsa–stained cytospin preparation of sorted c-Kit⁺αIIb⁺ cells from EB at day 6 is shown. Bar, 10 μm.