Early differentiation of CD105-deficient

(eng^−/−) ES cells in vitro. (A) Genotyping ofeng^−/− andeng^+/− ES cells was determined by multiplex PCR. DNA was isolated from ES cell clones, and gene-specific primers were used to amplify wild-type (300 bp) or targeted (476 bp) exon 1 and wild-type exon 2 (383 bp). (B) Flow cytometric analysis of Flk1 cell surface expression from day 6eng^−/− andeng^+/+ ES-cell/OP9 cocultures is shown. (C) Expression of tie-2, brachyury, andβ-actin transcripts was analyzed by RT-PCR from day-6 and day-9 ES-cell/OP9 cocultures. The cDNAs for day 6 analysis were prepared directly from sorted Flk1⁺ cells, and cDNAs for day-9 analysis were prepared after sorted Flk1⁺ cells were cocultured for an additional 3 days. Control RT-PCR reactions were performed with the use of RNA from OP9 cells and E13 fetal liver (FL). (D) Flk1⁺ cells were sorted from day-6 cocultures, reseeded onto OP9 cells, and analyzed by flow cytometry for the surface expression of CD105 and CD45 at day 9. CD105 expression is not detected in eng^−/− cocultures. The total number of cells obtained from each coculture is indicated in parentheses.