APLN augments hemangioblast colony formation. (A) Augmentation of day 3 hemangioblast colony formation in methylcellulose cultures supplemented with APLN. HES3 hESCs were differentiated as EBs for 3 days in BVS alone or in BVS with APLN added at day 2 (BVS/APLN). Dissociated cells were cultured further in methylcellulose (MC) supplemented with VEGF, SCF, and EPO (VSE) alone or in combination with APLN (VSE/APLN) as indicated. Error bars represent SEM for n = 4 independent experiments; *P < .01 for pairwise comparisons between BVS/APLN VSE/APLN and all other conditions. (B) Dark field images of hematopoietic colonies demonstrating the effect of APLN addition to both the initial mesoderm induction phase (EB) and the methylcellulose (MC) cultures. Original magnification, ×50. (C-D) Bright field images of replated hemangioblast colony demonstrating the presence of both hematopoietic cells and adherent cells (arrows). (E-F) Bright field and immunofluorescence overlay images demonstrating the presence of CD34⁺ (arrows) as well as CD34⁻ adherent cells (asterisks). (G) Immunofluorescence overlay demonstrating that adherent cells either expressed SMA or took up DiI-Ac LDL (arrows). (H) Flow cytometric analysis of nonadherent and adherent cell fractions from pooled hemangioblast colonies differentiated for 11 days. Samples were stained with Abs to GLYCOPHORIN A (GLY), CD45, and CD34. The percentage of cells falling into each quadrant is indicated in the top right of each plot. Nonadherent cells predominantly comprised GLYCOPHORIN A⁺ CD45⁻ erythroid cells with a small percentage of CD34⁻CD45⁺ myeloid cells whereas over 50% of the adherent cells were CD34⁺CD45⁻ cells, many of which are likely to be endothelial cells. (G) Approximately 40% of the adherent cells were CD34⁻CD45⁻ cells, consistent with the proportion of SMA⁺ smooth muscle cells observed by immunofluorescence (see also supplemental Figure 11). Original magnification: (C) ×50; (D) ×100; (E-G) ×200.