Development of myeloid, NK, and B cells from hES cell–derived CD34⁺ cells. CD34⁺ cells were cultured on MS-5 stromal cells as described in “Materials and methods.” (A) CD34⁺ cells gave rise to phase-dark cobblestone-shaped colonies underneath the stroma starting from day 7 of coculture. Image captured with an inverted Leica DM1RB microscope (Leica Microsystems), using 20 × objective with numerical aperture 0.3 and acquired through Spot RT camera and Spot software (Diagnostic Instruments). (B-F) Phenotype of the cells generated after 21 days of CD34⁺/MS-5 coculture. Flow cytometric analysis demonstrated the presence of CD14⁺HLA-DR+ macrophages (B) and CD66b⁺CD10⁺ mature granulocytes (C) within the CD45⁺ gated population. In addition, CD56⁺CD45⁺ NK cells (D) and CD19⁺CD45⁺ B-cell precursors (E) were evident. Numbers in the upper right corner indicate percentages of positive cells in the corresponding quadrant (mean ± SD of 4 experiments; H1 = 2, H9 = 2). (F) IL-15–induced expression of perforin in CD56⁺ cells. CD34⁺ cells were cultured on MS-5 cells without (left panel) or with IL-15 (right panel). Cells were stained with CD56-PE and CD45-APC mAbs followed by permeabilization and staining with perforin-FITC mAbs. Dot plots represent CD45⁺ gated cells. (G) Analysis of gene expression in isolated CD34⁺ cells and cells after 21 days of CD34⁺/MS-5 coculture by RT-PCR. Positive controls are as follows: human bone marrow (GATA-3); peripheral blood lymphocytes (CD3ϵ, CD3δ, CD3γ, CD3ξ); thymus (pre-Tα); fetal liver (mb-1, VpreB). Transcripts of the studied genes were not amplified from MS-5 cells alone. M indicates DNA markers (100-bp ladder).