Human CD34⁺CD1a⁻BMPRIA⁺ thymic precursors generate functional NK cells. Chimeric human-mouse FTOCs were performed with sorted human thymic CD34⁺CD1a⁻BMPRIA⁻ or CD34⁺CD1a⁻BMPRIA⁺ cells and cultured for 9 days. (A) Cells recovered from hybrid FTOCs were labeled with anti–human CD45, anti-CD3, anti-CD4, and anti-CD8 or anti–human CD45, anti-CD3, and anti-CD56 combined with anti-CD161, anti-CD94, or anti-NKp46 mAbs. Dot plots show the expression of CD4/CD8 and CD56/CD161 on gated human CD45⁺ cells from hybrid FTOCs. Histograms show the expression of CD94 and NKp46 in human CD3⁻CD56⁺ cells. Data are representative of 4 independent experiments wherein a pool of at least 3 thymic lobes per experiment was analyzed. In the experiment shown, human cell recoveries were 4 × 10⁴ and 1.4 × 10⁴ for FTOCs reconstituted with CD34⁺CD1a⁻BMPRIA⁻ or CD34⁺CD1a⁻BMPRIA⁺ cells, respectively. (B) Large numbers of NK cells develop in hybrid FTOCs reconstituted with CD34⁺CD1a⁻BMPRIA⁺ cell precursors. NK cells appear as round elements showing numerous electron-dense granules in the cytoplasm arranged close to the nucleus (arrow). Immature NK cells exhibit a well-developed Golgi complex (arrowhead) and moderately electron-dense secretory vesicles (asterisk). In contrast, thymocytes predominate in hybrid FTOCs reconstituted with CD34⁺CD1a⁻BMPRIA⁻ cell precursors. Scale bars represent 2 μm. (C) Equal numbers of total human cells were used to assay the cytolytic capacity and IFN-γ production by NK cells generated in hybrid FTOCs reconstituted with CD34⁺CD1a⁻BMPRIA⁺ or CD34⁺CD1a⁻BMPRIA⁻ precursors. The data represent the mean ± SD from 3 to 5 independent experiments (***P ≤ .001 by t test).