CD56⁺CD94^–CD117high cells are present in UCB. (A) Small numbers of CD56⁺CD94^–CD117^high cells can be detected in UCB. FACS staining for CD117 and CD94 (after gating on CD56) of UCB-derived NK cells (left). FACS analysis after 1 week of culture of UCBderived CD56⁺ cells with either cytokines alone (IL-7, IL-15, SCF, and FLT3L) or with cytokines and EL08.1D2 (middle and right, respectively). Results are representative of 3 or more experiments. (B) Sorting strategy to isolate CD56⁺CD94^–CD117^high cells from UCB. The shown population was first gated on CD56⁺ cells (not shown). (C) Average fold expansion (±SD) of freshly isolated UCB CD56⁺CD94^–CD117^high cells after culture for 1 week either in the presence or absence of the stromal cell line (EL08.1D2). (D) Phenotype of FACSsorted UCB CD56⁺CD94^–CD117^high cells after 1 week of culture either in the presence (left panel) or absence (right panel) of the stromal cell line (EL08.1D2). (E) Expression of granzyme B and perforin in cultured UCB cells after transition to the CD56⁺CD94⁺CD117^low/– phenotype. CD56⁺CD94^–CD117^high cells from UCB were FACS sorted and cultured on the stromal cell line for 1 week. Expression of granzyme B and perforin in either bulk (left), CD56⁺CD94^–CD117^high cells (middle), or CD56⁺CD94⁺CD117^low/– cells (right) was determined by FACS. Panels B to E are the representative results of 4 separate donors.