Abstract: Objective To detect the efficiency and function of NK cell differentiation from human umbilical cord hematopoietic stem cells (HSCs) in vitro. Methods CD34+ hematopoietic stem cells were isolated from human umbilical cord blood, and inoculated into SCGM medium containing 20 μg/L FMS like tyrosine kinase 3 ligand (Flt- 3L), stem cell factor (SCF), interleukin (IL) -7, IL-15 and IL-21. And CD34+ HSCs were differentiated into NK cells in directional inducing. The growth state of cells was observed. The expressions of CD56, NKG2D, NKp46, CD3, CD19 and CD34 were detected by flow cytometry in the differentiation of 7, 14, 21 and 28 d. In the differentiation of 21 d and 28 d, the differentiation cells were used as effector cells, and K562 cells as target cells. The ratios of effector cells and target cells were 8∶1, 4∶1, 2∶1 and 1∶1. The killing activity of the differentiated cells was detected by lactate dehydrogenase (LDH) cell toxicity assay and 7AAD/CFSE labeling method. Results CD34+ HSCs derived from human umbilical cord blood can proliferate in vitro under appropriate condition. There were no significant differences in the expression of CD3 and CD19 between different differentiation stages (7, 14, 21 and 28 d, P > 0.05). The expressions of CD56, NKG2D and NKp46 were significantly different (P < 0.05), and the ultimate expression amount was (72.57±1.60)%, (32.83±1.29)% and (29.53±2.40)%. The expression of CD34 decreased gradually, and the lowest was (12.13 ± 2.01)%. The maximum killing activity detected by LDH cell toxicity assay and 7AAD/CFSE labeling method reached(49.91±2.76)% and (40.87±1.12)%.The killing activity of NK cells was decreased in the order of 8∶1, 4∶1, 2∶1 and 1∶1 groups (P < 0.05). There was no significant difference in the killing activity between NK cells of 28 d and 21 d. Conclusion Human umbilical cord hematopoietic stem cells can differentiate into NK cells under appropriate conditions in vitro, and the NK cells induced from differentiation are with killing activity.

Key words: fetal blood, hematopoietic stem cells, killer cells, natural, immunophenotyping, cytotoxicity tests, immunologic, CD34+ hematopoietic stem cells, in vitro differentiation, cytotoxicity