Ex vivo expansion of hematopoietic stem cell and subsequent differentiation into mature neutrophils remains a challenge. Here, we have developed a three-stage culture system to produce efficiently functional neutrophils derived from cord blood CD34+ cells. A procedure of ex vivo expansion and differentiation in a large-scale was developed in a modified IMDM basal medium supplemented with transferrin, insulin, fetal bovine serum, and some other nutrients with selected cytokine combination that contained stem cell factor (SCF), Flt-3 ligand (FL), granulocyte-colony stimulating factor(G-CSF), granulocyte macrophage colony-stimulating factor(GM-CSF) and thrombopoietin (TPO) in stage I (days 0~6); SCF, FL, G-CSF, interleukin 3 (IL-3), and GM-CSF in stage II (days 6~9); SCF, FL, and G-CSF in stage III (days 9~18), respectively. Enriched CD34+ cells were firstly cultured and expanded in 25-T flasks. After 6 day-culture, the cells were transferred to a 2-L bottle with 500 ml of medium in the bottle-turning device system. During the differentiation process, neutrophil marker CD66b was evaluated by flow cytometry. Ex vivo generated neutrophils or medium only were incubated with E.coli overnight for its bacteria killing assay, and then the E.coli colony-forming units were counted separately. Matured neutrophils or vehicle were transplanted into NOD/SCID mice intravenously for chemotactic activity in vivo. The cells that accumulated in the pouch with chemoattractant were collected and subjected to flow cytometric analysis for human CD66b antigen. The ex vivo generated neutrophils/progenitors were then injected into sub-lethal irradiated NOD/SCID mice to monitor the viability and maturation in vivo. After the three-stage culture, proliferation fold of total cell reached 30013 ± 286.5 with 60.2% ± 2.4% for CD66b+ neutrophils. The calculated yield of matured neutrophils from each CD34+ cell was ranged from 1.8 × 104 to 1.87 × 104 for 18-day culture. There was no E.coli colony formed after incubation with neutrophils in bacteria killing assay, indicating that the generated neutrophils was functional. For in vivo chemotaxis assay, the neutrophils collected from 18-day culture were injected into mice and detected at 1.08% ±0.16% for human CD66b+ cells and no any CD66b+ cell observed for negative control group. In addition, the CD66b+ cells were extended for 4 days from a 15-day cultured neutrophil group in mouse peripheral blood (PB), while only for 2 days from a freshly isolated human PB neutrophils post injection, indicating that the ex vivo generated neutrophils/progenitors could further matured in vivo. Taken together, we have established a pilot-scale culture system to produce functional human neutrophils ex vivo. Considering that one neutrophil transfusion unit (100ml) contains 2×1010 cells, the CD34+ cells from one CB unit (80 ml) would generate 1.6×1011 neutrophils, which are equivalent to 8 unit doses of neutrophils in the clinical application.

Citation Format: Zhenwang Jie, Yu Zhang, Chen Wang, Bin Shen, Xin Guan, Zhihua Ren, Xinxin Ding, Wei Dai, Yongping Jiang. Large-scale ex vivo generation of human neutrophils from cord blood CD34+ cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2701. doi:10.1158/1538-7445.AM2017-2701