Knockdown of TET2 disturbs myeloid differentiation of cord blood CD34⁺ cells in vitro. Cord blood CD34⁺ cells were transduced by lentiviruses expressing GFP and either shRNA scramble or shRNA TET2. GFP⁺ cells were sorted 2 days after the end of the transduction procedure. (A) Sorted GFP⁺ cells were grown in MEM-α medium supplemented with SCF, FLT3-L, IL-3, and G-CSF. After 5-15 days of culture cells were harvested, DNA extracted, and spotted for 5-hmC dot blot assay. Membranes were stained with MB to assess equal spotting. Results are representative of 3 experiments, each performed at 3 time points of culture. (B) Sorted GFP⁺ CD34⁺CD38⁻ cells were seeded at one cell per well on a confluent layer of MS-5 cells in a specific medium supporting B/NK/GM differentiation. After 4-6 weeks of culture wells with significant cell growth were harvested and 450 clones from 3 independent experiments were tested for B, natural killer, and myeloid differentiations with the use of anti-CD19, anti-CD56, and anti-CD15 antibodies. Histograms show the percentages of lymphoid (CD15⁻ and CD56⁺ or CD19⁺), lympho/myeloid (CD15⁺CD19⁺ or CD15⁺CD56⁺ or CD15⁺CD19⁺CD56⁺), and myeloid (CD15⁺CD19⁻CD56⁻) clones. *P < .05, unpaired Student t test. Error bars indicate SEM. (C) Transduced CD34⁺ cells were seeded in methylcellulose in the presence of EPO, IL-3, SCF, and G-CSF. At day 14, colonies derived from shRNA scramble and shRNA TET2 BFU-E and CFU-G/GM were enumerated. Histograms show the number of colonies derived from transduced CD34⁺ cells (n = 5 independent experiments). *P < .05, unpaired Student t test. Error bars indicate SEM. (D) Photographs showing BFU-E– and CFU-G/GM–derived colonies. Colonies derived from shRNA TET2 BFU-E were smaller than those derived from shRNA scramble BFU-E. In contrast, CFU-G/GM–derived colonies appeared larger. (E) Transduced CD34⁺ cells were grown in serum-free medium supplemented with SCF, IL-3, and EPO. At day 14, CD34, CD36, and glycophorin-A (GPA) expression were analyzed by flow cytometry. Scattergrams from one representative of 4 experiments are shown. (F) Histograms represent the mean percentages of cells positive for GPA, CD36, CD34, and double-positive for GPA and CD36 antigens in the whole cell suspension after the 14-day culture. *P < .05, unpaired Student t test. Error bars indicate SEM (G) Transduced CD34⁺ cells were grown in a medium containing SCF, FLT3-L, IL-3, and G-CSF to monitor granulomonocytic differentiation in vitro. CD14 and CD15 immunophenotypic analysis at day 10 in 1 representative of 5 independent experiments is shown. (H) CD14, CD15, CD11b, and CD34 immunophenotypic analysis of cultured cells at day 10. Histograms represent the mean percentages of cells positive for each antigen in the whole cell suspension (5 independent experiments). *P < .05, unpaired Student t test. Error bars indicate SEM.