MN1 overexpression inhibits M-CSF or vitamin D₃-induced differentiation and enhances growth of CD34⁺ primary human hematopoietic cells. (A) Real-time RT-PCR analysis of endogenous MN1 expression in BM-derived human CD34⁺ cells (CD34-BM), peripheral blood monocytes, and granulocytes, normalized for endogenous HPRT expression (mean ± SEM of triplicates). ***P < .001. (B) Primary human CD34⁺ cells from peripheral blood of G-CSF mobilized healthy donors maintained in cultures containing progenitor, granulocyte, or monocyte cytokine cocktails, respectively. Marker analysis was performed using FACS of day 5 CD34⁺ stem cell/progenitors (progenitor cytokines) cultures and day 14 in vitro differentiated granulocytes (granulocyte cytokines; CD15) or monocytes (monocyte cytokines; CD14) cultures. Endogenous MN1 expression in these cells was analyzed using real-time RT-PCR. The graph shows MN1 expression normalized for endogenous HPRT expression (mean ± SEM of duplicates). *P = .01; **P = .008. (C) CD34⁺ cells were transduced with MIG (mock) or MIG-MN1 (MN1) retroviruses, and unsorted cells were cultured with monocyte-cytokines (M-CSF) in the absence or presence of vitamin D₃ (M-CSF + vitamin D₃). GFP⁺ and GFP⁻ cells in mock or MN1 cultures were analyzed for CD14 expression by FACS at day 14 (mean ± SEM of duplicates). **P < .002. In an independent experiment, GFP⁺ cells from M-CSF cultures were FACS-sorted and cytospin preparations were stained with May-Grünwald-Giemsa (top panel; original magnification ×600). (D) Human CD34⁺ cells were expanded 1 day and transduced 3 times in 2 consecutive days in media containing progenitor cytokines. One day after the last transduction, CD34⁺/GFP⁺ mock or MN1 cells were sorted into 96-well plates containing 100 μL of media with progenitor cytokines (2000 cells/well; triplicate). Cell proliferation was determined at indicated times using the MTT assay (mean ± SEM). **P < .007. (E) In an independent experiment, the percentage of GFP⁺ cells in unsorted CD34-mock and CD34-MN1 cultures were analyzed at 3, 12, and 30 days after transduction. Data represent one of 2 independent experiments. (F) Human CD34⁺ cells were transduced and sorted as in panel D, and 500 or 1000 cells were plated in 1 mL of methylcellulose medium in 35-mm dishes in duplicate. Eight days later, colonies were counted and GFP⁺ cells were replated following the same procedure (mean ± SEM). (G) GFP⁺ CD34-mock and MN1 cells were sorted 3 days after transduction. After 3-day expansion, cells were seeded at the same density (day 0; 1.2 × 10⁵ cells/well in 1 mL media, 24-well plate) in medium supplemented with progenitor cytokines and followed for 7 days. Media were refreshed every 3 days, and survival (Annexin V) and cell-cycle analysis (H) was performed in duplicate using FACS at day 7. Panel H represents the mean of duplicate ± SEM.