FIGURE 2.

Knockdown of VentX promotes expansion of human CD34⁺ cells ex vivo. A, real-time PCR showing the knockdown efficiency of VentX shRNA (shVentX) lentivirus in CD34⁺ cells. shGFP, GFP shRNA. B, VentX knockdown promotes human bone marrow CD34⁺ cell growth in liquid culture. Human CD34⁺ cells were transduced with either non-effective GFP shRNA or VentX shRNA lentivirus. Positively transduced cells (2 × 10⁴) were sorted by GFP and grown in stroma-free liquid cultures using a mixture of cytokines as described under “Experimental Procedures.” The cells were counted weekly for 4 weeks, and the mean ± S.D. of three independent experiments is shown. The number of cells at Day 0 represents the number of total transduced cells that were plated. The statistically significant difference (p < 0.05) is marked with asterisks. C–F, knockdown of VentX in CD34⁺ cells increases the colony formation in semisolid culture. Positively transduced CD34⁺ cells as described above were plated in triplicate in methylcellulose-based medium and allowed to form colonies for 2 weeks. C and D show the colony numbers of BFU-E/CFU-E and CFU-GM, respectively. E shows total progenitor cells from 2 × 10⁴ CD34⁺ cells after culturing for 2 weeks. The significant difference between the two groups (three independent experiments for each group) was determined by Student's t test (p < 0.05). F, representative images of CFC assays showing the erythroid colonies in GFP shRNA- or VentX shRNA-transduced CD34⁺ cells.