Comparative study of the human hematopoietic supportive ability of control MS5 cells and MS5 cells transduced with vectors expressing RNAi directed against Pitx2 mRNA. (A) Schematic representation of the TRIP ΔU3-H1Pitx2RNAi vector. (B) Quantitative RT-PCR was performed using RNA isolated from control MS5 or MS5 GFP cells and MS5 Pitx2RNAi cells in 3 independent transduction experiments. Histograms show the results expressed as the percentage of Pitx2 mRNA expression in control cells, MS5 (left), and MS5 GFP (right). The Hprt housekeeping gene was used as internal standard to normalize cDNA input. All samples were run in duplicate and gave identical results. (C) Results of representative quantitative LTC-IC assays performed by coculture of human CD34⁺ cord blood cells using the different MS5 cells described in panel B. The frequency of wells negative for the presence of LTC-ICs is plotted against the number of CD34⁺ cells seeded, and the LTC-IC frequency, indicated in boxes for each condition, is defined as the inverse of the number of seeded cells that corresponds to 37% negative wells (Poisson statistics²⁷ ).