Comparative study of the hematopoietic supportive ability of primary fetal liver stromal cells expressing or not Pitx2 mRNAs. (A) Schematic representation of the functional elements of the TRIPΔU3-EF1α-Pitx2c-IRES-GFP vector. (B) Quantitative RT-PCR was performed on (Pitx2^–/–, Pitx2c), (Pitx2^–/–, GFP), and control (Pitx2^+/+, GFP) stromal cells. Histograms show the results expressed as the percentage of Pitx2 mRNA expression obtained in the control stromal cells. The Hprt housekeeping gene was used as internal standard to normalize the results for cDNA input. All samples were run in duplicate and gave identical results. (C) Results of 1 of 2 representative quantitative CAFC assays performed by coculture of Pitx2^+/+ CD45⁺ fetal liver hematopoietic cells using the different stromal cells described in panel A: Pitx2^–/–, GFP (solid lines, ▴); Pitx2^–/–, Pitx2c (broken lines, □); and Pitx2^+/+, GFP (solid lines, ▪). The frequency of wells negative for the presence of CAFCs is plotted against the number of CD45⁺ cells seeded. The CAFC 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 ).