Comparative study of the hematopoietic supportive ability of Pitx2–/– and Pitx2+/+ fetal liver stromal cells. (A) Schematic diagram illustrating CAFC assays using cocultures between preestablished fetal liver stromal cells and CD45+ hematopoietic fetal liver cells and microscopic appearance of CAFCs when they are enumerated after 5 weeks of coculture. Phase-contrast image was obtained using a Nikon Eclipse TE2000S inverted microscope (Nikon, Champigny sur Marne, France) and a Nikon Plan Fluor 20×/0.50 numeric aperture objective, for a total magnification of 200×. Image was captured using a Nikon DXM1200F digital camera and Nikon EclipseNet software. (B) Results of 1 of 3 representative (Table 2, experiment 3) quantitative CAFC experiments generated using Pitx2–/– (solid lines) and Pitx2+/+ (broken lines) fetal liver stromal cells after coculture with Pitx2+/+ (▴) and Pitx2–/– (□) CD45+ hematopoietic cells, respectively. 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) is defined as the inverse of the number of seeded cells that corresponds to 37% negative wells (Poisson statistics27 ).
