Stromal-dependent clonogenic progenitors are inhibited in Wif1-expressing cocultures. The effect of Wif1 expression on the formation of primary and secondary LTC-derived CAFCs was assayed in cocultures on AFT024, AFT024-control, and AFT024-Wif1 monolayers. Cultures were initiated with sorted HSCs from normal C57Bl6 mice. The primary limiting-dilution frequency of characteristic CAFC was determined at week 4 (primary). For secondary CAFCs, cells were maintained in bulk LTCs on each type of monolayer for 4 weeks and then harvested for replating onto fresh naive AFT024 monolayers in limiting dilution for an additional week to determine frequency (secondary). To normalize data among all the in vitro experiments, results are expressed as the percentage ± SD of AFT024-control (n = 11). Significance as determined by a 2-tailed t test of AFT024-Wif1 versus AFT024-control or AFT024, respectively, was P = .0004 or P = .03 for primary and P = 4.8 × 10−12 or P = 7.2 × 10−6 for secondary assay CAFC assay.
