In vivo Opn is involved in the regulation of HSC proliferation. (A) BM was analyzed for progenitor cell content using an in vitro colony-forming assay 3 days after 5FU cytotoxic challenge. There was a significant reduction in the number of cells with HPP-CFC in the presence of 4 stimulatory growth factors from an Opn^–/– microenvironment (▪) compared with wt controls (▦). Data are the mean ± SEM of triplicate plates from 3 individual mice in 2 independent experiments. *P < .05. Furthermore, HSCs (LSK cells) cycle significantly faster in the absence of Opn in the hematopoietic microenvironment (B). Compared with HSCs isolated from a wt microenvironment (▦), significantly more HSCs isolated from an Opn^–/– microenvironment (▪) had undergone cell cycle and incorporated BrdU after 4 weeks of continual oral administration. Data are the mean ± SEM of 3 individual mice from 2 independent experiments. *P < .05.