Low levels of HOXA10 expand stem cells in vitro, whereas high levels block expansion. LSK cells from rtTA-HA10 mice and WT mice were purified and cultured for 13 days in TPO, FLT-3, and SCF with 6 different concentrations of doxycycline. (A) HOXA10 mRNA was detected using Taq-man PCR, and RI was calculated by comparing results using the formula 2^{−(CtHOXA10 − CtHPRT)}. (B) The diagram shows fold expansion of total cells after 13 days of liquid culture with SCF, FLT-3, and TPO in 6 different concentrations of doxycycline. Black bars represents inducible HOXA10 LSK cells and gray bars represent WT LSK cells (n = 7) (C) LSK cells were cultured as single cells in 3 different concentration of doxycycline to measure recruitment into proliferation. Clones were scored at day 12 as high proliferative clones (> 50 cells) or as low proliferative clones (< 50 cells); n = 3. (D) Two hundred freshly sorted LSK cells or the expansion equivalent (Ly^5.2/5.2) after 13 days of culture from panel A were transplanted with 200 000 unfractionated (Ly^5.1/5.1) competitor bone marrow cells into lethally irradiated recipients. Peripheral blood cells were analyzed by flow cytometry 16 to 20 weeks after bone marrow transplantation to evaluate reconstitution of transplanted cells. Shown here is the reconstitution in comparison to uninduced HOXA10 LSK cells cultured for 13 days. Black bars represent inducible HOXA10 LSK cells, and gray bars represent WT LSK cells. Nd indicates not done (n = 7); *P < .05. Data represent mean ± SEM.