Effects of overexpression of <i>Bmi1</i> on HSCs <i>in vitro.</i>

<p>(A) Colony formation by HSCs isolated from <i>Tie2-Cre</i> (Control) and <i>Tie2-Cre</i>;<i>R26Stop^FLBmi1</i> (Bmi1) mice. Single CD34^-LSK cells were sorted into 96-well microtiter plates containing the SF-O3 medium supplemented with 10% FBS and multiple cytokines (10 ng/ml SCF, 10 ng/ml TPO, 10 ng/ml IL-3, and 3 u/ml EPO) and allowed to form colonies. At day 14 of culture, the colonies were counted and individually collected for morphological examination. Absolute numbers of LPP and HPP-CFCs which gave rise to colonies with a diameter less and greater than 1 mm, respectively are shown as the mean ± S.D. for triplicate cultures (left panel). Absolute numbers of each colony types were defined by the composition of colonies (right panel). Colonies were recovered and examined by microscopy to determine colony types. Composition of colonies is depicted as n, neutrophils; m, macrophages; E, erythroblasts; and M, megakaryocytes. (B) Colony formation by HSCs cultured for 10 days. CD34^-LSK cells from <i>Tie2-Cre</i> (Control) and <i>Tie2-Cre;R26Stop^FLBmi1</i> (Bmi1) mice were cultured in the SF-O3 serum-free medium supplemented with 50 ng/ml of SCF and TPO. At day 10 of culture, the cells were counted (left panel) and plated in methylcellulose medium to allow formation of colonies in the presence of 20 ng/ml SCF, 20 ng/ml TPO, 20 ng/ml IL-3, and 3 u/ml EPO. Absolute numbers of LPP and HPP-CFCs (middle panel) are shown as the mean ± S.D. for triplicate cultures. Absolute numbers of each colony type are shown in the right panel. (C) Flow cytometric analysis of CD34^-LSK HSCs at day14 of culture. Representative flow cytometric profiles of LSK cells in cultures of CD34^-LSK HSCs from <i>Tie2-Cre</i> (Control) and <i>Tie2-Cre;R26Stop^FLBmi1</i> (Bmi1) mice are depicted. The proportion of Lin^- and LSK cells in total cells are indicated. (D) Quantitative RT-PCR analysis of the expression of <i>p19^Arf,</i> and <i>Bmi1</i> in <i>Tie2-Cre</i> (Control) and <i>Tie2-Cre;R26Stop^FLBmi1</i> (Bmi1) LSK cells. LSK cells were purified by cell sorting from CD34^-LSK cultures in (C) at day 14 of culture. Each value was normalized to <i>Hprt1</i> expression and the expression level of each gene in control cells was arbitrarily set to 1. Data are shown as the mean ± S.D. for triplicate analyses. * <i>p</i><0.05, **<i>p</i><0.01.