© 2006 Nature Publishing Group Pten dependence distinguishes haematopoietic stem cells from leukaemia-initiating cells O ¨ mer H. Yilmaz 1 , Riccardo Valdez 2 , Brian K. Theisen 2 , Wei Guo 3 , David O. Ferguson 2 , Hong Wu 3 & Sean J. Morrison 1 Recent advances have highlighted extensive phenotypic and functional similarities between normal stem cells and cancer stem cells. This raises the question of whether disease therapies can be developed that eliminate cancer stem cells without eliminating normal stem cells. Here we address this issue by conditionally deleting the Pten tumour suppressor gene in adult haematopoietic cells. This led to myeloproliferative disease within days and transplantable leukaemias within weeks. Pten deletion also promoted haematopoietic stem cell (HSC) proliferation. However, this led to HSC depletion via a cell-autonomous mechanism, preventing these cells from stably reconstituting irradiated mice. In contrast to leukaemia-initiating cells, HSCs were therefore unable to maintain themselves without Pten. These effects were mostly mediated by mTOR as they were inhibited by rapamycin. Rapamycin not only depleted leukaemia-initiating cells but also restored normal HSC function. Mechanistic differences between normal stem cells and cancer stem cells can thus be targeted to deplete cancer stem cells without damaging normal stem cells. Cancer stem cells have notable phenotypic and mechanistic simi- larities to normal stem cells in the same tissues 1–4 . Acute myeloid leukaemia (AML) is sustained by leukaemic stem cells that are also called leukaemia-initiating cells because they are defined by their ability to transfer disease on transplantation into irradiated mice 5–7 . Leukaemia-initiating cells express markers similar to normal HSCs 5,6 and depend on similar mechanisms to self-renew 8,9 . Brain cancer stem cells also express markers of normal neural stem cells and depend on similar pathways for their proliferation 4,10 . The Hedgehog, Wnt and Notch pathways that often promote cancer cell proliferation also promote normal stem cell self-renewal 1,2,11,12 . Conversely, tumour suppressors that inhibit cancer cell proliferation —such as p53, p16 INK4a and p19 ARF —also inhibit stem cell self-renewal 11,13,14 . Whether cancer stem cells arise from normal stem cells or other cells, their similarity to normal stem cells indicates that they inherit or acquire stem cell properties. This raises the question of whether it will be possible to identify therapies that eliminate cancer stem cells without eliminating normal stem cells in the same tissues. We have addressed this issue by examining the effect of Pten deletion on leukaemia-initiating cells and normal HSCs. PTEN (for phospha- tase and tensin homologue) is a phosphatase that negatively regulates signalling through the phosphatidylinositol-3-OH kinase (PI(3)K) pathway, inhibiting proliferation and survival 15,16 . Pten is commonly deleted or otherwise inactivated in diverse cancers 17 , including haema- topoietic malignancies 18–21 . Here we report that whereas Pten deletion causes the generation of transplantable leukaemia-initiating cells, it also causes the depletion of normal HSCs, thus identifying a mechanistic difference between the maintenance of normal stem cells and cancer stem cells. Pten deletion leads to leukaemogenesis Pten was conditionally deleted from 6-to-8-week-old Pten fl/fl ; Mx-1- Cre mice by administering seven doses of polyinosine-polycytidine (pIpC) over 14 days to induce Cre expression 22,23 . After 14 days, Pten seemed to be completely deleted from HSCs and other haemato- poietic cells (Supplementary Fig. 1). We analysed Pten fl/fl ; Mx-1-Cre mice, as well as Pten fl/þ ; Mx-1-Cre littermate control mice, five days after pIpC treatment. Almost all Pten fl/fl ; Mx-1-Cre mice (17 out of 19) developed myeloproliferative disease marked by a tenfold increase in spleen cellularity (Fig. 1c), complete histological effacement of the splenic architecture (Fig. 1b), reduced bone marrow cellularity (Fig. 1c), and increased blast cell frequency (Fig. 1d). The increased spleen cellularity was largely attributable to extramedullary haemato- poiesis (Supplementary Fig. 2c, d) with a prominent expansion in the number of immature myeloid cells (Supplementary Fig. 2e–g; Sup- plementary Table 1). None out of 20 Pten fl/þ ; Mx-1-Cre littermates showed these changes after pIpC treatment (Fig. 1c, a, d). Within 4 to 6 weeks after pIpC treatment, most Pten fl/fl ; Mx-1-Cre mice progressed to frank leukaemia 24 , including AML and acute lymphoblastic leukaemia (ALL), and died (for the criteria used to diagnose leukaemias, see Supplementary Table 2). AMLs were characterized by large numbers of chloroacetate-esterase-positive myeloid blasts in the spleen (Fig. 1e), and ALLs were characterized by large numbers of terminal deoxynucleotidyl transferase (TdT)- positive lymphoid blasts throughout the thymus, which was also enlarged and effaced (Fig. 1f). The bone marrow contained Mac-1 þ Gr-1 low CD4 2 myeloid blasts and CD4 þ CD8 þ CD3 þ Mac- 1 2 lymphoid blasts (Fig. 1h, i; data not shown). Karyotypic analysis of myeloid blasts from four Pten-deleted mice with AML revealed ARTICLES 1 Howard Hughes Medical Institute, Life Sciences Institute, Department of Internal Medicine, and Center for Stem Cell Biology, University of Michigan, Ann Arbor, Michigan 48109-2216, USA. 2 Department of Pathology, University of Michigan, Ann Arbor, Michigan 48109-2216, USA. 3 Molecular and Medical Pharmacology, UCLA School of Medicine, Los Angeles, California 90095-1735, USA. Vol 441|25 May 2006|doi:10.1038/nature04703 475