LETTERS 172 NATURE CELL BIOLOGY VOLUME 7 | NUMBER 2 | FEBRUARY 2005 Negative cell-cycle regulators cooperatively control self-renewal and differentiation of haematopoietic stem cells Carl R. Walkley 1,2,6 , Matthew L. Fero 4 , Wei-Ming Chien 4 , Louise E. Purton 1,5,6,7 and Grant A. McArthur 1,2,3,5,7 Haematopoietic stem cells (HSCs) are capable of shifting from a state of relative quiescence under homeostatic conditions to rapid proliferation under conditions of stress. The mechanisms that regulate the relative quiescence of stem cells and its association with self-renewal are unclear, as is the contribution of molecular regulators of the cell cycle to these decisions. Understanding the mechanisms that govern these transitions will provide important insights into cell-cycle regulation of HSCs and possible therapeutic approaches to expand HSCs. We have investigated the role of two negative regulators of the cell cycle, p27 Kip1 and MAD1, in controlling this transition. Here we show that Mad1 –/– p27 Kip1–/– bone marrow has a 5.7-fold increase in the frequency of stem cells, and surprisingly, an expanded pool of quiescent HSCs. However, Mad1 –/– p27 Kip1–/– stem cells exhibit an enhanced proliferative response under conditions of stress, such as cytokine stimulation in vitro and regeneration of the haematopoietic system after ablation in vivo. Together these data demonstrate that the MYC-antagonist MAD1 and cyclin-dependent kinase inhibitor p27 Kip1 cooperate to regulate the self-renewal and differentiation of HSCs in a context-dependent manner. In vivo HSCs have a cell division cycle in the order of 2–4 weeks, a char- acteristic considered to be critically important for their biological func- tion 1,2 . In addition to the slow time of the cell division cycle of HSCs in vivo, isolated stem cells in vitro exhibit a significant delay in commit- ting to their first cell division, but exhibit a high proliferative poten- tial 3–5 . Several families of genes have been found to have central roles in the regulation of the cell cycle during both the entry to and exit from quiescence, in particular the Max-network and the CIP/KIP family of CDK inhibitors 6–8 . As relative quiescence is a defining characteristic of the HSC, we sought to determine if, as for terminal differentiation, the 1 Research Division, Peter MacCallum Cancer Centre, Victoria 3002, Australia. 2 Department of Medicine, St. Vincent’s Hospital, University of Melbourne, Victoria 3065, Australia. 3 Department of Haematology and Medical Oncology, Peter MacCallum Cancer Centre, Victoria 3002, Australia. 4 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA. 5 These authors contributed equally to this work. 6 Present addresses: Department of Hematology- Oncology, Children’s Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA (C.R.M.) and Center for Regenerative Medicine and Technology, Massachusetts General Hospital, Boston, MA 02129, USA (L.E.P.). 7 Correspondence should be addressed to L.E.P (e-mail: lpurton@partners.org) or G.A.M. (e-mail: grant.mcarthur@petermac.org) Published online: 16 January 2005, DOI: 10.1038/ncb1214 MYC-antagonist MAD1 and the CDKi p27 Kip1 has a role in regulating the quiescence of the stem cell 6,9,10 . Analysis of the more mature progenitor compartment revealed a p27 Kip1 -dependent increase in the functional day-12 colony-forming-unit spleen cells (CFU-S 12 ) from whole bone marrow 11,12 (see Supplementary Information, Fig. 1a). The frequency of the common myeloid, granu- locyte/monocyte, megakaryocyte/erythroid and common lymphoid progenitor cells was not significantly different between genotypes 13,14 (see Supplementary Information, Fig. 1b and data not shown). To further study the effects of loss of p27 Kip1 and MAD1, the stem-cell- enriched lineage negative (lin – ), c-Kit + , Sca-1 + (lin – c-Kit + Sca-1 + or LKS + ) population was isolated 12,15 . The non-HSC-containing lin – c-Kit + Sca- 1 – (LKS – ) fraction was also isolated 15 . Quantitative real-time PCR with wild-type LKS + and LKS – populations revealed a marked difference in expression of Mad1, with the LKS + population expressing over 700-fold more Mad1 mRNA than the LKS – population (Fig. 1a). The p27 Kip1 tran- script was expressed in both LKS + and LKS – fractions, with fourfold more expression in the LKS – population (Fig. 1a). Loss of either p27 Kip1 or MAD1 alone did not alter the relative frequency of LKS + or LKS – cells relative to wild-type mice. Notably, however, loss of both MAD1 and p27 Kip1 resulted in a 3.3-fold increase in the frequency of the HSC-containing LKS + population (P ≤ 0.05; Fig. 1b). By contrast, despite the expanded progenitor populations in both the p27 Kip1–/– and the Mad1 –/– p27 Kip1–/– bone marrow, there was no increase in the relative frequency of the LKS – populations 6,9 (Fig. 1b). The discrepancy between the expanded CFU-S 12 progenitors and that of phenotypic evaluation of progenitors (CMP, LKS + , LKS – ) in the p27 Kip1–/– and Mad1 –/– p27 Kip1–/– mice may be accounted for by an increased proliferative capacity of p27 Kip1 - mutant progenitors despite an unaltered frequency as assessed phenotypi- cally, or due to different cell types being assessed in these assays 16 . We also determined the frequency of the highly HSC-enriched LKS + CD34 –/low population 17 . Loss of both MAD1 and p27 Kip1 significantly increased both Nature Publishing Group ©2005