TitleAuthorsAbstractMethods SummaryBrdU, CldU and IdU administrationBrdU segregation in cultured HSCsReferencesMethodsFlow cytometric isolation of HSCsBrdU stainingCldU and IdU double-labellingAnalysis of cell cycle distribution and cell death in HSCsMathematical models of BrdU uptake and retentionCase 1: random segregation of chromosomesCase 2: asymmetric chromosome segregationMethods ReferencesFigure 1 Contrasting predictions regarding stem cell labelling on the basis of the immortal strand model versus random chromosome segregation.Figure 2 Six per cent of HSCs stochastically enter the cell cycle each day.Figure 3 Few HSCs retain BrdU, and most BrdU-retaining bone marrow cells are not HSCs.Figure 4 HSCs segregate chromosomes randomly in vivo and in vitro.LETTERSHaematopoietic stem cells do not asymmetricallysegregate chromosomes or retain BrdUMark J. Kiel1, Shenghui He1, Rina Ashkenazi2, Sara N. Gentry2, Monica Teta3, Jake A. Kushner3, Trachette L. Jackson2& Sean J. Morrison1Stem cells are proposed to segregate chromosomes asymmetricallyduring self-renewing divisions so that older (‘immortal’) DNAstrands are retained in daughter stem cells whereas newly syn-thesized strands segregate to differentiating cells1–6. Stem cellsare also proposed to retain DNA labels, such as 5-bromo-2-deoxyuridine (BrdU), either because they segregate chromosomesasymmetrically or because they divide slowly5,7–9. However, thepurity of stem cells among BrdU-label-retaining cells has not beendocumented in any tissue, and the ‘immortal strand hypothesis’has not been tested in a system with definitive stem cell markers.Here we tested these hypotheses in haematopoietic stem cells(HSCs), which can be highly purified using well characterizedmarkers. We administered BrdU to newborn mice, mice treatedwith cyclophosphamide and granulocyte colony-stimulatingfactor, and normal adult mice for 4 to 10 days, followed by 70 dayswithout BrdU. In each case, less than 6% of HSCs retained BrdUand less than 0.5% of all BrdU-retaining haematopoietic cells wereHSCs, revealing that BrdU has poor specificity and poor sensitivityas an HSC marker. Sequential administration of 5-chloro-2-deoxyuridine and 5-iodo-2-deoxyuridine indicated that all HSCssegregate their chromosomes randomly. Division of individualHSCs in culture revealed no asymmetric segregation of the label.Thus, HSCs cannot be identified on the basis of BrdU-label reten-tion and do not retain older DNA strands during division, indi-cating that these are not general properties of stem cells.The immortal strand hypothesis was proposed as a mechanism bywhich stem cells could avoid accumulating mutations that arise dur-ing DNA replication2. Whereas most cells segregate their chromo-somes randomly1,10, it was argued that adult stem cells in steady-statetissues might retain older DNA strands during asymmetric self-renewing divisions, segregating newly synthesized strands to daugh-ter cells fated to differentiate (Fig. 1a). Evidence has supported thismodel in some epithelial stem cells1, neural stem cells3, mammaryepithelial progenitors4and muscle satellite cells5,6. A related idea isthat adult stem cells in steady-state tissues might consistently retainDNA labels. This could be because chromosomes segregate randomlybut stem cells divide more infrequently than other cells (Fig. 1b), oralternatively because the older DNA strand is labelled and segregatedasymmetrically (Fig. 1c). Tritiated thymidine8or histone7label-retaining cells from the hair follicle are enriched for epithelial stemcells, although the purity remains uncertain. Label-retaining cellshave also been identified in the haematopoietic system9,11, in mam-mary epithelium12, in intestinal epithelium1,13and in the heart14, butthe purity of stem cells among these label-retaining cells has not beentested. As a result, it remains unclear whether label retention canconsistently identify stem cells with specificity or sensitivity.Under steady-state conditions in adult bone marrow, all HSCsdivide regularly but infrequently15to sustain haematopoiesis and tomaintain nearly constant numbers of HSCs. As a result of this obser-vation, as well as the finding that HSC divisions yield asymmetricoutcomes in culture16, it has been proposed that adult HSCs divideasymmetrically16, although the rarity of HSCs in vivo and their1Howard Hughes Medical Institute, Life Sciences Institute, Department of Internal Medicine, and Centre for Stem Cell Biology,2Department of Mathematics, University of Michigan,Ann Arbor, Michigan 48109-2216, USA.3Division of Endocrinology, Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania19104, USA.aAsymmetricsegregationAsymmetricdivisionRandomsegregationAsymmetricdivisionAsymmetricsegregationInitial symmetricdivisionorororRandomsegregationInitial symmetricdivisionStem cellCell fatedto differentiateUnlabelledchromosomeBrdU-labelledchromosomeDividing stem cellin S/G2/M phasebcd+BrdU +BrdU +BrdU +BrdUFigure 1|Contrasting predictions regarding stem cell labelling on the basisof the immortal strand model versus random chromosome segregation.a, According to the immortal strand model2, stem cells divideasymmetrically under steady-state conditions and BrdU is incorporated intonewly synthesized DNA strands that are asymmetrically segregated intodifferentiating daughter cells with each round of division, such that stemcells retain only the unlabelled older DNA strands.b, In contrast, ifchromosomes segregate randomly, then BrdU-labelled chromosomes will bestochastically lost over multiple rounds of divisions.c, In the immortalstrand model, if stem cells divide symmetrically then BrdU can beincorporated into DNA strands that become the ‘older’ strands once stemcells resume asymmetric division. Under these circumstances, the BrdU1older strands would be retained indefinitely in stem cells. d, In contrast, ifchromosome segregation is random then BrdU1chromosomes arestochastically lost over time after BrdU is discontinued.Vol 449|13 September 2007|doi:10.1038/nature06115238Nature ©2007PublishingGrouprelative quiescence has made it impossible to confirm this directly.Nonetheless, if BrdU-label retention and/or asymmetric chromo-some segregation are general properties of adult stem cells, theneither or both of these characteristics should be evident in HSCs,depending on experimental conditions.To test the rate at which HSCs enter the cell cycle