Heterogeneity in the differentiation behavior of hematopoietic stem cells is well documented but poorly understood. To investigate this question at a clonal level, we isolated a subpopulation of adult mouse bone marrow that is highly enriched for multilineage in vivo repopulating cells and transplanted these as single cells, or their short-term clonal progeny generated in vitro, into 352 recipients. Of the mice, 93 showed a donor-derived contribution to the circulating white blood cells for at least 4 months in one of four distinct patterns. Serial transplantation experiments indicated that two of the patterns were associated with extensive self-renewal of the original cell transplanted. However, within 4 days in vitro, the repopulation patterns subsequently obtained in vivo shifted in a clone-specific fashion to those with less myeloid contribution. Thus, primitive hematopoietic cells can maintain distinct repopulation properties upon serial transplantation in vivo, although these properties can also alter rapidly in vitro.

The regulation of HSC proliferation and engraftment of the BM is an important but poorly understood process, particularly during ontogeny. Here we show that in mice, all HSCs are cycling until 3 weeks after birth. Then, within 1 week, most became quiescent. Prior to 4 weeks of age, the proliferating HSCs with long-term multilineage repopulating activity displayed an engraftment defect when transiting S/G 2 /M. During these cell cycle phases, their expression of CXC chemokine ligand 12 (CXCL12; also referred to as stromal cell-derived factor 1 [SDF-1]) transiently increased. The defective engrafting activity of HSCs in S/G 2 /M was reversed when cells were allowed to progress into G 1 prior to injection or when the hosts (but not the cells) were pretreated with a CXCL12 antagonist. Interestingly, the enhancing effect of CXCL12 antagonist pretreatment was exclusive to transplants of long-term multilineage repopulating HSCs in S/G 2 /M. These results demonstrate what we believe to be a new HSC regulatory checkpoint during development. They also suggest an ability of HSCs to express CXCL12 in a fashion that changes with cell cycle progression and is associated with a defective engraftment that can be overcome by in vivo administration of a CXCL12 antagonist.

IntroductionEach day, the hematopoietic system of the adult mouse produces billions of mature blood cells. The multistep process that underlies the production of these cells is controlled by complex mechanisms that enable changing physiologic demands to be met without overwhelming the system. It is now clear that a small population of cells known as hematopoietic stem cells (HSCs) are ultimately responsible for maintaining the lifelong output of new blood cells. 1 Nevertheless, a molecular understanding of what constitutes an HSC and how its key functions are maintained are still poorly understood.The existence of hematopoietic cells with the individual potential to produce large numbers of multiple blood cell types in vivo for prolonged periods was first suggested by retrospective clonal tracking experiments that used unique chromosomal, 2 and later, retroviral marking approaches. 3,4 The relatively short lifespan of many mature blood cell types and the contrasting longevity of some of the multilineage clones identified in these experiments implied an origin of the clones from undifferentiated cells with an extensive ability to divide and maintain a derivative population of similarly undifferentiated cells. Serial transplantation experiments demonstrated that such cell divisions did occur and thus provided the first definitive evidence of HSC self-renewal. 3,4 These findings prompted a search for functional end points that would allow HSCs to be specifically quantified independent of the presence of other cell types when assayed in limiting dilution transplantation strategies using suitably irradiated congenic hosts. 5,6 A sustained output of at least 1% of all the circulating white blood cells (WBCs) for at least 4 months is now widely assumed to be suitable for this purpose. 7 Interestingly, most analyses of individual pluripotent hematopoietic cells proliferating either in vivo or in vitro have generally found the self-renewal activity actually displayed to be highly variable. 3,4,8,9 How such a variable behavior is related to the molecular state of the initial cells is not well defined and remains a subject of intense interest and investigation. 10 Variations in external cues may be one contributing parameter, at least in vivo, because it is known that self-renewal responses can be directly and rapidly modulated in this way in vitro. 11,12 In addition, there is some evidence of predetermined heterogeneity in HSC self-renewal potential. This is exemplified by the differences in regenerative activity of HSCs from fetal and adult sources 13,14 and the finding of a consistent association of short-term and long-term multilineage WBC outputs with distinct phenotypes of adult bone marrow (BM) cells (eg, according to their expression of CD49b and CD34). 15,16 Taken together, these results suggest that some hematopoietic cells can remain pluripotent for several divisions even though they are already destined to undergo terminal differentiation within a finite period. This is the basis of the concept of durable versu...

Hematopoietic stem cells (HSCs) execute self-renewal divisions throughout fetal and adult life, although some of their properties do alter. Here we analyzed the magnitude and timing of changes in the self-renewal properties and differentiated cell outputs of transplanted HSCs obtained from different sources during development. We also assessed the expression of several ''stem cell'' genes in corresponding populations of highly purified HSCs. Fetal and adult HSCs displayed marked differences in their self-renewal, differentiated cell output, and gene expression properties, with persistence of a fetal phenotype until 3 weeks after birth. Then, 1 week later, the HSCs became functionally indistinguishable from adult HSCs. The same schedule of changes in HSC properties occurred when HSCs from fetal or 3-week-old donors were transplanted into adult recipients. These findings point to the existence of a previously unrecognized, intrinsically regulated master switch that effects a developmental change in key HSC properties.bone marrow transplantation ͉ development ͉ gene expression ͉ self-renewal

Understanding the intrinsic pathways that regulate hematopoietic stem cell (HSC) proliferation and self-renewal responses to external signals offers a rational approach to developing improved strategies for HSC expansion for therapeutic applications. Such studies are also likely to reveal new targets for the treatment of human myeloid malignancies because perturbations of the biological processes that control normal HSC self-renewal divisions are believed to drive the propagation of many of these diseases. Here, we review recent findings that point to the importance of using stringent functional criteria to define HSCs as cells with longterm repopulating activity and evidence that activation of the KIT receptor and many downstream effectors serve as major regulators of changing HSC proliferative and self-renewal behavior during development.

Fetal hematopoietic stem cells (HSCs) regenerate daughter HSCs in irradiated recipients more rapidly than do adult HSCs. However, both types of HSCs divide in vitro with the same cell-cycle transit times, suggesting different intrinsically determined self-renewal activities. To investigate the mechanism(s) underlying these differences, we compared fetal and adult HSC responses to Steel factor (SF) stimulation in vitro and in vivo. These experiments were undertaken with both wild-type cells and W(41)/W(41) cells, which have a functionally deficient c-kit kinase. In vitro, fetal HSC self-renewal divisions, like those of adult HSCs, were found to be strongly dependent on c-kit activation, but the fetal HSCs responded to much lower SF concentrations in spite of indistinguishable levels of c-kit expression. Fetal W(41)/W(41) HSCs also mimicked adult wild-type HSCs in showing the same reduced rate of amplification in irradiated adult hosts (relative to fetal wild-type HSCs). Assessment of various proliferation and signaling gene transcripts in fetal and adult HSCs self-renewing in vitro revealed a singular difference in Ink4c expression. We conclude that the ability of fetal HSCs to execute symmetric self-renewal divisions more efficiently than adult HSCs in vivo may be dependent on specific developmentally regulated signals that act downstream of the c-kit kinase.

Recent improvements in the development of methods for isolating functionally validated populations of nearly pure (>20%) murine hematopoietic stem cells (HSCs) have made it possible to analyze the molecular basis of the properties of these cells with increased precision. One intriguing feature of HSCs is the change they undergo in many of their key properties during development - a change that affects the control of their self-renewal, cycling status, differentiated progeny output and steel factor sensitivity. To investigate how these differences are mediated, we undertook a genome-wide analysis of the transcripts present in highly purified fetal and adult HSCs using an adaptation of the LongSAGE methodology that allows its application to small numbers of cells (10 ng of RNA) by inclusion of an initial PCR amplification step that preserves the transcript repertoire while excluding less than 0.25% of the transcripts. The LongSAGE methodology was adopted because it is sequence-based and thus quantitative and independent of prior knowledge of expressed genes or variations in their hybridization to matching or related cDNAs, ESTs or derived oligos. A suspension of 10,000 lin-Sca-1+CD43+Mac1+ fetal liver (FL) cells (∼20% pure HSCs as determined by 16-week limiting dilution and single cell transplantation experiments) was obtained from embryonic day 14.5 fetuses. From these cells, we generated a 160,000-tag LongSAGE library containing 7865 tags that map uniquely to the mouse genome (using the RefSeq database through DiscoverySpace; www.bcgsc.ca/DiscoverySpace). A suspension of 3700 CD45midlin-Rho-SP cells (∼30% pure HSCs) was isolated from adult mouse bone marrow (BM) and then used to generate a 37,000-tag LongSAGE library (956 uniquely mapped tags). Both of these libraries contained tags identifying transcripts that have been previously reported to be associated with HSCs from FL and/or adult BM, including c-kit, pbx-1, tgf-β, cul-4a, PrP, c-myc, robo1, sox17, as well as a number of Smarc transcripts, ubiquitin ligase transcripts and TNF-related transcripts. As a first test of the utility of the libraries, we looked for differences in the expression of genes that have been broadly associated with differences in cellular proliferative activity. This comparison identified many such tags in the FL HSC library that were absent from the adult BM HSC library, including multiple cyclins (A2, B2, C, D1, D2, D3, E1, F, H, I, J L1, L2), cdc20, cdc5b, and plk, as well as 34 of the top 50 “proliferation” genes identified by Venezia et al (PLoS Biology, 2004) to be selectively expressed in adult BM HSCs that had been stimulated to proliferate. Other transcripts that were present at significantly higher levels in the FL HSC library (95% C.I. using Audic Claverie statistics) included msl2, rbx1, lmo2, pfn1, and 16 members of the tripartite motif protein (trim) family. Conversely, many transcripts for components of the proteosome, involved in nucleic acid binding, and transcripts coding for proteins with receptor activity were present at higher levels (or uniquely) in the adult BM HSC library. Taken together, these findings establish the validity and potential of these permanent HSC transcriptome resources for further investigation of mechanisms that determine the different biology of fetal and adult HSCs.

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