Hematopoietic stem cells (HSCs) regenerated in vivo display sustained differences in their self-renewal and differentiation activities. Variations in Steel factor (SF) signaling are known to affect these functions in vitro, but the cellular and molecular mechanisms involved are not understood. To address these issues, we evaluated highly purified HSCs maintained in single-cell serum-free cultures containing 20 ng/mL IL-11 plus 1, 10, or 300 ng/mL SF. Under all conditions, more than 99% of the cells traversed a first cell cycle with similar kinetics. After 8 hours in the 10 or 300 ng/mL SF conditions, the frequency of HSCs remained unchanged. However, in the next 8 hours (ie, 6 hours before any cell divided), HSC integrity was sustained only in the 300 ng/mL SF cultures. The cells in these cultures also contained significantly higher levels of Bmi1, Lnk, and Ezh2 transcripts but not of several other regulators. Assessment of 21 first division progeny pairs further showed that only those generated in 300 ng/mL SF cultures contained HSCs and pairs of progeny with similar differentiation programs were not observed. Thus, SF signaling intensity can directly and coordinately alter the transcription factor profile and long-term repopulating ability of quiescent HSCs before their first division. (Blood. 2008;112:560-567) IntroductionThe hematopoietic system of the adult mouse is responsible for the daily production of billions of differentiated blood cells of various types. Because most of these cells have a limited lifespan and proliferative ability, they must be continuously generated from a population of more primitive cells that collectively have life-long self-sustaining ability. This function is restricted to a tiny subset of multipotent cells generally referred to as hematopoietic stem cells (HSCs). Historically, HSCs have been both defined and quantified retrospectively by their ability to generate clones containing both lymphoid and myeloid blood cells for at least 4 months when transplanted into irradiated recipients at limiting dilutions. 1 Analyses of such mice have also allowed the long-term differentiation activity of individual HSCs to be characterized. 2,3 Methods have now been developed for isolating purified populations in which 20% to 60% of the cells display this durability of reconstituting activity, indicating that intravenously injected HSCs can have very high seeding efficiencies in irradiated mice. [4][5][6][7] The ability to isolate such highly purified HSC populations has also permitted a more direct and complete analysis of their in vivo differentiation activity in single-cell transplant experiments. 6,[8][9][10][11] More recently, serial transplants of such clonally repopulated mice have been performed. Together, these experiments have revealed that HSCs possess one of 4 distinct differentiation programs that are propagated over many generations in vivo. 10,12 In addition, these studies have shown that extensive self-renewal ability in vivo is strongly associated with the display of either a...

Significant advances have been made in the development of methods for purifying murine hematopoietic cells with longterm (>4 months) in vivo reconstituting ability although these longterm repopulating cells (LTRCs) remain heterogeneous with regard to the self-renewal (SR) activity they display when transplanted into irradiated hosts. Furthermore our group has also identified cell culture conditions that differentially alter LTRC activity without immediate effects on their proliferation or survival. Here, we show that highly purified LTRCs with high and low SR properties can be prospectively isolated from normal adult mouse bone marrow (ABM) as 2 separate populations according to their expression of CD150 within the EPCR++CD48−CD45mid fraction of cells: 56% total LTRCs and 43% of the high SR type in the CD150+ subset vs. 39% total LTRCs and 32% of the low SR type in the CD150− subset (as determined from 62 and 28 single cell transplants, respectively). As a first test of whether these populations would likely be useful to search for new molecular differences associated with their different SR properties, we compared the level of expression in these 2 populations of a small set of genes previously reported to regulate LTRC SR activity: c-Kit, Bmi1, Gata3, Rae28, Ezh2 and Lnk by quantitative real-time PCR (Q-RT-PCR). This exercise revealed transcript levels of the first 4 of these genes to be significantly higher in the CD150+ subset that is selectively enriched in high SR LTRCs, thus validating the concept that they have a distinct molecular signature. Previous evidence shows that high SR LTRCs are present in both FL LTRCs and ABM LTRCs but they differ in some properties (i.e.: cell cycle status, regeneration kinetics). We therefore began a search for ontogeny-independent components of the SR machinery by comparing tags present in 2 LongSAGE libraries produced from CD45midlin−Rho−SP ABM cells and from lin−Sca1+CD43+Mac1+ embryonic day 14.5 fetal liver (FL) cells (each 20–30% total LTRCs and 12–20% of the high SR type, as determined by 132 (FL) and 352 (ABM) single cell transplants, respectively). From these comparisons and additional data in other publicly available datasets for primitive murine hematopoietic cells, we identified 28 genes not previously shown to have a functional role in LTRC SR control. We then compared the level of expression of these 28 genes between the CD150+ subsets of EPCR++CD48−CD45mid ABM cells and FL cells (24% total LTRCs and 12% high SR LTRCs in the FL subset) and their respective downstream lin− progeny. This comparison revealed 10 of these genes to be down-regulated in the lin− populations of both ABM and FL. Further comparison of the expression of these 10 genes between the high vs. low SR LTRCs (found in the CD150+ and CD150− subsets of EPCR++CD48−CD45mid) ABM cells showed the expression of 5 (Vwf, Rhob, Pld3, Prnp and Smarcc2) to be downregulated in the CD150− (low SR LTRC) subset. Interestingly, the first 4 of these genes, as well as 2 of the preliminary set of SR regulators (Bmi1 and Gata3), were also selectively down-regulated in EPCR++CD150+CD48−CD45mid ABM cells that had been incubated for 16 hours in 1 or 10 ng/ml Steel factor + 20 ng/ml IL-11 (conditions that decrease LTRC activity in vivo 4–5-fold before any of these divide or die). Taken together, these results point to the existence of more, although a rather small number of additional genes, including Vwf, Rhob, Pld3, and Prnp, whose products may be involved in controlling the SR potential of normal mouse LTRCs.

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.