Using a newly established HSC tracking method that utilizes in vivo CFSE dilution, we communicate three major findings on HSC turnover and contribution to blood formation in steady state and upon inflammation. The first finding is that in steady state, HSCs with equivalent life-long multilineage repopulation potential are contained in both frequently cycling cell populations that divide ≥5× in 7 wk, i.e., about every 1.4 wk, and in quiescent cells that do not divide over 14 wk. The finding is not consistent with the previous observations that only quiescent cells possess serial reconstitution capacity and fast-diving cells have limited self-renewal (Wilson et al., 2008
; Foudi et al., 2009
). This contradiction might arise from technical issues, as numbers of cells transferred into secondary transplants, and from sensitivity and resolution of divisional tracking methods. Another possibility would be that the dividing HSCs are not included in the population with LKS CD48−
phenotype as subsets of HSC might not express CD150 (Weksberg et al., 2008
). Although it was assumed that both BrdU incorporation and H2B-GFP transgenic animal models allow us to follow seven cellular divisions, our comparative analysis demonstrates that staining intensity for BrdU detection reaches a limit at two to three divisions and that BrdU staining is not linear, suggesting a lower divisional resolution of BrdU retention than expected, whereas, in contrast, CFSE dilution can distinguish at least five divisions with high resolution. Furthermore, the fact that BrdU has mitogenic activity, which has been shown in this paper and previously (Kiel et al., 2007
; Wilson et al., 2008
), has a substantial impact on the experimental readout because BrdU labeling changes cell cycle state as well as, potentially, consecutive function of cells. Also, most quiescent 0×-dividing HSCs are ignored in the BrdU assay, as our data shows remaining CFSE-high cells that do not incorporate BrdU. Thus, with some HSCs being deeply quiescent and inactive in DNA replication and protein synthesis, uniform labeling of all HSC by DNA labeling or marker protein expression might not be achieved at the starting point of chase. This contrasts with the CFSE labeling established in this paper that ensures highly uniform labeling and high-resolution divisional tracking of cells without impairing HSC function, independent of cell cycle activity during the labeling process.
The second finding is that steady-state fast-cycling populations can slow down over time in steady-state serial transplantation and that LKS CD150+
cells containing HSCs with extensive proliferative history—i.e., HSCs that have gone through extended proliferation in aging or after in vivo challenge by transplantation—are prone to return to quiescence. As demonstrated in this paper, divisional activity is not associated with HSC function in young adult mice. Furthermore, HSCs have the capacity to engraft and constitute long-term hematopoiesis over several serial transplantations, indicating that high divisional history does not lead to immediate loss of HSC function (Allsopp et al., 2001
). HSC cycling activity is a result of extrinsic and intrinsic regulation (Orford and Scadden, 2008
). Based on our data, we would like to suggest that steady-state fast-cycling or enhanced turnover with aging or irradiation- and transplantation-induced proliferation might activate an intrinsic HSC program that, based on divisional history, drives toward quiescence. Thus, an intrinsic cell memory effect to prevent HSC exhaustion might be counterbalanced by an extrinsic drive for proliferation. The underlying mechanisms will need to be determined in the context of environmental cues such as availability of adhesion molecules and growths factors in the putative BM HSC niche.
Third, we show that in vivo TLR4 agonist challenge recruits in vivo functional quiescent HSCs into proliferation and self-renewal with nonbiased lineage repopulation capacity. Although TLR ligation on HSCs has been shown to induce cellular division and myeloid lineage-skewed differentiation ex vivo, there was no direct evidence for enhanced self-renewal of HSC in BM (Nagai et al., 2006
; Massberg et al., 2007
). It is not clear from our experiments if LPS executes its effect on cell cycle regulation directly via TLR4 expressed on HSCs, via an indirect pathway, or via a combination of both. Hematopoietic and nonhematopoietic cell–secreted interferons have been recently identified by several studies to drive HSC in cycle upon artificial addition or in chronic infection (Essers et al., 2009
; Sato et al., 2009
; Baldridge et al., 2010
). Our study extends these findings and directly demonstrates that correlates of gram-negative infections, or possibly self-damage (Rakoff-Nahoum and Medzhitov, 2009
), can signal from the periphery to primary hematopoietic sites in BM and have an impact on divisional behavior of HSCs. This mechanism likely allows adequate hematopoietic responses and, at the same time, prevents loss of HSCs by differentiation.
Our mathematical simulation reveals that HSCs with different cycling activity can be contained in one HSC population with relatively broad cycling variation and that, on average, HSCs divide 18× during a 2-yr lifespan of a laboratory mouse. Two principle models have been posed for the maintenance of hematopoiesis by stem cells. The clonal maintenance model suggests that all HSCs give rise to mature blood cells continuously throughout life and, thus, all HSCs should divide similarly to produce cells that contribute to blood formation (Fig. S5 A
; Jordan and Lemischka, 1990
; Cheshier et al., 1999
; McKenzie et al., 2006
; Kiel et al., 2007
; Nygren and Bryder, 2008
). The clonal succession model proposes that some HSCs divide frequently, contribute to hematopoiesis, and fully differentiate or die subsequently and are followed by previously quiescent HSCs that then meet the same fate (Fig. S5 B; Kay, 1965
; Drize et al., 1996
; Wilson et al., 2008
; Foudi et al., 2009
). Based on our data, we suggest a “dynamic repetition” model, where some HSCs dominate blood formation for a time, subsequently enter a quiescent state in which other HSCs increase hematopoietic contribution, and get reactivated again and contribute to blood formation in repetitive cycles (Fig. S5 C). Our data do not suggest how long active and resting phases might last or how many HSC clones at any given time contribute to hematopoiesis. However, the model of steady-state reversible change between proliferation and quiescence in HSCs over time is consistent with virtual single cell–based simulation models (Glauche et al., 2009
). Furthermore, the dynamically changing cycling activity likely results in a similar turnover of the entire HSC pool, indicating a homogeneous divisional history for all HSCs at the end of life, a suggestion which would be coherent with linear telomere shortening observed in the human aging HSC pool (Rufer et al., 1999
). The findings reported in this paper might represent a biological principle that could hold true for other somatic stem cell–sustained organ systems and might have developed during evolution to ensure equal distribution of work load, efficient recruitment of stem cells during demand, and reduction of risk to acquire genetic alterations by alternating fractions of stem cells in quiescence at any given time.