The classical model of hematopoietic hierarchy which proposes that all the mature cells of the peripheral blood are the progeny of a single LT-HSC, the so-called clonal succession model (), has come under scrutiny recently as more evidence accumulates that the HSC pool actually consists of multiple HSC subtypes with distinct functional potentials. The work presented here provides further evidence for this clonal diversity model (), and the combination of SP and CD150 shows a clear gradient in HSC potential suggesting a continuum of HSC is present in the marrow. By competing the subtypes against each other, we demonstrated that in the presence of the other subtypes, the HSC subtypes default to perform almost exclusively their primary function (i.e. their biases are extreme). This suggests that in vivo, most stem cells may be highly biased and behave not at all like classically-defined multi-lineage HSC.
Model for HSC clonal diversity and its relation to the SP phenotype.
Initial studies that established this concept of biased HSC subtypes largely arose from analysis of the repopulation kinetics of mice transplanted with phenotypically identical populations (Muller-Sieburg et al., 2002
; Muller-Sieburg et al., 2004
; Dykstra et al., 2007
). Further understanding of the mechanisms that regulate these HSC subtypes requires the ability to prospectively isolate them. The Eaves group recently showed that CD150 could be used to distinguish myeloid- and lymphoid-biased HSC purified on the basis of CD45+
expression (Kent et al., 2009
), and were able to identify some differentially expressed genes.
Here, we show that Hoechst dye efflux and CD150 reveal a gradient of HSC self-renewal activity and myeloid bias correlating with the phenotypes (in order) CD150+
with an opposing gradient of proliferative potential and lymphoid bias. However, while the average activities of the clones point to these correlations, there was substantial variation between individual HSCs. This implies that either the populations are still a mixture of more functionally distinct cells, or that the SP actually represents a continuum of HSC activities, which hampers the absolute segregation of distinct HSC phenotypes (). Because the peculiar Hoechst staining of the SP results from differential efflux of the dye by HSCs due to high multi-drug-resistance (MDR)–type transporter activity (Goodell et al., 1996
; Zhou et al. 2001
), it will be interesting to determine whether the discrete efflux properties will prove to have biological significance for the HSC subtypes.
Previous studies have shown that aging HSCs have reduced ability to reconstitute lethally irradiated recipients in transplantation assays (Morrison et al., 1996
; Liang et al., 2005
), a vastly different gene expression profile (Rossi et al., 2005
; Chambers et al., 2007b
), and a myeloid-differentiation bias compared to their young counterparts (Sudo et al., 2000
; Kim et al., 2003
). However, these studies compared the entire HSC pools from old versus young mice, not taking into account potential population dynamics. One study suggested that the age-related changes reflect changes in the clonal composition of the HSC pool of old mice (Cho et al., 2008
). Indeed, we observed an accumulation of lower-SPKLS
in the bone marrow of old mice, yet lower- and upper-SPKLS
cells exhibit the same differentiation bias of their young counterparts. Thus, the apparent myeloid-bias of aging HSCs reflects a higher proportion of My-HSCs in the HSC pool, rather than alterations in the developmental potential of HSC with age. This could be due to differential responses to the aging cytokine milieu, the higher proliferative rate of Ly-HSCs which could lead to their exhaustion, and a greater self-renewal capacity of My-HSCs. Clearly, this dramatic proportional shift of these subpopulations with age has significant implications for the interpretation of studies that have described distinct molecular or biological properties of the aged HSC pool.
TGFβ-signaling has been implicated in maintaining HSC quiescence (Fortunel et al., 2000
; Chabanon et al., 2008
). Here, TGFβ1 stimulated My-HSCs to proliferate, whilst proving inhibitory to Ly-HSCs. This appears to be physiological relevant, as increased proliferation of My-HSCs was apparent in vitro
and in vivo
. Furthermore, when mice were co-transplanted with limited numbers of genetically distinguishable My- and Ly-HSCs, and then administered TGFβ1 after stable engraftment (), TGFβ1 acted directly on the transplanted My-HSC population, stimulating the proliferation of its daughter myeloid-biased HSCs and myeloid progenitors, while in the same animals simultaneously inhibiting the proliferation of transplanted Ly-HSC-derived myeloid progenitors. This highlights the unique responsiveness of distinct HSC subtypes, and their immediate progeny, to a growth factor and provides a potential mechanism for differential regulation of HSC subtypes.
One of the molecular mechanisms governing the proliferative response of My-HSCs to TGFβ1 appears to be downregulation of the G0 to G1 cell cycle inhibitors p18 and p19. Other differential responses were detected in HSC subtypes upon exposure to TGFβs, most notably the proto-oncogenes Evi1 (upregulated in Ly-HSCs) and c-jun (upregulated in My-HSCs). It is clear that My- and Ly-HSC subtypes have distinct molecular and cellular responses to TGFβ signaling, further enforcing their specific characteristics. It is possible that TGFβ signaling in the bone marrow niche functions as a “fine-tuning” mechanism for cross-talk between HSC subtypes to mediate the numbers of each actively engaged in hematopoiesis at any given point in time. We also speculate that the proportional increase of the My-HSC compartment with age could be due in part to differences in TGFβ ligand production, and the consequent response particularly of My-HSCs, in the inflammatory setting of an aging environment.
The My-HSCs exhibit the highest engraftment rate per mouse when single cells were transplanted, as well as the highest overall contribution to peripheral blood regeneration in each mouse. The secondary transplant data also suggest My-HSCs have higher self-renewal capacity. It is likely that if the contribution of the HSC subtypes were examined over a longer time-period (6 to 12 months), we would see further distinctions in the My- and Ly-HSCs with regard to their ability to sustain blood production. While clearly bona fide
HSCs according to the rigorous criteria imposed here, blood production from Ly-HSCs may prove less durable, consistent with their lower potency and higher proliferative rate. The recent report of a class of HSCs with intermediate-term durability underscores this key property (Benveniste et al., 2010
This visual continuum of HSCs with different dye efflux capacity, correlating with a functional gradient, makes it tempting to speculate that My-HSCs are the most primitive, generating the less-primitive, less quiescent Ly-HSC. While both HSC types clearly have the potential to generate each other (), each HSC subtype preferentially regenerated itself, arguing that these HSC pools operate largely independently of each other. This suggests a deterministic explanation for the data. Perhaps an omnipotent HSC present during development (conceivably still present in adult marrow) establishes a consortium of HSCs during seeding of the bone marrow. Their epigenetic state, or their different niches, may indelibly dictate their propensity to generate the downstream components of the blood in a biased fashion, as well as their self-renewal capacities. Thus, the properties of these HSC subtypes suggest a lineal relationship, but that may be a result of the limitations of the current assays and our historical interpretations.
Intriguingly, argument about the precursors of the hematopoietic systems can be traced back over 100 years when staining techniques enabled identification of different white blood cell lineages. “Unitarians” believed that erythrocytes, granulocytes and lymphocytes all came from a cell of common origin while “Dualists” argued that myeloid and lymphoid cells derived from committed precursors residing in distinct hematopoietic tissues (reviewed in Ramalho-Santos and Willenbring, 2007
). The Unitarian concept may still hold true, but the mounting evidence for clonal diversity in the HSC pool with distinct subtypes dedicated to regenerating particular compartments argues that revision of the long-held view of a unipotent stem cell pool generating the entire branching hematopoietic differentiation tree must come under scrutiny. The clonal diversity model has important implications for experimental and clinical HSC biology including the selection of appropriate HSC subtypes for transplantation or cell / gene therapy applications, the incidence of myeloproliferative disorders in the elderly and the cellular and behavioral heterogeneity seen in genetically similar leukemias. Here we prospectively isolate functionally distinct HSC subtypes, and provide the first mechanistic insight into the molecular regulation of myeloid- and lymphoid-biased HSC function.