In this paper, we demonstrate that the rare population of adult BM LT-HSC exists in two distinct states that are both equally important for their in vivo functions as stem cells: a numerically dominant quiescent state, which is critical for LT-HSC function in hematopoietic reconstitution, and a proliferative state, which represents almost one fourth of this population and is essential for LT-HSC functions in differentiation and self-renewal. We show that when LT-HSC exit quiescence and enter G1
as a prelude to cell division, at least two critical events occur: first, during the G1
and subsequent S-G2
/M phases, they temporarily lose efficient in vivo engraftment activity while retaining in vitro differentiation potential, and second, they select the particular cell cycle proteins that are associated with specific developmental outcomes (self-renewal vs. differentiation) and developmental fates (myeloid vs. lymphoid). A growing body of published literature already exists demonstrating the functional involvement of many of these cell cycle proteins in maintaining normal numbers of hematopoietic cells (9
). In this paper, we provide a global description of the precise stages of hematopoietic differentiation in which such regulators likely play a role and a comprehensive description of their relative expression levels that can directly be used to further understand these published studies and to design future experiments targeting the relevant cell populations.
Our results demonstrate that LT-HSC function in hematopoietic reconstitution after transplantation critically depends on maintenance of the quiescent state. Hence, actively cycling G1
/M phase LT-HSC display dramatically impaired long-term engraftment and multilineage reconstitution capabilities compared with G0
LT-HSC. Because purified G1
/M LT-HSC readily proliferate and differentiate in vitro, their functional deficiencies in vivo may result in part from impaired or altered homing after intravenous transplant. Such an explanation has some support from observations in human CD34+
BM progenitor cells, which in G0
phase show increased adhesiveness to stromal cells (28
) and appear to migrate to the BM of conditioned recipients with increased frequency over S-G2
/M cells (17
). The recent microarray analysis of quiescent and proliferative mouse HSC (15
) also suggests the importance of migratory molecules for establishing HSC quiescence and supports the hypothesis that at least a component of the enhanced engraftment capacity of G0
LT-HSC may relate to a superior BM homing efficiency. The demonstration that HSC cell cycle status directly and dramatically affects HSC engraftment function has important implications for human therapy because cycling HSC derived from mpb or from ex vivo–expanded HSC cultures are commonly used, or proposed for use, in clinical transplantation. It suggests that strategies designed to reestablish quiescence in HSC before transplantation should dramatically improve their efficiency. A detailed investigation of cycling and resting HSC is now essential to address completely the functional relationship between their proliferation, homing, and engraftment capacity. In this context, the analysis of HSC cell cycle progression and regulation during homeostatic HSC maintenance, induced HSC expansion, and HSC differentiation () presented here provides a molecular foothold from which such functional relationships can be further examined.
Figure 7. Molecular networks linking proliferation and cell fate decision in HSC and progenitor cells. Summary of the proliferation index, phase of the cell cycle, and status of the cell cycle machinery associated with differentiation, self-renewal, or long-term (more ...)
Because cycling LT-HSC display impaired function in hematopoietic reconstitution, one may wonder whether non-G0
LT-HSC should be called LT-HSC at all, as the name stands for long-term reconstituting cells. Historically, LT-HSC have been identified and isolated based on their ability to reconstitute the blood system of an irradiated host. With the evolution of the stem cell field, LT-HSC are now more precisely defined by their unique ability to balance self-renewal and differentiation division without depletion of the stem cell pool. This definition implies that LT-HSC must contain a proliferative component to fulfill both self-renewal and differentiation divisions. In fact, it has been shown that the only precursors for LT-HSC in mouse BM are the LT-HSC themselves (29
), and as all LT-HSC enter cell cycle at least once every 15–30 d (3
), the products of LT-HSC division must include G0
phase LT-HSC. It is also important to note that although our analysis does demonstrate a substantially more robust capacity for hematopoietic reconstitution in the G0
fraction of LT-HSC, these data do not exclude the existence of less potent engraftment potential within other cell cycle stages. In previous experiments (16
), though reduced compared with G0
cells, long-term multilineage engraftment function could be detected within the S-G2
/M population when large numbers of these cells were transplanted. Thus, LT-HSC at all cell cycle stages do retain some engraftment capacity and, for these reasons, we feel it is appropriate to continue to refer to the entire population as LT-HSC.
Our results indicate that LT-HSC are maintained in G0
phase of the cell cycle through the concerted action of high levels of Ckis, and predominant expression of p130–E2F4 complexes, which are thought to transcriptionally repress most of the E2F target genes involved in S phase progression (7
). Together, these factors preclude accumulation of high levels of cyclins (mostly mitotic cyclins) and limit LT-HSC proliferation rates. Although every member of the Ink4 and Cip/Kip families of Ckis is highly expressed in LT-HSC, they are not functionally equivalent in preserving HSC quiescence. Analyses of p21 and p18 knockout mice have demonstrated their critical functions in maintaining normal numbers of resting LT-HSC capable of long-term engraftment and multilineage reconstitution (8
). In contrast, p27-null mice have a normal HSC compartment but show increased numbers of progenitor cells (9
), whereas p16-null mice specifically develop thymic hyperplasia (31
) and p19- or p57-deficient mice do not show overt hematopoietic abnormalities (32
). These results suggest that these Ckis are either dispensable for HSC function or are highly specialized in controlling proliferation in subsets of differentiating hematopoietic cells. However, further investigation may uncover additional functions in controlling HSC proliferation. Hence, the recent study of bmi
-1 knockout mice demonstrated a novel and critical function for p16 in regulating adult HSC self-renewing proliferation (11
), which was not previously appreciated in p16-null mice (31
). Mice deficient for single Rb proteins or for E2F4 also show defects in various hematopoietic lineages (34
) but have not yet been analyzed specifically for changes in the stem cell compartment. Future investigations will therefore be required to assay their importance in controlling HSC quiescence.
Although analyses of genetically modified mice are usually performed to demonstrate the functional involvement of specific cell cycle components in regulating stem and progenitor cell proliferation, these in vivo approaches have also their limitations. A good example comes from analyses of the D cyclin–Cdk4-6 complexes, whose critical function during hematopoietic development has only recently been appreciated through the generation of D1/D2/D3 triple knockout (12
) and Cdk4/6 double knockout (13
) mice. Both mice have complete hematopoietic failure starting at the HSC level and die during late embryonic development due, in part, to severe anemia. However, none of the single or double D cyclins knockout mice previously analyzed display major hematopoietic defects or impaired HSC function. This result emphasizes the functional redundancy existing in vivo between the three D cyclins that limits the understanding of their specific function during hematopoiesis. Our analysis sheds new light on the involvement of each particular D cyclin in controlling stem and progenitor fates (). Hence, HSC differentiation specifically associates with cyclin D1 induction, whereas self-renewing HSC do not show induction of cyclin D1. Furthermore, myeloid differentiation only correlates with cyclin D2 induction, whereas lymphoid differentiation specifically associates with cyclin D3 induction. Together, these findings suggest that specific cellular outcomes may be effected by the induction of a particular D cyclins. In addition, differentiating HSC show specific induction of cyclins G2 and B1, whereas self-renewing HSC display specific induction of cyclin G1 and B2 and no change in cyclin G2 expression (). Hence, cyclin D1, cyclin G2, and, to a lower extent, cyclin B1 may be privileged mediators of HSC differentiation-associated proliferation, whereas cyclin G1 and cyclin B2 could be privileged mediators of HSC self-renewal–associated proliferation. HSC fate determination is controlled by many intrinsic and extrinsic signals, which act through regulation of different signaling pathways. Because all these pathways must eventually converge on cell cycle regulation, it is conceivable that one might drive HSC fate determination by directly manipulating the expression levels of specific components and regulators of the cell cycle machinery, as suggested by the recent demonstration that ex vivo targeting of the Cki p21 permits a relative expansion of human HSC (35
). Our results provide a molecular basis for the rational design of strategies to test whether inactivation or activation of specific sets of cyclins may prevent differentiation and favor self-renewal proliferation during ex vivo expansion of HSC.
In conclusion, understanding the particular transcriptional programs used by HSC as they exit quiescence and transit through the cell cycle are likely to reveal how HSC fate decisions are regulated at the molecular level and to provide ways to allow their manipulation for clinical applications. Moreover, because leukemia (and other cancer) stem cells also undergo similar self-renewal versus differentiation cell fate decisions (36
), identifying the key entities involved in these decisions could reveal new molecular targets for human therapies.