While the concepts of apoptotic versus necrotic cell death have been formulated a long time ago, the more recent molecular characterization of the genes and proteins involved has led to a major increase in the appreciation of their importance during normal and pathological development. Apoptosis plays an important role in hematopoiesis, and various transgenic animal models have been generated to study aspects of this. We have generated a transgenic mouse that overexpresses BCL-2, the archetypal protein that protects against different forms of apoptosis, in all hematopoietic cells, including HSC. This was previously demonstrated for radiation-induced cell death 30
, and is extended in this paper to cell death induced by growth factor deprivation; HSC from H2K-BCL
-2–transgenic mice are protected. We have used this transgenic model to study the role of apoptosis in the maintenance of HSC in vivo. To guide the discussion of our finding that HSC have among their options programmed cell death, is provided.
Model for regulation of HSC numbers. (A) HSC homeostasis through balancing self-renewal and differentiation. (B) HSC homeostasis by balancing self-renewal, differentiation, and apoptosis.
The cell surface markers that are used to delineate HSC, such as Thy-1.1low
, and c-Kithigh
are not essential for HSC function, nor are they invariably associated with it. Under specific conditions (e.g., during development) 34
, or after 5-FU treatment 32
, changes occur in these staining profiles. Similar changes cannot be excluded in transgenic mice. However, transplantation experiments show that the cells with HSC surface markers in H2K-BCL
-2–transgenic mice are indeed HSC, and that they are at least as efficient on a per-cell basis in repopulating lethally irradiated hosts as wild-type HSC.
BCL-2 overexpression in transgenic mice has been shown to increase population sizes of various cell types. This was initially demonstrated for lymphocytes 456
, but has been expanded to other cell populations, such as monocytes 7
and neurons 35
. The expansions tend to be most dramatic in resting cells. This would be in line with the expansion in transgenic HSC. Most stem cells are in the G1
phases of the cell cycle. The implication of this observation is that HSC under steady state conditions are subject to regulation by apoptosis.
The inability to maintain and expand long-term multilineage reconstituting HSC as such in vitro, despite all the factors and culture systems available, has led to the hypothesis that such maintenance in vivo is strictly dependent on particular microenvironments, and that the number of HSC reflects the number of microenvironments. The limited increase in HSC in the bone marrow of H2K-BCL
-2–transgenic mice indicates that HSC, despite their resistance to apoptosis, are not simply accumulating in ever larger numbers, but are still subject to regulation. Also, the variation in bone marrow HSC numbers between animals is larger in transgenic than in wild-type animals. This indicates that additional events play a role in determining HSC numbers. Since BCL-2 overexpression by itself does not prevent differentiation in hematopoietic progenitor cells in vitro 24
, the limits on the increase of HSC numbers probably reflect a limit in the HSC-supporting microenvironment. The variation in HSC numbers observed between different transgenic animals may reflect variations in the number of niches available. However, it remains unclear what the regulatory events are that set these limits. More insights may come from some of the genetic analyses that are currently being undertaken (reviewed in reference 8). The fact that ST-HSC from H2K-BCL
-2–transgenic mice have a limited life span similar to that of wild-type HSC shows that the functional life span of short-term stem cells is not limited seriously by apoptosis. Commitment to further differentiation, rather than apoptosis, seems to limit clonal life span.
At each HSC cell division, the two daughter cells choose their cell fate, self-renewal or differentiation. This can be done symmetrically (both cells adopt the same fate) or asymmetrically (different fates). The balance determines the number of stem cells. The cells that start differentiation regulate their cell numbers by extensive proliferation and apoptosis 36
. Cell death rates seem to be higher at earlier stages of differentiation (CFU-S level) than at later stages (CFC) 37
. Progress in characterizing the signaling pathways involved in differentiation has been made; e.g., the notch
family of receptors 383940
. However, it is still unclear what determines whether a HSC self-renews or undergoes differentiation. In traditional models of hematopoiesis, with most HSC quiescent and only one or a few actively contributing, limited regulation is necessary. However, more recent insights into HSC dynamics [all HSC are cycling, albeit slowly 12
, and all can be made to cycle rapidly under certain conditions such as mobilization 41
] means that the fate of HSC has to be decided continuously. Under steady state conditions, ~8% of the LT-HSC in C57Bl/Ka mice complete cell division every day 2
. Assuming 3 × 108
bone marrow cells per mouse, and 0.01–0.02% 31
() of these cells are LT-HSC, this means that, every day, 2–5 × 103
LT-HSC complete cell division, producing 4–10 × 103
cells. Under steady state conditions, half of these remain LT-HSC, the remaining 2–5 × 103
cells either differentiate or undergo apoptosis. The regulation of HSC numbers (the decision to self-renew) has to be strictly regulated and enforced to prevent an unwanted expansion of HSC, with all the risks of transformation that entails. In this paper, we demonstrate that in addition to self-renewal and differentiation, a third option is open to HSC under steady state conditions in vivo: apoptosis.
It should be pointed out that while BCL
-2 is a proto-oncogene 424344
, the increase in HSC frequency in H2K-BCL
-2–transgenic mice (, ) and the increased competitive reconstitution of transgenic HSC upon transfer to irradiated hosts, did not result from the expansion of malignant clones; no stem-cell leukemias have been seen in these mice. However, in line with other BCL
-2–transgenic mouse models, some of the H2K-BCL
-2–transgenic mice do develop myeloid or lymphoid leukemias late in life.
BCL-2 overexpression increases the length of the G1
phase of the cell cycle 2245
. In H2K-BCL
-2–transgenic mice a similar phenomena is seen when transgenic HSC are compared with wild-type HSC with respect to the number of cells with >2n DNA. 5-Bromo-2′-deoxyuridine labeling experiments in vivo confirm that HSC in H2K-BCL
-2–transgenic mice cycle more slowly (Cheshier, S.H., J. Domen, and I.L. Weissman, manuscript in preparation). The decrease in the percentage of HSC undergoing cell division is accompanied by increased numbers of HSC, thus increasing the number of HSC offspring to slightly greater than wild-type levels. This could indicate a homeostatic adaptation, assuming that all dividing HSC contribute to the peripheral pool, an assumption supported by the observation that donor ratios in HSC and peripheral myeloid cells are similar. However, the fact that the proliferative potential present in even a fraction of the normal HSC number is sufficient to produce all the mature cells necessary would seem to argue against the need for such a feedback. Also, as shown, there is no inverse correlation between the percentage of HSC with >2n DNA and the percentage of HSC in bone marrow in either transgenic or wild-type mice that would be expected if such a feedback existed. If anything, especially in ST-HSC, the reverse is true. One study addressing the correlation between progenitor cell cycle kinetics and HSC numbers between different mouse strains fails to find a clear correlation for HSC, but reports a negative correlation for progenitors 46
. However, this may differ from regulation of numbers within an inbred strain. The reduction in cycling cells in tissues such as thymus in the transgenic mice is similar to other transgenic models 45
The difference in engraftment between H2K-BCL-2–transgenic and wild-type HSC in competitive repopulations shows that transgenic stem cells have a competitive advantage. The peripheral expansion of transgenic cells seen in reconstituted animals reflects increased survival of both HSC and more differentiated cells. However, direct determination of HSC ratios in reconstituted animals shows that the increase, and competitive advantage, starts at the level of HSC. This relative increase in BCL-2 overexpressing HSC does not only occur in the immediate post-reconstitution expansion period, when HSC are expanding rapidly, but continues gradually over a prolonged period of time. While there are minor antigenic differences between the CD45-congenic strains that are used for these competitive reconstitutions, the fact that this increase is not seen when wild-type CD45.2 cells are competed against wild-type CD45.1 cells argues strongly against the selective advantage being at this level. Consequently, overexpression of BCL-2, and thus protection against apoptosis, causes this expansion. This demonstrates that, after reconstitution, apoptosis plays an important role in regulating and limiting stem cell numbers. The main remaining challenge in this and other systems is the identification of the signals that control cell fate decisions of HSC.