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Reconstruction of the Hematopoietic System After Stem Cell Transplantation
Cell Transplantation, Vol. 7, No. 4, pp. 339 –344, 1998 © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0963-6897/98 $19.00 1 .00
PII S0963-6897(98)00012-8
Original Contribution RECONSTRUCTION OF THE HEMATOPOIETIC SYSTEM AFTER STEM CELL TRANSPLANTATION M.Y. GORDON
AND
N.M. BLACKETT
Department of Haematology, Imperial College School of Medicine, Hammersmith Hospital, London, UK
M Abstract — The practice of hematopoietic stem cell transplantation to rescue patients from the myeloablative effects of chemo- or radiotherapy, or to replace defective hematopoiesis, is based on the assumption that hematopoietic stem cells in the graft have sufficient proliferative potential to supply mature blood cells for the remainder of the recipient’s lifespan. However, the mechanism(s) whereby this is achieved are not well understood. Here we address the reconstruction of the hematopoietic system by considering the effects of stem cell and progenitor cell renewal and differentiation. We conclude that stem cell self-renewal is necessary for hematological recovery and that infused committed progenitor cells (CFU-GM) may contribute to the neutrophil count in the early posttransplant period. © 1998 Elsevier Science Inc.
cell populations ex vivo in order to increase the numbers available for transplantation. The most important early result of stem cell transplantation is the provision of mature neutrophils to combat infection, but the source of this initial recovery is not well understood. One view is that it results from the activity of lineage-restricted progenitors, such as granulocyte-macrophage colony-forming cells (CFU-GM), and that this is followed by engraftment of oligopotent progenitors and finally by stem cell engraftment (12). This hypothesis predicts that CFU-GM produce sufficient mature cells after transplantation to contribute to recovery. Another hypothesis (7) suggests that a seemingly uniform population of primitive hematopoietic stem cells might be able to provide hematopoietic repopulation in the short as well as in the long term.
M Keywords — Hematopoietic system; Stem cell transplantation.
INTRODUCTION HOW MANY STEM CELLS ARE THERE? DO STEM CELLS SELF-RENEW?
The ability of transplanted hematopoietic tissue to reconstitute hematopoiesis in myeloablated animals was demonstrated in 1956 (3) and by 1960, Lajtha and Oliver (14) had shown that recognizable granulocytic and erythroid cells are a transit population. These two pieces of information led to the realization that primitive hematopoietic stem cells must exist, and the debate about stem cell function began. In view of the long history, it may be surprising that the debate continues. Unanswered questions include how many stem cells are there?; do stem cells self renew?; do more mature progenitor cells self renew?; how much recovery is necessary? Clearly, the answers to these questions are relevant not only to stem cell recovery after transplantation but also to the success of gene therapy protocols and to efforts to expand stem
These two questions are interdependent because fewer stem cells will be needed to sustain haemopoiesis if they self-renew than will be needed if they do not. In essence, there are two alternative hypotheses concerning the ability of stem cells to self-renew: either that sufficient stem cells are produced during embryogenesis to supply the needs of the adult animal throughout life (Hypothesis 1); or that adult animals contain only a small number of stem cells that can self-replicate to produce more stem cells (Hypothesis 2) (15). In the context of stem cell transplantation these hypotheses mean either that sufficient stem cells are infused to supply a patient’s needs for the rest of his or her life or, alternatively, stem cell
ACCEPTED 1/9/98. Correspondence should be addressed to M.Y. Gordon, Im-
perial College School of Medicine, Department of Haematology, Hammersmith Hospital, London, UK W12 0NN. 339
Cell Transplantation ● Volume 7, Number 4, 1998
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Table 1. Assumptions 8
5
Stem cell frequency (see text) 4/10 –4/10 Daily neutrophil requirement 1011 9 Normal marrow cellularity (20,22) 14 3 10 /kg (1012/70/kg) Typical number of cells 1 3 108/kg (0.7%) transplanted ‘‘Seeding efficiency’’ 10% Effective transplanted cell dose 1 3 107/kg (0.07%) Optimal recovery factor 1400-fold Total body weight 70 kg
renewal occurs and expands the stem cell population after transplantation. Hypothesis 1 According to this hypothesis, stem cells are quiescent until they are needed to supply mature blood cells. If the hypothesis is correct, it has implications for both donor and recipient, unless normally there is a vast excess of stem cells in the marrow. One prediction of the hypothesis is that stem cells will be gradually used up during a person’s lifetime. Some insight into these implications may be obtained by simple ‘‘order of magnitude’’ calculations. First, the number of cell doublings needed to produce the required number of neutrophils per day (1011; Table 1) can be calculated assuming different rates of activation in the stem cell pool. If one stem cell is activated per day the number of cell doublings would be 36 (236 5 1011); if 1000 stem cells are activated per day the number of cell doublings would be 26. Using these estimates as boundaries, the minimum number of stem cells required for an entire lifetime of 100 years (36,500 d) would be 4 3 104 and 4 3 107, respectively. The corresponding concentrations of stem cells in 1012 marrow cells (Table 1) would be 4/108 and 4/105. Neither of these estimated concentrations of stem cells is unacceptable. Of far more significance to this hypothesis is the success of bone marrow transplantation. It can be estimated that a typical bone marrow harvest for allogeneic transplantation removes approximately 1% of the marrow (Table 1). It seems inescapable, based on hypothesis 1, that this amount will correspond to 1 yr of marrow function denied to a donor who expected to live to be 100. More seriously, with a marrow ‘‘seeding efficiency’’ of 10%, the recipient would be supplied with sufficient marrow function for only 0.1 yr. This scenario could be avoided if people are born with sufficient stem cells for 100,000 years of life (i.e., stem cells 1000-fold in excess of normal requirements). This would require a normal stem cell frequency of between 4/105 and 4/102. Of even more significance is the probably unique clinical occurrence of a father being the bone marrow
donor for his son and later the son being the donor for his father (21). Both the father and his son remain alive and well today (R. Powles, personal communication). In this highly unusual situation it is reasonable to assume, on the basis of effective transplanted marrow cell dose, that the father would have received a very small fraction of his own marrow (i.e., 0.1 3 0.1 3 0.1 5 0.001%) via his son. This calculation would raise the required frequency of stem cells a further 1000-fold. This reasoning is similar to the argument by Lord and Dexter (15), who calculated that the bone marrow of a single mouse has the potential to repopulate 1015 recipients.
Hypothesis 2 This hypothesis considers that stem cells can divide and that not all of the daughter cells differentiate. For steady-state hematopoiesis it is necessary for 50% of the daughter cells to differentiate and for the remaining 50% to replenish the stem cell pool. If the proportion of daughter cells that do not differentiate could change from 50% the number of stem cells could be increased (or decreased) under different demands for hematopoietic cell production. In consequence, not only would fewer stem cells be required but also more rapid hematological recovery would be possible in transplant recipients. The magnitude of regeneration needed to achieve full recovery can be estimated as shown in Table 1. Thus, a transplant of 1 3 108 marrow cells per kilogram is equivalent to 0.7% of the normal marrow cellularity, and will contain 0.7% of the normal stem cell number. This implies that the transplanted stem cells need to expand ;140-fold to return to normal levels. However, if only 10% of the stem cells reach the marrow, as indicated by studies of ‘seeding efficiency’ in mice (9) the required expansion factor will be 1400-fold. This introduces a discussion of the nature and proliferation kinetics of stem cells and their progeny which will form the remainder of this article.
WHAT IS A STEM CELL?
Hematopoietic stem cells are generally defined as predominantly quiescent cells that are capable of selfrenewal and giving rise to eight separate lineages of differentiated progeny. In contrast, progenitor cells are seen as more actively proliferating cells that are restricted in their self-renewal and lineage differentiation potentials. These descriptions of stem and progenitor cells raise several further questions that require definition: what maintains stem cell quiescence?; What is self-renewal?; How does lineage selection occur?
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Fig. 2. Model of hematopoietic cell development.
Fig. 1. Diagrammatic representation of the steps necessary for the derivation of eight hematopoietic cell lineages from a single stem cell.
Quiescence The microenvironment of the bone marrow cavity has for long been the candidate of choice for the negative regulation of stem cell activity. Hematopoietic stem cells are normally found almost exclusively in the marrow cavity, and primitive hematopoietic stem cells are known to be able to bind to marrow stromal cells in vitro (16,23). This association was the basis for the development of the long-term bone marrow culture system (2), which established the importance of stromal cells for hematopoietic cell regulation. Recently we were able to demonstrate that stromal cells, at least in vitro, exert a powerful influence in preventing primitive quiescent cells from entering the cell cycle. In other words, the primitive cells are more likely to remain quiescent when they are in the presence of stromal cells than when they are not (8). Self-Renewal and Lineage Commitment Self-renewal is what happens when cells divide and one or both of their progeny do not differentiate or die by apoptosis. In the case of the earliest hematopoietic stem cells, where there is no input of cells from a more primitive compartment, the population will be selfmaintaining if 50% of the stem cell progeny do not differentiate or die. In other words, the probability of self-renewal is 0.5, and the probability of differentiation is 0.5. In these terms, increased self-renewal would be apparent if fewer cells differentiated or fewer cells died. Consequently, self-renewal may be influenced in several ways and no special mechanism can be implied. Stem cells are held to be ultimately responsible for producing up to eight lineages of mature functional cells. For this to be accomplished by a single stem cell, the stem cell progeny that are destined for differentiation must divide a further three times to produce the requisite eight cells (Fig. 1). One perception is that these divisions may be associated with progressive loss of lineage
potential (18). An alternative perception is that these divisions occur without differentiation and that the eight cells available for differentiation are identical prior to lineage commitment. This view is in keeping with the observations of Greaves and colleagues (4 – 6,9,11), who report that several lineage specific genes are concurrently ‘‘primed’’ for transcription in uncommitted cells. It is also consistent with Ogawa’s hypothesis (17,19) that lineage selection is a stochastic process. The scheme in Fig. 1 allows differentiation to occur after one, two or three poststem cell divisions, but the number of lineages that can be derived from a single stem cell will be commensurately reduced. If, as the above discussion implies, poststem cells are capable of self-renewal, the distinction between stem cells and progenitor cells becomes less clearcut. Moreover, if self-renewal is a product of the positive influences of cellular input and proliferation balanced by the negative influences of differentiation and apoptosis, there seems to be no a priori reason to ascribe the capacity for self-renewal to stem cells alone. An aid to considering the implications is to think in terms of proliferating and maturational cell cycles. By proliferating cell cycles we mean a cell division at a given stage of maturation that does not result in further maturation; a maturational cell cycle, on the other hand, results in the progeny entering the next maturational stage. These concepts are illustrated diagrammatically in our working model of hematopoietic stem cell kinetics (Fig. 2). The model is restricted to the production of a single lineage (granulocyte production in the present context) for convenience and consists of four cell compartments. The first compartment (C1) represents the stem cell population. By definition, there will be no input into this compartment from a prior population and all of the cell gain is the result of self-renewal (i.e., cell production by mitosis minus loss by differentiation and apoptosis). The second and third compartments (C2 and C3) represent progenitor cells that are not self-maintaining and depend on input from the stem cell population. The final compartment (C4) consists of mature end cells where all of the gain is provided by input from earlier populations and all of the loss consists of cell death by apoptosis.
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cell recovery to normal levels as a result of reduced apoptosis, it would be necessary for 99.9% of stem cell daughter cells to die during normal steady-state hematopoiesis. While there is no formal proof that this is not the case, it seems counterintuitive that hematopoiesis should be so wasteful. The proliferating fraction of stem cells and their proliferation rate can influence the rate of hematopoietic cell production. However, it is not known whether the ability of stromal cells to maintain stem cell quiescence, or the response of the stem cells to proliferative restraint, alters after transplantation. An increase in proliferation rate will not, per se, increase cell production but will reduce the time taken for a certain level of recovery to be achieved. Fig. 3. Required number of stem cell generations for the complete regeneration of the stem cell compartment following transplantation, according to the probability of stem cell selfrenewal.
Changes in the numbers of cells in any compartment will depend on the balance between cell production and cell loss and self-renewal can now be seen as a balance between four processes (inflow, cell division, differentiation, and apoptosis). WHAT ARE THE DETERMINANTS OF STEM CELL REGENERATION?
The size of the stem cell population will increase only if the effective probability of self-renewal is greater than 0.5. Figure 3 shows the number of stem cell generations needed to generate the required 1400-fold amplification in stem cell number for complete restoration of the stem cell pool (see Table 1). It shows that the minimum number of stem cell generations is 10 if the probability of self-renewal is 1.0. However, in these circumstances the corresponding probability of differentiation is zero, and no differentiated cells will be produced. If the PSR is reduced, the number of cell generations needed to achieve normality increases (Fig. 3). It has been proposed that changes in the level of stem cell death (apoptosis) might be responsible for controlling changes in the size of the stem cell population (13). There is some evidence that this might be the case at some stages during erythroid maturation. To evaluate whether or not this mechanism might also operate at the stem cell level we need to evaluate the magnitude of apoptosis in normal hematopoiesis that would be required to account for recovery. Table 1 suggests that the effective transplanted stem cell dose is ;0.1% of the normal stem cell number. Consequently, to explain stem
WHAT DETERMINES THE TIME TAKEN FOR NEUTROPHIL RECOVERY?
One of the most important indications that a transplant has been successful is the time taken to achieve a certain level of neutrophils in the blood, 0.5 3 109/L being the value that is frequently used. It is reasonable to assume that eventually all of the neutrophils will originate from the stem cell pool, but there is less consensus concerning the origin of neutrophils in the short term. The frequency of stem cells has implications for the time taken for them to produce mature neutrophils. As we have seen from the ‘‘order of magnitude’’ calculations above, it would take 26 –36 days for a cell cycle time of 24 h and 13–18 days for a cycle time as short as 12 hs. Because, posttransplant, there is a conflicting need to repopulate the stem cell pool, from what is a small fraction of the normal stem cell number, other mechanisms are likely to contribute to posttransplant neutrophil recovery. It has been suggested that initially after transplantation neutrophils are derived from CFU-GM. However, calculations based on the results of in vitro cultures suggest at first sight that this is unlikely. The frequency of granulocyte macrophage colony-forming cells (CFU-GM) in bone marrow is about 1 in 103. Referring to Table 1, a transplant of 1 3 108 cells/kg (total 7 3 109) would be expected to contain ;7 3 106 CFU-GM. Given the frequency of CFU-GM, and the fact that the myeloid series accounts for some 75% of the nucleated marrow cellularity, it follows that CFU-GM, on average, are capable of producing 750 mature cells. Consequently, 7 3 106 CFU-GM can potentially generate ;5 3 109 neutrophils, which is much less than the normal daily rate of production (1011). Conversely, a transplant containing more than 108 CFU-GM, equivalent to a total nucleated cell dose of 1011, would be needed for the infused CFU-GM to supply neutrophils for 1 day.
Hematopoietic system after stem cell transplantation ● M.Y. GORDON
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reaches a new steady state (indicated by the plateau on the curve) because, with increasing numbers of cell generations, the contribution made by input from C1 (stem cells) becomes relatively minor compared with cell production by mitosis within C2. OVERVIEW
Fig. 4. Influence of the probability of self-renewal by poststem cells on their numbers relative to the size of the stem cell population.
Another possibility is that intermediate cell populations, such as CFU-GM, have a variable capacity to amplify through ‘‘self-renewal’’ that is increased when demand for neutrophils is high. This suggestion is quite acceptable if, as discussed earlier, self-renewal is simply the result of changes in cell division, differentiation, and cell death. We have used a CFU-GM replating assay to evaluate the amplification potential of CFU-GM, and have found that this ability can be increased by the addition of cytokines such as interleukin 3 (10). This result demonstrates that amplification of cell numbers within the CFU-GM population may be under exogenous control and responsive to demand. A further implication of variable self-renewal by CFU-GM relates to the relative sizes of the stem and progenitor cell populations. Estimates of stem cell frequency suggest that they may be 100 –1000 times less numerous than CFU-GM. This difference is equivalent to 6 –10 population doublings and implies either that 6 –10 maturational stages lie between the stem cells and the CFU-GM, or that some intermediate stage(s) along the maturation pathway are capable of expanding in number through self-renewal. To test the influence of such amplification we modeled the effects of renewal in C2 as well as C1 (see Fig. 1). For this purpose, the probability of self-renewal in C1 was 0.5, so that stem cell numbers did not change. Then, self-renewal probabilities of 0 to 0.5 were attributed to C2. As shown in Fig. 4, the result predicts that renewal at an intermediate stage is required for creation of the differential cell numbers, and that the magnitude of the differential achieved is reliant on the probability of intermediate cell renewal. The size of C2
In spite of the well-established clinical role of hematopoietic stem cell transplantation, many questions about the processes underlying hematological reconstitution remain to be fully answered. The same processes are likely to be essential for the successful expansion of stem cells ex vivo, with and without genetic manipulation. Consequently, the need to continue research into the regulation of hematopoietic cell kinetics in vivo and in vitro should not be underestimated. Finally, it is likely that the application of cell kinetic principles will be an essential component of the future success of ex vivo stem cell expansion protocols and of stem cell-mediated gene therapy. Acknowledgment — M.Y.G. was supported by the Leukaemia Research Fund of Great Britain.
REFERENCES
1. Coggle, J.E.; Gordon, M.Y. Quantitative measurements on the haemopoietic systems of three strains of mice. Exp. Hematol. 3:181–186; 1975. 2. Dexter, T.M.; Allen, T.D.; Lajtha, L.G. Conditions controlling the proliferation of hemopoietic stem cells in vitro. J. Cell Physiol. 91:335–344; 1977. 3. Ford, C.E.; Hamerton, J.L.; Barnes, D.W.H.; Loutit, J.F. Cytological identification of radiation chimeras. Nature 177:452– 454; 1956. 4. Ford, A.M.; Bennett, C.A.; Healy, L.E.; Towatari, M.; Greaves, M.F.; Enver, T. Regulation of the myeloperoxidase enhancer binding proteins Pu1, C-EBP alpha, -beta and -delta during granulocyte lineage specification. Proc. Natl. Acad. Sci. USA 93:10838 –10843; 1996. 5. Ford, A.M.; Healy, L.E.; Bennett, C.A.; Navarro, E.; Spooncer, E.; Greaves, M.F. Multilineage phenotypes of interleukin-3-dependent progenitor cells. Blood 79:1962– 1971; 1992. 6. Ford, A.M.; Bennett, C.A.; Healy, L.E.; Navarro, E.; Spooncer, E.; Greaves, M.F. Immunoglobulin heavy chain and CD3 delta chain gene enhancers are DNase I-hypersensitive in hemopoietic progenitor cells. Proc. Natl. Acad. Sci. USA 89:3424 –3428; 1992. 7. Gordon, M.Y.; Blackett, N.M.; Lewis, J.L.; Goldman, J.M. Evidence for a mechanism that can provide both short-term and long-term haemopoietic repopulation by a seemingly uniform population of primitive human haemopoietic precursor cells. Leukemia 9:1252–1256; 1995. 8. Gordon, M.Y.; Lewis, J.L.; Marley, S.B.; Grand, F.H.;
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9.
10.
11.
12.
13. 14.
15.
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Goldman, J.M. Stromal cells negatively regulate primitive haemopoietic progenitor cell activation via a phosphatidylinositol-anchored cell adhesion/signalling mechanism. Br. J. Haematol. 96:647– 653; 1977. Gordon, M.Y.; Ford, A.M.; Greaves, M.F. Cell interactions and gene expression in early hematopoisis. Int. J. Cell Cloning 8:11–25; 1990. Gordon, M.Y.; Blackett, N.M.; Marley, S.B.; Lewis, J.L.; Davidson, R.J.; Goldman, J.M. Evidence for amplification within committed granulocytic (CFU-GM) and erythroid (BFU-E) progenitor cell compartments and its regulation by interleukin-3 (IL-3) (submitted). Jiminez, G.; Griffiths, S.D.; Ford, A.M.; Greaves, M.F.; Enver, T. Activation of the beta-globin locus control region precedes commitment to the erythroid lineage. Proc. Natl. Acad. Sci. USA 89:10618 –10622; 1992. Jones, R.J.; Celano, P.; Sharkis, S.J.; Sensennbrenner, L.L. Two phases of engraftment established by serial bone marrow transplantation in mice. Blood 73:397– 401; 1989. Koury, M.J. Programmed cell death (apoptosis) in hematopoiesis. Exp. Hematol. 20:391–394; 1992. Lajtha, L.G.; Oliver, R. Studies on the kinetics of erythropoiesis. In: Wolstenholme, G.E.W. and O’Connor, M., Eds. Ciba Foundation Symposium on Haemopoiesis: Cell Production and its Regulation. London: J & A Churchill; 1960:289 –324. Lord, B.I.; Dexter, T.M. Which are the hematopoietic stem cells (or: Don’t debunk the history!). Exp. Hematol. 23:1237–1241; 1995.
16. McGlave, P.; Verfaillie, C.; Miller, J. Interaction of primitive human myeloid and lymphoid cells with the marrow microenvironment. Blood Cells 20:121–128; 1994. 17. Nakahata, T.; Gross, A.J.; Ogawa, M. A stochastic model of self-renewal and committment to differentiation of the primitive hemopoietic stem cells in culture. J. Cell Physiol. 113:455; 1982. 18. Ogawa, M.; Porter, P.N.; Nakahata, T. Renewal and commitment to differentiation of hemopoietic stem cells (an interpretive review). Blood 61:823; 1982. 19. Ogawa, M. Differentiation and proliferation of hematopoietic stem cells. Blood 81:2844 –2853; 1993. 20. Ryabov, S.I.; Shostka, G.D.; Vinogradova, T.V. Androgen therapy for anemia in renal failure. Int. Urol. Nephrol. 12:161–167; 1980. 21. Powles, R.L.; Parikh, P.M.; Helenglass, G.; Aboud, H.H.; Smith, C.L.; Mrazek, I.A.; Shepherd, V.; Milliken, S.T.; Treleaven, J.; Sharrock, C.; Feary, S.W.; Meller, S.T.; Lawler, S. Bone-marrow transplant from father to son and subsequent graft from son to father. Lancet 335:999 –1000; 1990. 22. Shostka, G.D. Bone marrow blood formation and iron stores in patients with chronic renal failure on maintenance dialysis. Folia Haematol. Int. Mag. Morphol. Blutforsch. 108:769 –777; 1981. 23. Siczkowski, M.; Clarke, D.; Gordon, M.Y. Binding of primitive hematopoietic progenitor cells to marrow stroma cells involves heparan sulfate. Blood 80:912–919; 1992.
PII S0963-6897(98)00012-8
Original Contribution RECONSTRUCTION OF THE HEMATOPOIETIC SYSTEM AFTER STEM CELL TRANSPLANTATION M.Y. GORDON
AND
N.M. BLACKETT
Department of Haematology, Imperial College School of Medicine, Hammersmith Hospital, London, UK
M Abstract — The practice of hematopoietic stem cell transplantation to rescue patients from the myeloablative effects of chemo- or radiotherapy, or to replace defective hematopoiesis, is based on the assumption that hematopoietic stem cells in the graft have sufficient proliferative potential to supply mature blood cells for the remainder of the recipient’s lifespan. However, the mechanism(s) whereby this is achieved are not well understood. Here we address the reconstruction of the hematopoietic system by considering the effects of stem cell and progenitor cell renewal and differentiation. We conclude that stem cell self-renewal is necessary for hematological recovery and that infused committed progenitor cells (CFU-GM) may contribute to the neutrophil count in the early posttransplant period. © 1998 Elsevier Science Inc.
cell populations ex vivo in order to increase the numbers available for transplantation. The most important early result of stem cell transplantation is the provision of mature neutrophils to combat infection, but the source of this initial recovery is not well understood. One view is that it results from the activity of lineage-restricted progenitors, such as granulocyte-macrophage colony-forming cells (CFU-GM), and that this is followed by engraftment of oligopotent progenitors and finally by stem cell engraftment (12). This hypothesis predicts that CFU-GM produce sufficient mature cells after transplantation to contribute to recovery. Another hypothesis (7) suggests that a seemingly uniform population of primitive hematopoietic stem cells might be able to provide hematopoietic repopulation in the short as well as in the long term.
M Keywords — Hematopoietic system; Stem cell transplantation.
INTRODUCTION HOW MANY STEM CELLS ARE THERE? DO STEM CELLS SELF-RENEW?
The ability of transplanted hematopoietic tissue to reconstitute hematopoiesis in myeloablated animals was demonstrated in 1956 (3) and by 1960, Lajtha and Oliver (14) had shown that recognizable granulocytic and erythroid cells are a transit population. These two pieces of information led to the realization that primitive hematopoietic stem cells must exist, and the debate about stem cell function began. In view of the long history, it may be surprising that the debate continues. Unanswered questions include how many stem cells are there?; do stem cells self renew?; do more mature progenitor cells self renew?; how much recovery is necessary? Clearly, the answers to these questions are relevant not only to stem cell recovery after transplantation but also to the success of gene therapy protocols and to efforts to expand stem
These two questions are interdependent because fewer stem cells will be needed to sustain haemopoiesis if they self-renew than will be needed if they do not. In essence, there are two alternative hypotheses concerning the ability of stem cells to self-renew: either that sufficient stem cells are produced during embryogenesis to supply the needs of the adult animal throughout life (Hypothesis 1); or that adult animals contain only a small number of stem cells that can self-replicate to produce more stem cells (Hypothesis 2) (15). In the context of stem cell transplantation these hypotheses mean either that sufficient stem cells are infused to supply a patient’s needs for the rest of his or her life or, alternatively, stem cell
ACCEPTED 1/9/98. Correspondence should be addressed to M.Y. Gordon, Im-
perial College School of Medicine, Department of Haematology, Hammersmith Hospital, London, UK W12 0NN. 339
Cell Transplantation ● Volume 7, Number 4, 1998
340
Table 1. Assumptions 8
5
Stem cell frequency (see text) 4/10 –4/10 Daily neutrophil requirement 1011 9 Normal marrow cellularity (20,22) 14 3 10 /kg (1012/70/kg) Typical number of cells 1 3 108/kg (0.7%) transplanted ‘‘Seeding efficiency’’ 10% Effective transplanted cell dose 1 3 107/kg (0.07%) Optimal recovery factor 1400-fold Total body weight 70 kg
renewal occurs and expands the stem cell population after transplantation. Hypothesis 1 According to this hypothesis, stem cells are quiescent until they are needed to supply mature blood cells. If the hypothesis is correct, it has implications for both donor and recipient, unless normally there is a vast excess of stem cells in the marrow. One prediction of the hypothesis is that stem cells will be gradually used up during a person’s lifetime. Some insight into these implications may be obtained by simple ‘‘order of magnitude’’ calculations. First, the number of cell doublings needed to produce the required number of neutrophils per day (1011; Table 1) can be calculated assuming different rates of activation in the stem cell pool. If one stem cell is activated per day the number of cell doublings would be 36 (236 5 1011); if 1000 stem cells are activated per day the number of cell doublings would be 26. Using these estimates as boundaries, the minimum number of stem cells required for an entire lifetime of 100 years (36,500 d) would be 4 3 104 and 4 3 107, respectively. The corresponding concentrations of stem cells in 1012 marrow cells (Table 1) would be 4/108 and 4/105. Neither of these estimated concentrations of stem cells is unacceptable. Of far more significance to this hypothesis is the success of bone marrow transplantation. It can be estimated that a typical bone marrow harvest for allogeneic transplantation removes approximately 1% of the marrow (Table 1). It seems inescapable, based on hypothesis 1, that this amount will correspond to 1 yr of marrow function denied to a donor who expected to live to be 100. More seriously, with a marrow ‘‘seeding efficiency’’ of 10%, the recipient would be supplied with sufficient marrow function for only 0.1 yr. This scenario could be avoided if people are born with sufficient stem cells for 100,000 years of life (i.e., stem cells 1000-fold in excess of normal requirements). This would require a normal stem cell frequency of between 4/105 and 4/102. Of even more significance is the probably unique clinical occurrence of a father being the bone marrow
donor for his son and later the son being the donor for his father (21). Both the father and his son remain alive and well today (R. Powles, personal communication). In this highly unusual situation it is reasonable to assume, on the basis of effective transplanted marrow cell dose, that the father would have received a very small fraction of his own marrow (i.e., 0.1 3 0.1 3 0.1 5 0.001%) via his son. This calculation would raise the required frequency of stem cells a further 1000-fold. This reasoning is similar to the argument by Lord and Dexter (15), who calculated that the bone marrow of a single mouse has the potential to repopulate 1015 recipients.
Hypothesis 2 This hypothesis considers that stem cells can divide and that not all of the daughter cells differentiate. For steady-state hematopoiesis it is necessary for 50% of the daughter cells to differentiate and for the remaining 50% to replenish the stem cell pool. If the proportion of daughter cells that do not differentiate could change from 50% the number of stem cells could be increased (or decreased) under different demands for hematopoietic cell production. In consequence, not only would fewer stem cells be required but also more rapid hematological recovery would be possible in transplant recipients. The magnitude of regeneration needed to achieve full recovery can be estimated as shown in Table 1. Thus, a transplant of 1 3 108 marrow cells per kilogram is equivalent to 0.7% of the normal marrow cellularity, and will contain 0.7% of the normal stem cell number. This implies that the transplanted stem cells need to expand ;140-fold to return to normal levels. However, if only 10% of the stem cells reach the marrow, as indicated by studies of ‘seeding efficiency’ in mice (9) the required expansion factor will be 1400-fold. This introduces a discussion of the nature and proliferation kinetics of stem cells and their progeny which will form the remainder of this article.
WHAT IS A STEM CELL?
Hematopoietic stem cells are generally defined as predominantly quiescent cells that are capable of selfrenewal and giving rise to eight separate lineages of differentiated progeny. In contrast, progenitor cells are seen as more actively proliferating cells that are restricted in their self-renewal and lineage differentiation potentials. These descriptions of stem and progenitor cells raise several further questions that require definition: what maintains stem cell quiescence?; What is self-renewal?; How does lineage selection occur?
Hematopoietic system after stem cell transplantation ● M.Y. GORDON
AND
N.M. BLACKETT
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Fig. 2. Model of hematopoietic cell development.
Fig. 1. Diagrammatic representation of the steps necessary for the derivation of eight hematopoietic cell lineages from a single stem cell.
Quiescence The microenvironment of the bone marrow cavity has for long been the candidate of choice for the negative regulation of stem cell activity. Hematopoietic stem cells are normally found almost exclusively in the marrow cavity, and primitive hematopoietic stem cells are known to be able to bind to marrow stromal cells in vitro (16,23). This association was the basis for the development of the long-term bone marrow culture system (2), which established the importance of stromal cells for hematopoietic cell regulation. Recently we were able to demonstrate that stromal cells, at least in vitro, exert a powerful influence in preventing primitive quiescent cells from entering the cell cycle. In other words, the primitive cells are more likely to remain quiescent when they are in the presence of stromal cells than when they are not (8). Self-Renewal and Lineage Commitment Self-renewal is what happens when cells divide and one or both of their progeny do not differentiate or die by apoptosis. In the case of the earliest hematopoietic stem cells, where there is no input of cells from a more primitive compartment, the population will be selfmaintaining if 50% of the stem cell progeny do not differentiate or die. In other words, the probability of self-renewal is 0.5, and the probability of differentiation is 0.5. In these terms, increased self-renewal would be apparent if fewer cells differentiated or fewer cells died. Consequently, self-renewal may be influenced in several ways and no special mechanism can be implied. Stem cells are held to be ultimately responsible for producing up to eight lineages of mature functional cells. For this to be accomplished by a single stem cell, the stem cell progeny that are destined for differentiation must divide a further three times to produce the requisite eight cells (Fig. 1). One perception is that these divisions may be associated with progressive loss of lineage
potential (18). An alternative perception is that these divisions occur without differentiation and that the eight cells available for differentiation are identical prior to lineage commitment. This view is in keeping with the observations of Greaves and colleagues (4 – 6,9,11), who report that several lineage specific genes are concurrently ‘‘primed’’ for transcription in uncommitted cells. It is also consistent with Ogawa’s hypothesis (17,19) that lineage selection is a stochastic process. The scheme in Fig. 1 allows differentiation to occur after one, two or three poststem cell divisions, but the number of lineages that can be derived from a single stem cell will be commensurately reduced. If, as the above discussion implies, poststem cells are capable of self-renewal, the distinction between stem cells and progenitor cells becomes less clearcut. Moreover, if self-renewal is a product of the positive influences of cellular input and proliferation balanced by the negative influences of differentiation and apoptosis, there seems to be no a priori reason to ascribe the capacity for self-renewal to stem cells alone. An aid to considering the implications is to think in terms of proliferating and maturational cell cycles. By proliferating cell cycles we mean a cell division at a given stage of maturation that does not result in further maturation; a maturational cell cycle, on the other hand, results in the progeny entering the next maturational stage. These concepts are illustrated diagrammatically in our working model of hematopoietic stem cell kinetics (Fig. 2). The model is restricted to the production of a single lineage (granulocyte production in the present context) for convenience and consists of four cell compartments. The first compartment (C1) represents the stem cell population. By definition, there will be no input into this compartment from a prior population and all of the cell gain is the result of self-renewal (i.e., cell production by mitosis minus loss by differentiation and apoptosis). The second and third compartments (C2 and C3) represent progenitor cells that are not self-maintaining and depend on input from the stem cell population. The final compartment (C4) consists of mature end cells where all of the gain is provided by input from earlier populations and all of the loss consists of cell death by apoptosis.
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cell recovery to normal levels as a result of reduced apoptosis, it would be necessary for 99.9% of stem cell daughter cells to die during normal steady-state hematopoiesis. While there is no formal proof that this is not the case, it seems counterintuitive that hematopoiesis should be so wasteful. The proliferating fraction of stem cells and their proliferation rate can influence the rate of hematopoietic cell production. However, it is not known whether the ability of stromal cells to maintain stem cell quiescence, or the response of the stem cells to proliferative restraint, alters after transplantation. An increase in proliferation rate will not, per se, increase cell production but will reduce the time taken for a certain level of recovery to be achieved. Fig. 3. Required number of stem cell generations for the complete regeneration of the stem cell compartment following transplantation, according to the probability of stem cell selfrenewal.
Changes in the numbers of cells in any compartment will depend on the balance between cell production and cell loss and self-renewal can now be seen as a balance between four processes (inflow, cell division, differentiation, and apoptosis). WHAT ARE THE DETERMINANTS OF STEM CELL REGENERATION?
The size of the stem cell population will increase only if the effective probability of self-renewal is greater than 0.5. Figure 3 shows the number of stem cell generations needed to generate the required 1400-fold amplification in stem cell number for complete restoration of the stem cell pool (see Table 1). It shows that the minimum number of stem cell generations is 10 if the probability of self-renewal is 1.0. However, in these circumstances the corresponding probability of differentiation is zero, and no differentiated cells will be produced. If the PSR is reduced, the number of cell generations needed to achieve normality increases (Fig. 3). It has been proposed that changes in the level of stem cell death (apoptosis) might be responsible for controlling changes in the size of the stem cell population (13). There is some evidence that this might be the case at some stages during erythroid maturation. To evaluate whether or not this mechanism might also operate at the stem cell level we need to evaluate the magnitude of apoptosis in normal hematopoiesis that would be required to account for recovery. Table 1 suggests that the effective transplanted stem cell dose is ;0.1% of the normal stem cell number. Consequently, to explain stem
WHAT DETERMINES THE TIME TAKEN FOR NEUTROPHIL RECOVERY?
One of the most important indications that a transplant has been successful is the time taken to achieve a certain level of neutrophils in the blood, 0.5 3 109/L being the value that is frequently used. It is reasonable to assume that eventually all of the neutrophils will originate from the stem cell pool, but there is less consensus concerning the origin of neutrophils in the short term. The frequency of stem cells has implications for the time taken for them to produce mature neutrophils. As we have seen from the ‘‘order of magnitude’’ calculations above, it would take 26 –36 days for a cell cycle time of 24 h and 13–18 days for a cycle time as short as 12 hs. Because, posttransplant, there is a conflicting need to repopulate the stem cell pool, from what is a small fraction of the normal stem cell number, other mechanisms are likely to contribute to posttransplant neutrophil recovery. It has been suggested that initially after transplantation neutrophils are derived from CFU-GM. However, calculations based on the results of in vitro cultures suggest at first sight that this is unlikely. The frequency of granulocyte macrophage colony-forming cells (CFU-GM) in bone marrow is about 1 in 103. Referring to Table 1, a transplant of 1 3 108 cells/kg (total 7 3 109) would be expected to contain ;7 3 106 CFU-GM. Given the frequency of CFU-GM, and the fact that the myeloid series accounts for some 75% of the nucleated marrow cellularity, it follows that CFU-GM, on average, are capable of producing 750 mature cells. Consequently, 7 3 106 CFU-GM can potentially generate ;5 3 109 neutrophils, which is much less than the normal daily rate of production (1011). Conversely, a transplant containing more than 108 CFU-GM, equivalent to a total nucleated cell dose of 1011, would be needed for the infused CFU-GM to supply neutrophils for 1 day.
Hematopoietic system after stem cell transplantation ● M.Y. GORDON
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N.M. BLACKETT
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reaches a new steady state (indicated by the plateau on the curve) because, with increasing numbers of cell generations, the contribution made by input from C1 (stem cells) becomes relatively minor compared with cell production by mitosis within C2. OVERVIEW
Fig. 4. Influence of the probability of self-renewal by poststem cells on their numbers relative to the size of the stem cell population.
Another possibility is that intermediate cell populations, such as CFU-GM, have a variable capacity to amplify through ‘‘self-renewal’’ that is increased when demand for neutrophils is high. This suggestion is quite acceptable if, as discussed earlier, self-renewal is simply the result of changes in cell division, differentiation, and cell death. We have used a CFU-GM replating assay to evaluate the amplification potential of CFU-GM, and have found that this ability can be increased by the addition of cytokines such as interleukin 3 (10). This result demonstrates that amplification of cell numbers within the CFU-GM population may be under exogenous control and responsive to demand. A further implication of variable self-renewal by CFU-GM relates to the relative sizes of the stem and progenitor cell populations. Estimates of stem cell frequency suggest that they may be 100 –1000 times less numerous than CFU-GM. This difference is equivalent to 6 –10 population doublings and implies either that 6 –10 maturational stages lie between the stem cells and the CFU-GM, or that some intermediate stage(s) along the maturation pathway are capable of expanding in number through self-renewal. To test the influence of such amplification we modeled the effects of renewal in C2 as well as C1 (see Fig. 1). For this purpose, the probability of self-renewal in C1 was 0.5, so that stem cell numbers did not change. Then, self-renewal probabilities of 0 to 0.5 were attributed to C2. As shown in Fig. 4, the result predicts that renewal at an intermediate stage is required for creation of the differential cell numbers, and that the magnitude of the differential achieved is reliant on the probability of intermediate cell renewal. The size of C2
In spite of the well-established clinical role of hematopoietic stem cell transplantation, many questions about the processes underlying hematological reconstitution remain to be fully answered. The same processes are likely to be essential for the successful expansion of stem cells ex vivo, with and without genetic manipulation. Consequently, the need to continue research into the regulation of hematopoietic cell kinetics in vivo and in vitro should not be underestimated. Finally, it is likely that the application of cell kinetic principles will be an essential component of the future success of ex vivo stem cell expansion protocols and of stem cell-mediated gene therapy. Acknowledgment — M.Y.G. was supported by the Leukaemia Research Fund of Great Britain.
REFERENCES
1. Coggle, J.E.; Gordon, M.Y. Quantitative measurements on the haemopoietic systems of three strains of mice. Exp. Hematol. 3:181–186; 1975. 2. Dexter, T.M.; Allen, T.D.; Lajtha, L.G. Conditions controlling the proliferation of hemopoietic stem cells in vitro. J. Cell Physiol. 91:335–344; 1977. 3. Ford, C.E.; Hamerton, J.L.; Barnes, D.W.H.; Loutit, J.F. Cytological identification of radiation chimeras. Nature 177:452– 454; 1956. 4. Ford, A.M.; Bennett, C.A.; Healy, L.E.; Towatari, M.; Greaves, M.F.; Enver, T. Regulation of the myeloperoxidase enhancer binding proteins Pu1, C-EBP alpha, -beta and -delta during granulocyte lineage specification. Proc. Natl. Acad. Sci. USA 93:10838 –10843; 1996. 5. Ford, A.M.; Healy, L.E.; Bennett, C.A.; Navarro, E.; Spooncer, E.; Greaves, M.F. Multilineage phenotypes of interleukin-3-dependent progenitor cells. Blood 79:1962– 1971; 1992. 6. Ford, A.M.; Bennett, C.A.; Healy, L.E.; Navarro, E.; Spooncer, E.; Greaves, M.F. Immunoglobulin heavy chain and CD3 delta chain gene enhancers are DNase I-hypersensitive in hemopoietic progenitor cells. Proc. Natl. Acad. Sci. USA 89:3424 –3428; 1992. 7. Gordon, M.Y.; Blackett, N.M.; Lewis, J.L.; Goldman, J.M. Evidence for a mechanism that can provide both short-term and long-term haemopoietic repopulation by a seemingly uniform population of primitive human haemopoietic precursor cells. Leukemia 9:1252–1256; 1995. 8. Gordon, M.Y.; Lewis, J.L.; Marley, S.B.; Grand, F.H.;
344
9.
10.
11.
12.
13. 14.
15.
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Goldman, J.M. Stromal cells negatively regulate primitive haemopoietic progenitor cell activation via a phosphatidylinositol-anchored cell adhesion/signalling mechanism. Br. J. Haematol. 96:647– 653; 1977. Gordon, M.Y.; Ford, A.M.; Greaves, M.F. Cell interactions and gene expression in early hematopoisis. Int. J. Cell Cloning 8:11–25; 1990. Gordon, M.Y.; Blackett, N.M.; Marley, S.B.; Lewis, J.L.; Davidson, R.J.; Goldman, J.M. Evidence for amplification within committed granulocytic (CFU-GM) and erythroid (BFU-E) progenitor cell compartments and its regulation by interleukin-3 (IL-3) (submitted). Jiminez, G.; Griffiths, S.D.; Ford, A.M.; Greaves, M.F.; Enver, T. Activation of the beta-globin locus control region precedes commitment to the erythroid lineage. Proc. Natl. Acad. Sci. USA 89:10618 –10622; 1992. Jones, R.J.; Celano, P.; Sharkis, S.J.; Sensennbrenner, L.L. Two phases of engraftment established by serial bone marrow transplantation in mice. Blood 73:397– 401; 1989. Koury, M.J. Programmed cell death (apoptosis) in hematopoiesis. Exp. Hematol. 20:391–394; 1992. Lajtha, L.G.; Oliver, R. Studies on the kinetics of erythropoiesis. In: Wolstenholme, G.E.W. and O’Connor, M., Eds. Ciba Foundation Symposium on Haemopoiesis: Cell Production and its Regulation. London: J & A Churchill; 1960:289 –324. Lord, B.I.; Dexter, T.M. Which are the hematopoietic stem cells (or: Don’t debunk the history!). Exp. Hematol. 23:1237–1241; 1995.
16. McGlave, P.; Verfaillie, C.; Miller, J. Interaction of primitive human myeloid and lymphoid cells with the marrow microenvironment. Blood Cells 20:121–128; 1994. 17. Nakahata, T.; Gross, A.J.; Ogawa, M. A stochastic model of self-renewal and committment to differentiation of the primitive hemopoietic stem cells in culture. J. Cell Physiol. 113:455; 1982. 18. Ogawa, M.; Porter, P.N.; Nakahata, T. Renewal and commitment to differentiation of hemopoietic stem cells (an interpretive review). Blood 61:823; 1982. 19. Ogawa, M. Differentiation and proliferation of hematopoietic stem cells. Blood 81:2844 –2853; 1993. 20. Ryabov, S.I.; Shostka, G.D.; Vinogradova, T.V. Androgen therapy for anemia in renal failure. Int. Urol. Nephrol. 12:161–167; 1980. 21. Powles, R.L.; Parikh, P.M.; Helenglass, G.; Aboud, H.H.; Smith, C.L.; Mrazek, I.A.; Shepherd, V.; Milliken, S.T.; Treleaven, J.; Sharrock, C.; Feary, S.W.; Meller, S.T.; Lawler, S. Bone-marrow transplant from father to son and subsequent graft from son to father. Lancet 335:999 –1000; 1990. 22. Shostka, G.D. Bone marrow blood formation and iron stores in patients with chronic renal failure on maintenance dialysis. Folia Haematol. Int. Mag. Morphol. Blutforsch. 108:769 –777; 1981. 23. Siczkowski, M.; Clarke, D.; Gordon, M.Y. Binding of primitive hematopoietic progenitor cells to marrow stroma cells involves heparan sulfate. Blood 80:912–919; 1992.