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Stem Cells, Vol. 16, No. 2, 112-119, March 1998
© 1998 AlphaMed Press

Stem Cell Factor Improves the Repopulating Ability of Primitive Hematopoietic Stem Cells after Sublethal Irradiation (and, to a Lesser Extent) after Bone Marrow Transplantation in Mice

Renee V. Gardnera, Peter Olivera, C.M. Astleb

a LSU Medical Center, New Orleans, Louisiana, USA;
b The Jackson Laboratory, Bar Harbor, Maine, USA

Key Words. Cytokines • Marrow transplantation • Radiation • Radioprotection

Dr. Renee V. Gardner, Department of Pediatrics, LSU Medical Center, 1542 Tulane Avenue, New Orleans LA 70112, USA.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Bone marrow transplantation (BMT) and sublethal irradiation (XRT) cause profound long-term damage to hematopoietic stem cells. We used the competitive repopulation assay in mice to test the ability of granulocyte-macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF), cytokines given in clinical settings to enhance marrow recovery after XRT or BMT and to protect the marrow repopulating ability of primitive hematopoietic stem cells (PHSC) after these modalities. The repopulating ability of exhaustible multilineage progenitors (EMP) was also tested after these modalities, with or without cytokines. Repopulating abilities of EMP and PHSC were significantly reduced after XRT or BMT; PHSC were preferentially affected. Administration of SCF to C57B6/J mice after XRT resulted in improved EMP and PHSC repopulating ability, although progenitor numbers—repopulating units—were not completely returned to control levels. Whether given as a single dose or multiple doses, GM after XRT did protect PHSC function from the deleterious effects of XRT, but this was not a significant effect. SCF caused an increase in PHSC repopulating ability after BMT, but this too was not a significant difference. GM after BMT had little effect. SCF administration before XRT led to severe impairment of PHSC function with very little or no stem cell activity observed. Therefore, timing of its administration is an important consideration since preadministration of the cytokine before XRT can be extremely harmful to PHSC function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The proliferative and self-renewal capacities of hematopoietic stem cells (HSC) are impaired after bone marrow transplantation (BMT) [1-5] and sublethal irradiation (XRT) [6-10]. Primitive hematopoietic stem cells (PHSC), responsible for life-long marrow stem cell reconstitution, are most significantly impacted by these treatment modalities. That PHSC have a diminished ability to repopulate the marrow of lethally irradiated recipients after BMT is demonstrated by both serial transplantation [1] and competitive repopulation [2, 4, 6]. This effect is apparently unrelated to aging by HSC or to the number of stem cells transplanted [2-4]. Nor does this effect appear to be strictly due to radiation-derived stromal damage [6]. The effects of stem cell manipulation with ensuing numerical losses, aberrant homing of transplanted stem cells, ablation of progenitors responsible for the initial phase of engraftment [11], or the damage of stem or stromal cells by free radicals [9] after irradiation are hypotheses advanced to explain this phenomenon.

Fortunately, clinically apparent stem cell damage after BMT is rare, but Champlin, Feig, and Gale have reported the findings of delayed incomplete recovery of hematopoiesis in a patient with aplastic anemia who had undergone BMT and initially had sustained engraftment [12]. The cause of the engraftment failure was apparently damage to stem cells. Torok-Storb et al. reported several patients who had undergone BMT and subsequently developed persistent post-engraftment pancytopenia with no evidence of donor-host chimerism or rejection [13]. In another study, of 122 patients who received HLA-identical marrow, 34% developed late marrow failure [14]. More recently, poor graft function after allogeneic BMT was noted in two patients who had no evidence of rejection and required supplemental infusions of peripheral blood progenitor cells before achieving adequate engraftment [15]. However, the more common clinical manifestations of bone marrow defect after BMT are isolated cytopenias such as chronic thrombocytopenia [16] or neutropenia [17].

Irradiation also damages stem cells and their microenvironment [18, 19], causing structural damage to DNA, damage which may not be repaired completely [20, 21]. Irradiation causes damage to cell surface membranes, cytoplasmic organelles, and DNA synthesis and repair, perhaps partially as a result of free-radical generation [19]. Although PHSC are relatively radioresistant, functional impairment and stem cell losses may be observed within this compartment as well as among more committed cells [22, 23].

Cytokines such as recombinant human granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been used in BMT to accelerate hematopoietic recovery and treat cases of bone marrow failure [24-27]. They have also been reported to enhance survival in mice after their exposure to lethal and sublethal doses of irradiation [28, 29]. We have performed the competitive repopulation assay [30] using C57Bl6/J mice to provide both a qualitative and quantitative assessment of late and early precursor function after BMT and XRT use with cytokines. Results showing that both stem cell factor (SCF) and GM-CSF improve PHSC repopulating ability after XRT, SCF significantly so, while only SCF leads to increased repopulating ability after BMT are described.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
Congenic strains of mice, C57Bl6/J (B6) and B6— Hbbd—Gpi-1a (GPI) (The Jackson Laboratory; Bar Harbor, ME) were used in all experiments. All the mice used were females aged 8 to 12 weeks old.

B6 and competitor B6—Hbbd—Gpi-1a mice have different alleles at their Hbb and glucose phosphate isomerase loci. B6 mice are homozygous for the Hbbs locus specifying {alpha}2ß2s (single) Hb, the Hb migrating rapidly on Hb electrophoresis. They also produce Gpi-1b glucose phosphate isomerase isoenzyme. The GPI congenic strain is homozygous for the Hbbd locus, having {alpha}2ß2dmaj and {alpha}2ß2dmin (diffuse) Hb, migrating at intermediate and slow speeds on electrophoresis. They produce Gpi-1a isoenzyme, which has a different electrophoretic pattern from the B6 isoenzyme. While the mice also differ at the H-1 locus, this difference has been shown to have no significant bearing on BMT outcome when the two congenic strains are used [31].

Cytokines
Murine recombinant GM-CSF was obtained from two sources: Genzyme (Cambridge, MA) with a specific activity of <5 x 106 units/mg and R & D Systems (Minneapolis, MN), specific activity of 3.5 x 105 units/mg. These preparations have >90% purity having been tested for and found free of pyrogens. We are indebted to Amgen (Thousand Oaks, CA) and Dr. K. Zsebo for her gift of pegylated recombinant rat SCF (lot RS5) which was supplied in a concentration of 1.6 mg/ml. In some experiments, recombinant mouse SCF was alternatively used (R & D Systems) with an ED50 of 5-10 ng/ml or 1.33 x 105 U/mg. Purity again was high and pyrogen was absent. All cytokines were in buffered solution with bovine serum albumin.

Transplantation and Irradiation
A representative experiment can be seen in Figure 1. For one group of animals, B6 female mice 8 to 12 weeks old were lethally irradiated (1,100 cGy). Twenty-four hours later, they received 106 cells of same-sex B6 bone marrow through tail-vein injection. After 24 h, transplanted mice either received no cytokine, or were given 1 mg GM-CSF s.c. as a single dose (BMT-GM-1), five daily doses (BMT-GM-5), or five daily doses of 0.68 mg SCF s.c. (~100 mg/kg/dose) (BMT-SCF). Other groups included the following: mice which received 500 cGy only XRT; XRT plus GM-CSF as a single dose of 1 mg s.c. after irradiation (XRT-GM-1), or five daily doses of 1 mg s.c. (XRT-GM-CSF-5); XRT plus five daily s.c. doses of SCF post-irradiation (XRT-SCF), or XRT with a dose of SCF (0.68 mg) given 20-24 h beforehand (SCF-XRT). Control mice were untreated B6 females. Mice receiving XRT with/without cytokine were allowed to recover over four to eight weeks from time of treatment before sacrifice. Transplanted mice were allowed four to eight weeks for marrow reconstitution and were then sacrificed. Previous experience has shown no significant differences in PHSC cellular function whether the animals have been sacrificed at four or eight weeks.



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  		Figure 1. Schematic representation of experiments examining the effects of cytokine after XRT or BMT on PHSC.

 
Competitive Repopulation
The competitive repopulation assay was used, as previously described [30]. The bone marrow from the mice in each donor group was obtained by flushing the femurs and tibias of the mice with 1-{alpha}-MEM or IMDM (GIBCO; Grand Island, NY). Cellular concentrations were adjusted to allow 106 cells of B6 donor type to mix with an equal number of competitor cells from unmanipulated B6-Hbbd-Gpi-1a mice. Equal portions or aliquots of these mixtures were then injected into lethally irradiated same-sex B6 recipients. Marrow reconstitution was complete at 90 days postinfusion. Blood was obtained by retro-orbital puncture at 30 and 150 days and subjected to glucose phosphate isomerase electrophoresis. Densitometric analysis was performed to give the percentage of cells having donor (Gpi 1b or B6) markers.

Statistical Analyses
Precursor numbers can be referred to as relative repopulating units (RU) and can be estimated by using the following formula [32]:


RU= (%) (# of 105 untreated competitor marrow cells)/(100-%), where % = percentage donor type cell.

 

One RU is defined as the repopulating ability of 105 untreated competitor marrow cells. If the donor cell population functions normally, 105 untreated donor cells should contain approximately 1 RU and should repopulate as well as 105 untreated competitor marrow cells. If, however, fewer RU are present it suggests that the number of cells available among donor cells which are capable of marrow reconstitution is fewer than that found in a comparable pool of competitor cells, that repopulating abilities per cell are reduced, or both.

Two to three replicates of each experiment were performed. Levels of significance for comparison of results were determined by analysis of variance (ANOVA), with results expressed as means ± standard error of the means.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Marrow Cell Counts
Marrow cell counts are recorded as the average of the values from each experiment and are shown in Table 1. The greatest reduction in marrow cellularity was seen in mice receiving SCF before or after XRT, with cell numbers falling to about 40%-70% of those seen for the untreated or control group. No decline in marrow cell numbers was seen in any of the BMT treatment groups. The use of GM after treatment with BMT and XRT was associated with numbers greater than those seen in the control group; we are unable to offer an analysis of statistical significance.


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  		Table 1. Results of competitive repopulation at 30 days' assessment of EMP
 
Exhaustible Multilineage Precursor (EMP) Function
Measurement of donor marrow precursor input to circulating blood cells in recipients at 30 days permits a study of populations of marrow precursors which lack the long-term repopulating capacity of PHSC but which give rise to both myeloid and lymphoid lineages [33]. The precursors measured at this early date are considered exhaustible. We admit that it is impossible to measure the relative proliferative contribution of PHSC at this time. We also acknowledge that other cells, including a subset of CFU-S at day 12, have been shown to display transient marrow repopulating ability and may contribute to early marrow repopulation [10, 34, 35]. However, we feel that the early EMP has a major input in the early repopulation of both lymphoid and myeloid systems [33]. These cells have been shown to be separable from PHSC on the basis of limiting dilution assays and cell surface antigenic characteristics and are not identical with CFU-S [36-39]. Measurement of donor percentages at or beyond 90 days gives us determinations of donor marrow PHSC input.

Results of experiments examining EMP function are recorded in Table 1. Cells which were exposed to XRT or which had undergone previous BMT suffered significant damage to their early marrow repopulating ability. Thirty days after competitive repopulation, donor cell percentages of 20% ± 3% and 23% ± 3% were recorded for irradiated and previously transplanted cells, respectively. These values were less than half of the control value of 49% ± 3%, a significant difference at p < 0.01. Partial restoration of EMP occurred when SCF was administered 24 h after XRT or BMT. The use of SCF resulted in increases of repopulating ability to 34% ± 1% after XRT and to 42% ± 8% after BMT for both treatment comparisons, while a single dose of GM after XRT or BMT led to donor cell percentages of about 30%. These were not, however, significant differences from treatment-only results. On the other hand, the administration of SCF 24 h before XRT resulted in significant reductions in donor cell percentages to 7% ± 2% (p < 0.001).

RU concentrations were reduced from control level for all treatment groups, although this decrease was less after the addition of SCF after BMT or XRT. Still, concentrations of RU were only 50%-70% of control. Total RU numbers for EMP were calculated using the marrow cell numbers from respective treatment groups. Total EMP RU for the control group was 375/donor. Total RU after XRT and BMT declined to respective values of 115 and 165. Use of SCF after XRT resulted in improved EMP function with regard to percentage of donor cells, there being no improvement in total RU numbers, 79. Total RU rose to 60%-75% of control numbers once either cytokine (given in multiple doses) was administered after BMT. At least 50% of control RU numbers was achieved with GM use after XRT. Administration of SCF before XRT caused an even greater decline in EMP RU than was seen with XRT alone, with total RU/donor being 16; RU concentration was only 6% of the control value.

Long-Term Repopulating Ability
Results of long-term PHSC function as competitive repopulation at 150-180 days are shown in Table 2. Neither cytokine, when administered to untreated animals, led to increases in marrow stem cell content or repopulating ability (data not shown). XRT and BMT were both very deleterious to PHSC repopulating ability. Donor marrow percentages of 6% ± 2% and 9% ± 2% were seen, with the control percentage being 54% ± 4%. Administration of SCF after XRT led to a seven-fold increase in PHSC repopulating ability (44% ± 5% [p< 0.001]) over XRT only. GM did not significantly increase PHSC repopulating ability, although donor percentages increased fourfold to about 23%-24% after XRT. Significantly, preadministration of SCF before XRT was a lethal treatment combination since no donor cells survived.


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  		Table 2. Results of long-term competitive repopulation at 150-180 days: assessment of PHSC function
 
The impact of cytokines on PHSC function after BMT was less striking. GM after BMT had no significant effect, but SCF when given after BMT led to a slightly greater than three-fold restoration of PHSC function than with BMT alone, with a donor percentage of 30% ± 9%.

The PHSC RU concentrations after the addition of SCF to XRT treatment were improved 13-fold over the XRT only group and were almost 70% of control values. No comparable rise in RU concentration was seen in any of the other cytokine-treated animals, although SCF after BMT led to four times the concentration of RU as seen with BMT only.

RU/donor numbers were drastically reduced for the XRT only treatment group (RU = 23, as compared to control numbers of 450), while again no RU survived with preadministration of SCF. GM after XRT resulted in increases in RU/donor number from 23 to 146 for a single dose and 195 total RU for multiple doses. SCF given after XRT led to a fivefold increase in RU numbers to 126, 28% of control numbers.

BMT led to a drop in RU numbers from a control value of 450 to 55, but administration of SCF after BMT increased total RU to 167, a threefold difference. Total RU after a single dose of GM increased to 97, while multiple doses of GM led to an expansion of RU numbers to 231 (>50% of control values).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Stem cell numbers, as estimated by total RU numbers, are drastically reduced, falling to between 3% and 12% of control levels after XRT and BMT have been administered in murine experiments. These declines in function and stem cell number occur irrespective of donor cell numbers. Transplantation may occur in two distinct phases, the first being an unsustained period of engraftment leading to early hematologic support and immediate recipient survival and the second being a more permanent sustainable engraftment for which PHSC are primarily responsible [11, 40]. It has been suggested that one of the reasons for the repopulating defect seen in PHSC after BMT may be loss of those progenitors responsible for the initial engraftment phase [11]. Our data point to reductions in donor cell percentages of >50% during the early post-engraftment period (27 days). Then, the majority of the replicative effort appears to be made by a distinct EMP population, although there may be an indeterminable contribution by PHSC and other progenitors at this early time point, as well. The stem cell numbers, as calculated by RU, are diminished with XRT resulting in a decrease in EMP RU numbers to 30% of control and with BMT to less than 50% of control. Still, comparison of short- and long-term repopulation results reveals a disproportionate deleterious impact upon PHSC rather than EMP, a result consistent with previous experience [5].

Clinically, cytokines have been reported as effective to varying extent in the treatment of engraftment failure [25, 41]. Survival of bone marrow grafts and concomitant improvement in animal survival have been observed in recipients of BMT after SCF alone [42]. Animals have also been rescued from anti-MHC class II antibody-mediated marrow graft failure by SCF [43]. Mardiney and Malech have postulated that administration of growth factors may result in stromal activation making the stroma more receptive to incoming stem cells [44]. Other investigators have also demonstrated that activation of SCF receptor-induced signaling does not appear to be a rate-limiting step in the control of hematopoietic production [45]. So, stromal receptivity may not be a factor here for BMT results; our experiments, in contrast with those of Mardiney and Malech, demonstrated enhanced PHSC survival with post-administration of cytokine only.

GM was not effective in improving the repopulating ability of previously transplanted marrow stem cells; the repopulating ability of both early and late engrafting precursors was not significantly greater than that of cells subjected to transplantation alone. However, despite nonsignificant changes in repopulating ability as measured by donor percentage, marrow cell count, and, accordingly, total RU or precursor numbers (231) were increased relative to the BMT alone values (55) by almost fourfold when GM was given as multiple doses after BMT. Total marrow cell counts were also elevated relative to both control and BMT-only cell numbers. In this set of experiments, only marrow precursor function was assayed; we did not examine the effects of GM or SCF on hematopoietic precursor function in the spleen or liver. Therefore, active hematopoiesis could conceivably be extant after BMT in those organs. If so, the impact of GM after BMT (or even XRT) on PHSC function or hematopoiesis as a whole may be underestimated.

Our data show that SCF was able to partially restore both stem cell function and numbers with a threefold increase in total RU numbers for PHSC. A lesser but still impressive increment in RU for the exhaustible progenitors over the BMT-only treatment group was also documented. Unfortunately, this latter difference is not statistically significant, but the data are consistent with results reported by other investigators of enhancement of survival of transplanted marrow after SCF administration [46-49]. In other reports, in vivo administration of SCF has been reported to lead to increases in primitive stem cells and other progenitors, as well as more differentiated cell types within the marrow [50]. Exogenous, pharmacologic doses of SCF could supplement and enhance the already-present production or expression of cytokines such as SCF, normally seen in marrow after irradiation [51], accelerating repair of hypoplastic injury and recovery of hematopoiesis.

GM after XRT brought about a fourfold increase in PHSC repopulating ability than after XRT alone. This was not, however, a statistically significant change. Nevertheless, while total RU or precursor numbers were not completely restored to control levels, they increased from 23 after XRT alone to 146 and 195 when GM was administered once or for five days after XRT, probably reflecting increases in donor marrow cell count. SCF did offer significant protection to PHSC after XRT, with a return to untreated or control levels of donor cell percentage. The percentage of donor cells increased from 6% with XRT alone to 44% after using SCF. This improvement in functional performance was not, however, accompanied by a proportionate improvement in stem cell numbers. Although stem cell numbers rose to almost six times those of the XRT-only group, they still remained at a level which was one-third of the control group. Improvement in individual cells' replicative ability is then inferred, with the replicative burden placed on each cell being decreased after cytokine administration. Increases in repopulating ability in bone marrow cells after SCF usage have been previously reported; marrow repopulating ability was higher than normal for up to six weeks after termination of treatment with SCF, an increase confirmed by injection of marrow into unconditioned recipients where engraftment was improved sevenfold over that of untreated marrow [48]. Use of GM after XRT resulted in rises in donor cell percentages to about 24%, an insignificant change, and increased both EMP and PHSC numbers.

Administration of SCF before lethal irradiation has previously been reported as being associated with an increase in progenitor cell numbers and LD50 among mice [47, 52, 53]. For this reason, we were surprised that SCF when administered prior to XRT resulted in complete loss of PHSC activity and numbers. The enhanced sensitivity to XRT could result from the induction of accelerated cycling of stem cells by SCF [54-56], with resultant vulnerability to XRT damage.

Our data validate the current practice of using cytokines to enhance post-engraftment recovery and hematologic performance, although the superior effect of SCF after BMT may lend impetus to the exploration of alternative cytokines other than the currently used GM. Further work is needed to confirm this benefit. It is also possible that combinations of SCF and GM may have an additive beneficial effect on stem cell function; further testing may be warranted.


   Acknowledgments
 
The authors wish to thank Mrs. Jessye Hilliard for her invaluable assistance in manuscript preparation, Mrs. Bea Stork and Avis Silva for performance of electrophoreses and densitometric analysis, and Dr. David Harrison for his generous provision of mice and technical advice.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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accepted for publication December 23, 1997.



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