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Stem Cells, Vol. 18, No. 2, 120-127, March 2000
© 2000 AlphaMed Press

Granulocyte-Colony Stimulating Factor Impedes Recovery from Damage Caused by Cytotoxic Agents through Increased Differentiation at the Expense of Self-Renewal

Ronald van Os, Simon Robinson, Tara Sheridan, Peter M. Mauch

Department of Radiation Oncology, Brigham and Women's Hospital and the Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachustetts, USA

Key Words. Hematopoiesis • Primitive stem cells • G-CSF • Cytotoxic agents • BCNU • Cobblestone area-forming cell

Correspondence: Ronald van Os, Ph.D., Department of Hematology, Leiden University Medical Center, Building 1, C2-R, PO Box 9600, 2300 RC Leiden, The Netherlands. Telephone: 31-71-5262271; Fax: 31-71-5266755; e-mail: rvanos{at}lumc.nl


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
G-CSF is routinely used to hasten recovery from chemotherapy-induced neutropenia. We have recently shown that G-CSF, when combined with stem cell-damaging cytotoxic agents, results in enhanced stem cell damage and loss of marrow reserve. To investigate the mechanisms of stem cell damage caused by G-CSF, we gave C57BL/6 (B6) mice repeated doses of cyclophosphamide ([CY] 84 mg/kg) or carmustine ([BCNU] 13.2 mg/kg) and G-CSF (250 µg/kg/day) for either four days or eight days. Two different regimens of G-CSF were chosen to study the influence of increased proliferation on hematopoiesis which was measured at the end of the first, third and sixth 14-day cycle of each cytotoxic agent and 7 and 20 weeks after completion of all cycles. A spectrum of hematopoietic indices was measured including WBC, bone marrow cellularity, granulocyte/macrophage-colony-forming cells (GM-CFC), colony-forming cells with high proliferative-potential (HPP-CFC), cobblestone area-forming cells ([CAFC]-day 7 and CAFC-day 28), and long-term marrow repopulating ability in vivo.

Despite the absence of differences in peripheral blood cell counts or bone marrow cellularity 14 days after each dose, progenitor cell levels (HPP-CFC, GM-CFC, and CAFC-7) were increased up to 2.5-fold with cytotoxic agent and G-CSF administration compared with cytotoxic agent administration alone. Mice given G-CSF for eight days had the greatest number of progenitors suggesting a dose-response relationship for G-CSF administration. G-CSF resulted in a decrease in hematopoietic stem cell (CAFC-28) content when measured two weeks after each cycle of saline, CY, and BCNU. Twenty weeks after six cycles of BCNU, the reduction in stem cell levels persisted and was further decreased when G-CSF was added to BCNU for four or eight days.

Data from this study suggest that the most likely explanation for the damaging effects of G-CSF is that G-CSF directly or indirectly induces stem cells to differentiate into more committed hematopoietic cells resulting in a loss of marrow reserve. This effect is enhanced in animals with an already compromised hematopoietic stem cell compartment as seen with repeated doses of BCNU.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In experimental studies, it has been shown that certain cytotoxic agents can permanently damage the stem cell compartment. This can occur even when peripheral blood and marrow counts remain normal. For example, busulfan, carmustine (BCNU) and cyclophosphamide (CY) (at high doses) can lead to a permanent loss in marrow reserve capacity [1-6]. Other experimental settings where there is a loss of marrow reserve include transplantation of marrow previously exposed to certain cytotoxic agents [6] and extensive proliferative stress to the marrow [7-9]. In the clinic, the use of autologous donor cells previously exposed to multiple courses of multiagent chemotherapy, or higher doses of chemotherapy and G-CSF, has been associated with a prolonged time to engraftment after marrow transplantation [10-12]. All these studies suggest that the bone marrow has a limited reserve capacity.

G-CSF has been increasingly used to circumvent the acute dose-limiting myelotoxicity of treatment with cytotoxic agents both in conjunction with escalated doses of chemotherapy and autologous bone marrow transplantation [13-16]. However, it has recently been shown in experimental models by our group and others that G-CSF under certain circumstances (i.e., when combined with multiple doses of cytotoxic agents that by themselves damage primitive stem cells) may have adverse effects on the stem cells responsible for long-term hematopoiesis [5, 17]. Several possible mechanisms have been proposed for the detrimental effects of G-CSF when given following multiple doses of cytotoxic agents. The most likely mechanism is an increased proliferation of stem cells that results from G-CSF-enhanced repopulation of the progenitor cell pool. Thus, G-CSF may activate stem cells to differentiate at the expense of self-renewal causing a permanent loss of stem cells. Other potential mechanisms include direct damage to stem cells by G-CSF, and activation of proliferation or migration making stem cells more susceptible to subsequent chemotherapy.

To further study the potential mechanisms of long-term stem cell damage following cytotoxic agents and G-CSF, we measured the content of stem and progenitor cell subpopulations in the bone marrow during and after six courses of saline, CY or BCNU with and without G-CSF. Increased progenitor cell numbers were seen during courses of chemotherapy and G-CSF suggesting increased proliferative demand on stem cells. This effect was enhanced with increased (eight days versus four days) administration of G-CSF. Factors that predict a permanent loss of marrow reserve were also evaluated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
C57BL/6J (B6)-mice purchased from Jackson Laboratories (Bar Harbor, ME) were used as recipients of cytotoxic agents and cytokines and for competitive repopulation studies. Congenic C57BL/6J-Gpi-1a/Gpi-1a (B6-Gpi-1a) mice purchased from Jackson Laboratories were used as a source of normal untreated marrow in the competitive repopulation assays.

Cytotoxic Agent Studies
Experiments were designed to expose groups of three-month-old B6-mice to six every-other-week i.p. doses of saline, CY or 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) in a total volume of 0.2 ml per mouse. The doses of CY and BCNU were determined as the dose which resulted in an equal fractional day-8 colony-forming-unit spleen survival of 0.37 (Do) at 24 h after a single i.p. administration [6]. CY (Bristol Myers; Princeton, NJ) was used at 84 mg/kg and BCNU (Bristol Myers) at 13.2 mg/kg. These doses were chosen to have equal effects on progenitor cells but different effects on primitive stem cells. CY was chosen because it has little long-term damaging effects on primitive stem cells when given at moderate doses. In contrast, BCNU is known to permanently damage primitive stem cells [6, 18]. Saline was used in control mice. Mice were treated with recombinant human G-CSF (250 µg/kg/day, s.c., b.i.d) for zero, four, or eight days, starting two days after saline, CY or BCNU. Treatment schedules are shown in Figure 1Go.



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  		Figure 1. Schematic representation of experimental setup.

 
At the end of the first, third, and sixth cycle, as well as 7 and 20 weeks later, animals were analyzed for WBC, marrow cellularity, marrow progenitor cell content (day 7 cobblestone area-forming cell [CAFC] frequencies, granulocyte/macrophage-colony forming cells [GM-CFC], colony-forming cells with high proliferative-potential [HPP-CFC]), and primitive stem cell content (stem cells with long-term repopulating ability [LTRA] and day-28 CAFC content). These different stem and progenitor cell assays were used because they represent the continuum from primitive stem cells to functional differentiated cells. We chose to test stem and progenitor cell content at the end of one, three, or six cycles to measure damage to the marrow at the time of the next dose of cytotoxic agent.

Progenitor Cell Assays
Three different assays were performed to measure progenitor cell content in the bone marrow: first, the GM-CFC assay that is used routinely to measure progenitor cells and second, the HPP-CFC assay, which measures a slightly more primitive progenitor cell. The CAFC assay measures both progenitor cells (CAFC-day 7) and more primitive stem cells (CAFC-day 28) in one assay system.

GM-CFC Assay and HPP-CFC Assay
GM-CFC and HPP-CFC assays were performed in 0.3% (weight by volume) melted agarose (Difco; Detroit, MI) cultures as previously described [19]. GM-CFC were cultured in the presence of interleukin 3 (IL-3) alone (conditioned medium from the WEHI 3B murine myelomonocytic leukemic cell line). HPP-CFC were grown in the presence of IL-3 and M-CSF (conditioned medium from the L929 murine fibroblast cell line). Four replicate dishes were prepared for each sample and a mean colony count determined to obtain total numbers of GM-CFC or HPP-CFC per hind limb.

CAFC Assay for Progenitors (CAFC Day 7)
In vitro determination of hematopoietic progenitor cell frequencies was performed by limiting dilution analysis (LDA) of CAFC in microcultures according to the method previously described [20, 21]. Nine dilutions of bone marrow cells were plated at 20 wells per dilution. Cultures were scored at day 7 by scanning each well under an inverted microscope for the presence or absence of cobblestone areas (CA). CA are colonies of immature hematopoietic cells (at least six cells per colony) residing within a pre-established stromal layer. The proportion of negative wells at each dilution was used in a Poisson-based LDA calculation to determine the CAFC frequency [20, 22].

CAFC Assay for Stem Cells (CAFC Day 28)
In vitro determination of hematopoietic stem cell frequencies was performed by LDA of CAFC as described above. Cultures were scored at day 28 to determine stem cell frequencies.

In Vivo Primitive Stem Cell Assay (Competitive Repopulation)
The competitive repopulation assay measuring the LTRA of a test cell population (Gpi-1b) relative to normal bone marrow cells (Gpi-1a) in vivo [23] was used as described [21]. The number of repopulating units (RU), a measure of long-term repopulating cells, was calculated according to the formula:

where C is the number of RU in control (Gpi-1a) marrow, and 1 RU is defined as the repopulating ability of 105 normal bone marrow cells [24].


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The mice used in this experiment were treated with every-other-weekly doses of saline, CY, or BCNU. Overall survival was high (>95%). At the end of each cycle (14 days after administration of cytotoxic agents at the time for the next dose of the cytotoxic agent), peripheral blood cell and bone marrow cellularity were normal, indicating that the period of neutropenia was shorter than 14 days. Thus, the doses of cytotoxic agents used in these experiments did not cause prolonged neutropenia or bone marrow aplasia.

Effects of Cytotoxic Agents with and without G-CSF on Progenitors (CAFC-7, GM-CFC, HPP-CFC)
In contrast to the lack of effect on peripheral cell counts, G-CSF significantly increased progenitor cell numbers in the marrow at the end of each cycle. Figure 2Go shows the changes in CAFC-7 per femur over time in mice treated with saline, CY or BCNU and G-CSF given for four or eight days. After one and three cycles, there was an increase in CAFC-7 content in the bone marrow for all groups (saline, CY, or BCNU) receiving G-CSF when compared with cytotoxic agent alone. This increase was greater for eight days of G-CSF than with four days of G-CSF, suggesting a dose-response relationship (Fig. 2Go). The same relative changes in marrow GM-CFC and HPP-CFC content were observed in mice given G-CSF compared with no G-CSF (not shown). Twenty weeks after the end of six cycles of cytotoxic agent ± G-CSF, progenitor cell levels (CAFC-7, GM-CFC, and HPP-CFC) had returned to normal in saline and CY-treated mice, but in BCNU-treated mice the progenitor cell content in the marrow continued to be depressed (Table 1Go).



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  		Figure 2. Progenitor cell numbers (CAFC-7) during six courses of cytotoxic agents and G-CSF. Mice were treated biweekly with saline, CY (84 mg/kg) or BCNU (13.2 mg/kg). G-CSF was given twice daily on days 3-6 (four days) or on days 3-10 (eight days) at a dose of 250 µg/kg/day. The figure shows CAFC-7 numbers per hind limb at different times after the start of treatment tested after one cycle, three cycles, six cycles of cytotoxic agents. Error bars represent 95% confidence intervals. Significant differences are indicated as {ddagger} for p < 0.05 when compared with saline, * for p < 0.05 when compared with cytotoxic agent alone, and ** for p < 0.01 when compared with cytotoxic agent alone.

 

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  		Table 1. Hematopoietic progenitor cell (CAFC-7, CFU-GM, and HPP-CFC) numbers 20 weeks after completion of six courses of saline, CY, or BCNU. Effect of G-CSF given for four or eight days.
 
Effects of Cytotoxic Agents with and without G-CSF on Stem Cells (CAFC-28, RU)
At the end of the first, third and sixth course of saline, CY or BCNU, the increase in progenitor cell numbers described above coincided with a decrease in the stem cell (CAFC-28) content in the marrow (Fig. 3Go). Additionally, for all three groups (saline, CY, and BCNU), mice given G-CSF for eight days in each cycle had a lower level of CAFC-28 per hind limb than four days of G-CSF or cytotoxic agent alone. Fourteen days after the last dose of each cytotoxic agent, G-CSF-treated mice exhibited a decrease in LTRA measured by RU when compared with saline or BCNU alone (Fig. 4Go). Seven weeks after completion of the six courses, the G-CSF-induced reduction in stem cell (RU) content persisted. At 20 weeks the G-CSF-dependent decrease in primitive stem cell content (RU) had recovered in saline-treated mice. In mice treated with CY and four days of G-CSF, the RU content was not permanently decreased, but with eight days of G-CSF the reduction in RU content was still present at 20 weeks. At 20 weeks after completion of six cycles of BCNU, the RU content was significantly reduced in mice given G-CSF when compared with BCNU alone (Fig. 4Go). Thus, when combined with BCNU, G-CSF was detrimental on stem cells when given for four or eight days. Finally, we found no factors that could predict for long-term detrimental effects of G-CSF administration on permanent primitive stem cell damage.



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  		Figure 3. Primitive stem cell numbers (CAFC-28) during six courses of cytotoxic agents and G-CSF. CAFC-28 numbers are shown in this figure in a similar manner as in Figure 2Go. Significant differences are also shown similarly.

 


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  		Figure 4. Effect of cytotoxic agents (CA) and G-CSF on hematopoietic stem cell (RU) numbers. Mice were given six every-other-weekly courses of saline, CY (84 mg/kg) or BCNU (13.2 mg/kg). After six cycles, and 7 or 20 weeks later, stem cell content was determined in bone marrow pooled from two to five mice. Bone marrow was tested using classical competitive repopulation assay [23] and measured by Gpi-phenotyping in 3-20 recipients per group (shown for each group above the bar). BCNU significantly reduced RU numbers when compared with saline (p = 0.03 at six cycles, p < 0.01 at 7 and 20 weeks after six cycles). After six cycles, G-CSF significantly reduced RU numbers in saline- and BCNU-treated mice (p = 0.04 and p = 0.01, respectively). After six cycles and seven weeks, stem cell content was reduced in all groups given G-CSF (four days or eight days; p < 0.05). Twenty weeks after completion of six cycles, the RU content remained reduced in CY given G-CSF for eight days and in BCNU-treated mice given G-CSF for either four or eight days. Significant differences are shown similarly as in Figure 2Go. {ddagger}{ddagger} for p < 0.01 when compared with saline.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To study the effect of G-CSF in combination with cytotoxic agents, on the hematopoietic stem and progenitor cell compartments, we treated mice with six every-other-weekly doses of saline, CY, or BCNU. Fourteen days was chosen as the length of each course because it was known that with the cytotoxic agent alone, progenitor cell content and peripheral counts would be back to normal at the start of the next cycle [25]. This makes this treatment schedule an excellent model system for multiple cycle chemotherapy in clinical settings. Although G-CSF is expected to cause increased recovery of progenitor cell content and peripheral blood count, the effect of G-CSF on primitive stem cells is not known. In this paper, measurements of marrow subpopulations are obtained at the end of each cycle of cytotoxic agent and G-CSF to further study the stem cell deficit caused by G-CSF when given after stem cell-damaging agents [17]. Our results reaffirm that the measurement of normal peripheral blood counts, used in the clinic as guidance to proceed with additional chemotherapy, is not a good indicator for primitive stem cell damage [9, 17].

In these experiments, we found that administration of G-CSF after one, three, and six doses of saline, CY, or BCNU resulted in increased levels of progenitors (CAFC-7) but a decrease in stem cell content (CAFC-28). It is therefore likely that G-CSF administration leads to increased progenitor cell numbers to rapidly recover peripheral blood and marrow cellularity, but hematopoietic stem cell numbers decrease as a result of the rapid G-CSF-induced proliferation and differentiation. In addition, G-CSF will only enhance the long-term stem cell deficit when the marrow reserve has been compromised by prior cytotoxic treatment, such as with BCNU. However, it remains uncertain whether the highest dose of G-CSF (eight days) will also cause a permanent depression of stem cell numbers when combined with saline or CY, or that without severe damage to primitive stem cells, both stem and progenitor cell populations will ultimately recover.

The changes in stem and progenitor cell numbers during and after cytotoxic agent and G-CSF administration indicate that CAFC-7, GM-CFC, and HPP-CFC all belong to an expandable progenitor cell population, whereas CAFC-28 represents a stem cell population replenishing the progenitor pool upon growth factor stimulation. However, the CAFC-28 appears not to represent the most primitive stem cell population because its numbers are able to recover following a 90% drop in CAFC-28 numbers in the marrow caused by excessive G-CSF stimulation. Our results suggest that CAFC-28 numbers can recover following significant reduction in numbers, and this recovery is likely to be dependent on the marrow reserve and therefore, on the magnitude of reduction caused by the cytotoxic agent used (direct damage to the stem cell pool) and the dose of G-CSF (indirect damage of the stem cell pool). Since CAFC-28 does appear to be a replenishable stem cell population, in vivo long-term repopulation studies remain the most accurate assay for primitive stem cells in murine models. In these studies, in vivo transplantation experiments confirmed enhancement of the long-term stem cell deficit by G-CSF when combined with BCNU (Fig. 4Go).

In accordance with our studies, it has been proposed that the proliferative capacity and therefore the number of divisions of hematopoietic stem cells is finite [26, 27]. One more recent theory proposed by Van Zant et al. [28] is partly based on studies measuring CAFC frequencies in old and young mice of various strains [29]. They suggest that the primitive stem cell pool, as measured by functional assays, consists of stem cells of high or low "quality," depending on their proliferation history. During aging and possibly also after cytotoxic or radiation injury, high "quality" stem cells have to undergo a number of divisions to replenish the progenitor compartment. Experimental data indeed have shown that following radiation or proliferative stress, the self-renewal capacity of primitive stem cells is limited [8, 9]. Stimulation with hematopoietic growth factors may accelerate the aging process by causing direct or indirect proliferation of primitive stem cells. In combination with damage to the primitive stem cell pool by radiation or chemotherapy, this may lead to loss of long-term repopulation potential as seen in our experiments with BCNU and G-CSF. This theory appears to be supported by previous clinical reports on stem cell "quality" in patients having undergone multiple cycles of chemotherapy. First, the number of cycles of chemotherapy has been found to correlate with the decrease in the stem cell pool as measured by late-appearing (week 6) CAFC [30]. Second, combining G-CSF with an increased dose of cytotoxic agents prior to bone marrow harvesting for autologous bone marrow transplantation has compromised neutrophil and platelet recovery to suggest exhaustion of the primitive stem cell pool [12]. Moreover, prior chemotherapy may also adversely affect stem cell mobilization [10, 31], which in one clinical study was shown to be associated with poor bone marrow quality [32]. Thus, hematopoietic growth factors may contribute to accelerated reduction of the size and quality of the primitive stem cell pool when used in combination with cytotoxic agents that damage primitive stem cells.

In conclusion, we show that G-CSF combined with certain cytotoxic agents may enhance stem cell damage through stimulation of differentiation at the cost of self-renewal. Although, we demonstrate a G-CSF dose-response effect for the increase in progenitors during administration of G-CSF and cytotoxic agents, further studies are required to reveal whether the enhancement of long-term damage depends on the dose and length of G-CSF administration. To reduce the potential detrimental effect of G-CSF on marrow reserve when combined with certain cytotoxic agents, it may be preferable to limit its use to cytotoxic agents that do not significantly damage primitive stem cells when that option is available. Measurement of stem cell quality in patients undergoing chemotherapy with or without G-CSF support may provide additional information on the effects of G-CSF on long-term hematopoiesis in the clinic.


   Acknowledgments
 
This research was supported by the NIH RO1 Grant-CA 10941-28.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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accepted for publication on February 17, 2000.




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