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Stem Cells, Vol. 18, No. 2, 93-101, March 2000
© 2000 AlphaMed Press

Reduction of Marrow Hematopoietic Progenitor and Stem Cell Content Is Not Sufficient for Enhanced Syngeneic Engraftment

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

Joint Center for Radiation Therapy, Department of Radiation Oncology, Harvard Medical School, Boston, Massachusetts, USA

Key Words. Syngeneic engraftment • Long-term engraftment • Hematopoietic progenitor cells • Hematopoietic assays

Peter Mauch, M.D., JCRT, 330 Brookline Avenue, Boston, Massachusetts 02215, USA. Telephone 617-632-4116; Fax: 617-632-4115; e-mail: mauch{at}speedy.jcrt.harvard.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The mechanisms regulating long-term engraftment of primitive stem cells are largely unknown. Most conditioning strategies use myeloablative agents for experimental or clinical hematopoietic stem cell transplantation. Host conditioning regimens, in part, have been designed on the assumption that transplanted cells home to specific marrow sites and if these sites are occupied by host stem cells, engraftment will not take place. However, there is now evidence that stable long-term syngeneic engraftment may occur in the absence of host marrow stem cell depletion. To further study the association of engraftment with stem cell depletion, we investigated whether the marked egress of hematopoietic progenitor and stem cells from the marrow into the peripheral blood in C57BL6 mice following a single dose of cyclophosphamide (day 1) and four days of G-CSF (days 3-6) afforded an increased opportunity for long-term syngeneic donor engraftment. During and after mobilization, glucose phosphate isomerase (GPI)-1b mice received 30 x 106 GPI-1a marrow cells without further myeloablation. The level of donor/recipient chimerism was assessed in cell lysates after six months. Increased long-term syngeneic donor engraftment was observed prior to mobilization (before day 6), during a period of active hematopoietic regeneration following the administration of cyclophosphamide. Hematopoietic regeneration was evidenced by a reduced but rapidly increasing marrow cellularity and an increased proportion of hematopoietic progenitors in S-phase. In contrast, long-term syngeneic donor engraftment was not increased over controls during the period of maximum progenitor and stem cell mobilization (after day 5). At this time there were minimal numbers of progenitor and stem cells in the marrow. These data suggest that in the absence of host stem cell ablation, maximal engraftment does not occur during marrow progenitor or stem cell depletion, suggesting that the presence of "open" marrow sites is not a prerequisite for engraftment. The mechanisms for increased engraftment during progenitor cell regeneration following cyclophosphamide need further investigation. Understanding the mechanisms for engraftment without host stem cell ablation may allow strategies for improved long-term engraftment of syngeneic or autologous stem cells with reduced post-transplant toxicity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Most conditioning strategies for experimental or clinical hematopoietic stem cell transplantation with syngeneic or autologous marrow have required host preparation with myeloablative agents. Host conditioning regimens, in part, have been designed on the assumption that there are specific marrow sites to which transplanted cells home, and if these sites are occupied by host stem cells, engraftment will not take place. However, a number of studies have challenged the assumption that myeloablation is required for syngeneic or autologous engraftment [1-3]. Long-term engraftment of male donor marrow into nonconditioned female mice has been demonstrated when large numbers of syngeneic marrow cells (40 x 106) were transplanted for each of five consecutive days [3]. It was estimated that the 200 x 106 marrow cells transplanted represented approximately two-thirds the normal complement of marrow cells per mouse. By estimating the dilution with endogenous marrow cells, it was proposed that if all cells engrafted, maximum donor chimerism would be 40%. As mean engraftment experimentally at 10-12 months was approximately 40%, the results suggest that infused normal marrow competed at least equally with host marrow for long-term engraftment.

Other investigators have evaluated syngeneic engraftment in nonconditioned or minimally conditioned hosts using lower cell doses given as a single dose [2, 4]. In one report, eight weeks after transplantation of a single dose of 20 x 106 syngeneic marrow cells, donor engraftment of 2.5%-10% was measured in the marrow, but not in the spleen, thymus, or peripheral blood [2]. In another report, a similar cell dose (15 x 106) was transplanted into mice conditioned with doses of TBI up to 300 cGy [4]. Although temporary engraftment was seen with TBI, long-term engraftment occurred only in recipients conditioned with 150 cGy or greater. Furthermore, the ability to successfully achieve long-term donor engraftment was lost if transplantation of donor cells was delayed for seven days or more after conditioning.

The current experiments further explore mechanisms of syngeneic engraftment with minimal conditioning techniques. Prior work from our laboratory has demonstrated a marked egress of hematopoietic progenitor cells from the marrow to the blood after administration of cyclophosphamide followed by G-CSF [5]. On day 7 after initiation of the protocol, the marrow content for primitive (day 28 cobblestone area forming cells [CAFC-28]) and more mature progenitor cells (CAFC-7) was 11% and 9%, respectively. The current experiments were undertaken to determine if long-term syngeneic engraftment could be enhanced during this period of maximum mobilization of hematopoietic progenitor and stem cells from the marrow to the blood.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and Treatment Protocol
Eight to 12-week-old male C57BL6 mice (C57BL6, glucose phosphate isomerase [GPI]-1b, Jackson Laboratories; Bar Harbor, ME) were injected i.p. with 200 mg/kg cyclophosphamide (Cytoxan, Bristol-Myers Squibb Co.; Princeton, NJ) (day 1) followed on days 3-6 by administration of G-CSF (recombinant human [rHuG-CSF], Filgrastim, Amgen Inc.; Thousand Oaks, CA) given s.c. at 250 µg/kg/day in two equal fractions approximately 8 h apart as detailed in Figure 1Go [5].



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  		Figure 1. Hematopoietic mobilization protocol: C57Bl6 mice received 200 mg/kg cyclophosphamide on day 1 and 250 µg/kg/day G-CSF s.c. in equal doses twice per day, 8 h apart, on days 3-6 of the protocol.

 
To investigate the potential for engraftment of syngeneic donor marrow at different times during the mobilization protocol, cyclophosphamide and G-CSF-treated C57BL6, GPI-1b mice received an i.v. injection of 30 x 106 congenically labeled C57BL6, GPI-1a marrow cells (C57BL6 CAST-GPI-1a mice, Jackson Laboratories). Thirty million transplanted marrow cells constitute approximately one-tenth of the normal complement of marrow cells per mouse [3]. Following transplantation, animals received no further treatment (no G-CSF was given after transplantation even if marrow was given before the end of the mobilization protocol).

Measurement of Long-Term Syngeneic Donor Engraftment
Donor engraftment was evaluated using electrophoresis to measure GPI-1a/1b chimerism in lysates of erythrocytes and bone marrow cells in individual recipient animals >=6 months after transplantation. Lysates of known numbers of erythrocytes and nucleated marrow cells were prepared in water and stored (-20°C) until analyzed. Electrophoresis of lysates was performed on cellulose acetate strips and presence of the GPI enzyme was documented as previously described [6]. The proportion of donor-derived GPI-1a in the lysates was calculated using a scanning densitometer and analysis software (Hoeffer Scientific Instruments; San Francisco, CA) as follows. The area under the curve of the GPI-1a (donor) peak was calculated as a percentage of the total area under the GPI-1a (donor) and 1b (recipient) peaks. These data gave a measurement of the contribution of donor-derived GPI-1a to the total GPI in each individual animal. Two reference samples were included during each analysis (50% and 95% volume by volume [v/v] GPI-1a in GPI-1b). GPI-1a/1b chimerism present at >=6 months post-transplant in the individual animals was considered a measurement of stable long-term engraftment. Statistical comparison of experimental data with controls was performed using the Student's two sample t-test assuming unequal variances.

Measurement of Hematopoietic Indices
At various times in the mobilization protocol, the number of hematopoietic progenitors was measured in the peripheral blood and marrow. Control data were obtained from age-matched, untreated mice (receiving no cyclophosphamide or G-CSF). At sacrifice, individual mice were injected i.p. with heparin and anesthetized by methoxyflurane inhalation (Metofane, Mallinckrodt Veterinary Inc; Mundelein, IL). From each mouse 40 µl of blood were drawn from the retro-orbital sinus using a calibrated micropipet and the number of nucleated blood cells per ml of blood determined using a Coulter Counter (Coulter Corp.; Hialeah, FL). An additional drop of blood was used to prepare a blood smear for differential staining. Prepared blood smears were air-dried and differentially stained (Diff-Quik Stain Set, Baxter Scientific Products, Dade Division; Miami, FL). Differential counts were performed on >200 nucleated cells per animal under oil immersion at x400 magnification. Finally, approximately 1 ml of blood was withdrawn from the retro-orbital sinus using a sterile micropipet and collected into a sterile EDTA-treated blood collection tube. Within a treatment group, blood samples were pooled to yield a sufficiently large sample to perform the hematopoietic assays. Pooled blood was diluted 50% (v/v) with sterile saline, and mononuclear cells were isolated by centrifugation through an equivalent volume of a density separation medium (1-STEP 1.007/265 animal, Accurate Chemical & Scientific Corp.; Westbury, NY). Cells present at the interface after centrifugation were collected and washed once in Dulbecco's phosphate buffered saline ([DPBS], BioWhittaker; Walkersville, MD) containing 2% (v/v) fetal bovine serum ([FBS], GIBCO BRL; Grand Island, NY) (DPBS-2% FBS). Cells were resuspended in a known volume of DPBS-2% FBS and cell numbers determined using a hemocytometer and a 0.01% (weight by volume) solution of crystal violet in water containing 2% (v/v) acetic acid. Blood cells were prepared for hematopoietic assays by dilution in appropriate culture media.

After bleeding, mice were euthanized by cervical dislocation. The bones of both hind limbs from each mouse in each group were removed and ground using a mortar and pestle under sterile conditions to harvest marrow cells. Marrow cells were collected in a known volume of ice-cold Hank's balanced salt solution (BioWhittaker), filtered through a wire mesh to remove bone fragments, and cellularity determined using a hemocytometer. Marrow cells were subsequently prepared for hematopoietic assays by dilution in appropriate culture media.

Hematopoietic Assays

Granulocyte-Macrophage Colony-Forming Cells (GM-CFC) and High Proliferative Potential Colony-Forming Cell (HPP-CFC) Assays   GM-CFC and HPP-CFC populations were assayed in 0.3% semisolid agarose culture as previously described [7]. Four replicate dishes were prepared for each sample. GM-CFC were cultured in 35 mm culture dishes (Falcon, Becton Dickinson; Lincoln Park, NJ) in the presence of WEHI 3B (murine myelomonocytic leukemic cell line)-conditioned medium, a source of interleukin 3 (IL-3), and assayed after seven days as previously described [7]. HPP-CFC were cultured in 60 mm culture dishes (Falcon, Becton Dickinson) in the presence of a combination of L929 (murine fibroblast cell line)-conditioned medium, a source of M-CSF and WEHI 3B-conditioned medium. HPP-CFC were assayed after 14 days as previously described [7]. At assay, a mean number of GM-CFC and HPP-CFC colonies were determined for the four replicate dishes. Since these colonies were from a known number of cells plated, a frequency of GM-CFC and HPP-CFC per 106 nucleated cells plated could be calculated.

The proportion of the HPP-CFC population in S-phase in the marrow of mice at various time points during the mobilization protocol was determined in a limited number of experiments using an S-phase suicide assay as previously described [7]. At specific times in the mobilization protocol, a single cell suspension of marrow cells was prepared and divided into two equal volumes. One volume was incubated for 1 h at 37°C with 25 µg/ml cytosine ß-D-arabinofuranoside ([Ara-C], Sigma; St. Louis, MI). The second volume of cells was not exposed to Ara-C. After incubation both samples were identically washed and prepared for HPP-CFC culture. Since cells in S-phase during the 1-h exposure to Ara-C are killed, the difference between the numbers of HPP-CFC observed in the sample not exposed to Ara-C and the number of HPP-CFC observed in the sample exposed to Ara-C, allows an estimation of the proportion of the HPP-CFC population in S-phase in the marrow at the time of assay.

CAFC Assay   CAFC were assayed as previously described [8-11]. For each time point in the mobilization protocol, nine, 2.5-fold serial dilutions of marrow cells and six, 2.5-fold serial dilutions of isolated mononuclear blood cells were prepared and plated in flat-bottom 96-well plates over preestablished confluent layers of FBMD-1 cells (a murine preadipocyte cell line generously provided by Dr. S. Neben, Genetics Instititute; Cambridge, MA). Twenty wells were plated per dilution and 100 µl of medium per well (total volume 200 µl) were replaced at weekly intervals. Wells were analyzed for the presence of cobblestone areas at day 7 (CAFC-7) and day 28 (CAFC-28) in culture. Cobblestone areas are defined as groups of six or more phase-distinct cells persisting beneath the confluent FBMD-1 cell layer. CAFC-7 and CAFC-28 have been shown to be relatively mature and relatively primitive cell populations of the hematopoietic system, respectively [8-11]. Wells are scored either positive or negative for the presence of cobblestone areas. From the number of cells plated in the wells and the incidence of "negative" wells it is possible to use limiting dilution analysis to calculate the frequency of CAFC in the marrow or mononuclear blood cells plated, as previously described [10, 12].


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Hematopoiesis in Blood and Marrow During and After Cyclophosphamide and G-CSF
Hind limb marrow cellularity was determined from pooled marrow suspensions for each group of animals, with five to eight individual animals per pool. A mean of 48.6 ± 13.3 x 106 cells/hind limb (± standard deviation, n = 3 pools) was determined for untreated animals. Hind limb cellularity was 8% of normal untreated mice at day 3 of the mobilization protocol (after cyclophosphamide and just at the start of the G-CSF administration). From day 3 through day 7, the hind limb cellularity increased steadily and was 70% and 80% of normal on days 6 and 7, respectively. By day 11 hind limb cellularity was 88% of normal (Table 1Go).


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  		Table 1. Number of hematopoietic progenitors per hind limb and proportion of marrow HPP-CFC in S-phase at various points during the mobilization protocol
 

Hematopoietic Progenitor and Stem Cells in the Marrow   The number of progenitor cells (CAFC-7, GM-CFC, HPP-CFC) and stem cells (CAFC-28 ) per hind limb was calculated by multiplying the progenitor or stem cell frequency by the hind limb cellularity (Table 1Go and Fig. 2Go). For example, in control mice a frequency of 1,660 (1,170-2,360, 95% confidence interval, [CI]) CAFC-7/106 marrow cells was determined. The hind limb cellularity was 48.6 x 106 marrow cells. Multiplied together, these data yield a total of 80.7 (56.9-114.7, 95% CI) x 103 CAFC-7 per hind limb. Similarly, in normal control animals there were approximately 121.0 ± 5.8 x 103 GM-CFC, 46.2 ± 5.3 x 103 HPP-CFC, and 729 (486-1,069, 95% CI) CAFC-28 per hind limb (Table 1Go). The proportion of HPP-CFC in S-phase in control mice in our laboratory and from other work has been found to be <20% [13].



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  		Figure 2. Number of progenitor and stem cells per hind limb as a percentage of control values during the mobilization protocol.

 
Between days 3 and 5, the number of CAFC-7, GM-CFC, HPP-CFC, and CAFC-28 per hind limb remained between 20% and 156% of pretreatment levels (Fig. 2Go), and the percent of marrow HPP-CFC in S-phase increased to 45%-61%. After day 5, we observed a significant reduction in the number of CAFC-7, GM-CFC, HPP-CFC, and CAFC-28 per hind limb. By day 6, CAFC-7 and GM-CFC marrow content was reduced to 1% and 70% of pretreatment levels and by day 7, CAFC-7, HPP-CFC, and CAFC-28 were at 3%, 62%, and 0.4% of pretreatment levels, respectively. By this time the proportion of the marrow HPP-CFC population in S-phase had returned to normal (17%). After day 7, the numbers of progenitor and stem cells per hind limb gradually increased although they were still less than pretreatment levels at day 11. Notably, at day 9, CAFC-7, GM-CFC, HPP-CFC, and CAFC-28 content per hind limb was 8%, 31%, 43%, and 1%, respectively, of control values; at day 11 CAFC-7, GM-CFC, HPP-CFC, and CAFC-28 content per hind limb was 16%, 41%, 88%, and 9%, respectively (Fig. 2Go).

Peripheral Blood Cellularity and Differential Counts   There were 9.5 ± 1.8 x 106 nucleated cells per ml blood in control animals; 17% were granulocytes (Table 2Go). Following cyclophosphamide, the number of nucleated cells in the blood decreased significantly on days 3-5, then steadily increased on days 6-9 corresponding to G-CSF administration. During this time the percentage of granulocytes on peripheral smears also increased (Table 2Go). On days 6 and 7, the number of nucleated cells per ml was 2.7 and 7.1 times normal, respectively (Table 2Go). By day 9 blood cellularity had decreased and was no longer significantly different from control animals (p = 0.376).


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  		Table 2. Blood composition and approximate frequency of hematopoietic progenitors at various points during the mobilization protocol
 

Hematopoietic Progenitor and Stem Cells in the Blood   The reduction in the number of progenitor and stem cells in the hind limb marrow corresponded with an increase in the frequency of hematopoietic progenitor and stem cells in the peripheral blood (Table 2Go). No significant increase in peripheral blood progenitor and stem cells was observed until after day 4. At day 5, CAFC-7 and CAFC-28 frequencies were increased 850-fold and 169-fold, respectively, and at day 6, GM-CFC and HPP-CFC frequencies were increased 303-fold and 85-fold, respectively, compared to controls. At day 9, the frequencies of CAFC-7, GM-CFC, HPP-CFC, and CAFC-28 in the peripheral blood were 2,280-, 162-, 165-, and 97-fold, respectively, compared to controls. At day 11, the frequencies of CAFC-7, GM-CFC, HPP-CFC, and CAFC-28 were declining but were still elevated at 275-, 29-, 75-, and 60-fold, respectively, compared to controls.

Syngeneic Donor Engraftment
A total of 30 x 106 congenically marked (GPI-1a) marrow cells were transplanted into recipient (GPI-1b) mice at various times during the mobilization protocol. Long-term donor engraftment was measured >6 months after transplantation by measuring the proportion of donor-derived GPI-1a in lysates of bone marrow cells and erythrocytes of recipient, GPI-1b mice.

Syngeneic donor engraftment in untreated control animals was 16.3% ± 1.2% and 21.5% ± 1.4% (mean ± standard error [SE], n = 20 animals) as measured in lysates of erythrocytes and bone marrow, respectively (Table 3Go). Analysis of lysates from erythrocytes and marrow in animals transplanted at days 3, 4, and 5 showed a significant increase in long-term syngeneic donor engraftment when compared to untreated controls. We observed long-term donor engraftment of 23.0 ± 1.1% (n = 7, p = 0.0004), 24.1 ± 1.7% (n = 5, p = 0.006), and 23.1 ± 1.4% (n = 12, p = 0.0008) in lysates of erythrocytes, and 30.3 ± 1.3% (n = 8, p = 0.0001), 28.2 ± 3.2% (n = 6, p = 0.102), and 27.0 ± 1.7% (n = 11, p = 0.021) for lysates of bone marrow for days 3, 4, and 5, respectively. Transplantation after day 5 of the mobilization protocol did not achieve levels of long-term syngeneic engraftment (measured in both marrow and erythrocyte lysates) that were significantly higher than those seen in untreated, control (day 0) animals.


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  		Table 3. Donor marrow engraftment measured by proportion of GPI-1a in erythrocyte and nucleated bone marrow lysates of GPI-1b recipients >6 months after transplantation
 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Host preparation with TBI or with certain cytotoxic agents allows successful long-term donor marrow engraftment in murine models of syngeneic hematopoietic transplantation [14-20]. In part, host conditioning regimens have been designed with the assumption that there are specific marrow sites to which transplanted cells home, and if these sites are occupied, engraftment will not take place. Increased donor engraftment can be achieved with higher preparative doses of TBI or cytotoxic agents, and with transplantation of larger numbers of hematopoietic cells [18]. The most effective preparative regimens include TBI or the cytotoxic agent, busulfan. Both regimens are known to damage primitive stem cells suggesting that the loss of host stem cells favors donor hematopoietic stem cell engraftment and progenitor cell repopulation. Other non-TBI, nonbusulfan regimens that damage primitive stem cells, such as BCNU and melphalan or combinations of 5-fluorouracil (5-FU) and kit ligand, can also aid engraftment in experimental models [11, 20]. Down and colleagues evaluated syngeneic donor engraftment in B6 mice transplanted with 107 bone marrow cells 24 h after a single dose of cyclophosphamide (200 mg/kg), or 5-FU (agents which do not damage primitive stem cells) and BCNU, melphalan, or busulfan (agents which do damage stem cells) using congenic markers [19, 20]. Mixed donor-recipient chimerism was present in all recipient animals, and percent long-term donor chimerism appeared stable. The percentage donor engraftment of the bone marrow 36 weeks post-transplantation was 2.3% after cyclophosphamide, 1.4% after 5-FU, 12.9% after melphalan, 22.4% after BCNU, and 73.8% following busulfan, suggesting a relationship between extent of host stem cell ablation and degree of donor engraftment.

A number of studies have challenged the assumption that myeloablation is required for syngeneic or autologous engraftment by transplanting very large numbers of syngeneic cells into nonconditioned mice and demonstrating long-term engraftment [1-3]. These experiments suggest that donor marrow may compete effectively with host cells for long-term engraftment when large numbers of cells are transplanted. In the current experiments we used somewhat higher donor marrow doses (30 x 106 marrow cells) to enhance engraftment under minimal conditioning. Thirty million syngeneic marrow cells were transplanted as a single dose into mice at various times after cyclophosphamide and G-CSF to investigate engraftment into nonmyeloablated recipients. In prior experiments we documented long-term engraftment of 17% at eight months in nonconditioned animals given this cell dose [11]. The 16.3% long-term engraftment in the current study in control animals is similar to these previous results.

In the current experiments we prepared mice with cyclophosphamide and G-CSF, a regimen known to mobilize primitive stem cells into the blood. As previous data are limited, we measured the extent of mobilization of progenitor and primitive stem cells at various times after cyclophosphamide and G-CSF. The degree of mobilization correlated with previous data from our laboratory [5]. Increases in blood progenitor and primitive stem cell frequencies were observed starting on day 5, and correlated with their decrease in the marrow. The fewest numbers of marrow progenitor and primitive stem cells were noted on days 6 and 7 after cyclophosphamide, however, significant decreases were seen at least out to 11 days. The greatest decrease was seen in CAFC-28 marrow content; on day 7 the hind limb content of CAFC-28 was 0.4% of normal controls.

Significant enhancement of long-term donor engraftment was observed when mice were transplanted at days 3, 4, and 5 following the cyclophosphamide. This increased engraftment occurred prior to the mobilization of stem and progenitor cells into the blood and correlated with a significant increase in the proportion of HPP-CFC in S-phase. By day 6, both the proportion of HPP-CFC in S-phase and long-term engraftment returned to baseline levels. This suggests that increased engraftment correlates with increased proliferation of HPP-CFC and not with the administration of the G-CSF (which continued until day 7) or with the absolute number of marrow hematopoietic progenitor or stem cells present (which were lowest at days 6 and 7). Others have demonstrated a temporal relationship between conditioning and transplantation that affects long-term engraftment. In one study, successful long-term donor engraftment was lost if the time between sublethal TBI and transplantation of 15 x 106 cells was greater than seven days [4]. Further study of the time between irradiation and transplantation and achievement of long-term engraftment may help elucidate some of the mechanisms for engraftment.

No improvement in engraftment was seen compared to controls when animals were transplanted at day 6 or greater in the mobilization protocol. As the numbers of marrow progenitor and stem cells were the lowest on days 6 and 7, the results suggest that marrow engraftment is not enhanced by low numbers of progenitor or stem cells in the marrow. Although low levels of marrow progenitor and stem cells also are present after myeloablation, a circumstance in which enhanced engraftment does occur, many other factors may account for this increase in engraftment including simultaneous low levels of cells in the peripheral blood, damage to the microenvironment with TBI or busulfan, and the differential release of cytokines which results in regenerative hematopoiesis after myeloablation.

Factors affecting primitive stem cell engraftment are undoubtedly complex and not well studied. Although the current experiments provide a model for study of differential engraftment, many questions remain. Increased donor engraftment during increased hematopoietic proliferation seen in the current study suggests that the increased levels of cytokines, activation of cytokine cascades and changes in the expression of cell-cell adhesion molecules on hematopoietic and stromal cells seen during regenerating hematopoiesis may all enhance engraftment independent of myeloablation [21]. This is further supported by the increased engraftment seen both during regenerating hematopoiesis with myeloablation (with TBI) and without myeloablation or loss of marrow stem cell content (in the current experiments). In contrast, it has been shown that active hematopoietic proliferation within recipient mice (stimulated by either repeated bleeding or the administration of phenylhydrazine) does not in itself increase long-term syngeneic donor engraftment [22]. However, little is known of the environmental differences between recovery following acute loss of progenitor or stem cells and the increased hematopoiesis after a more chronic stimulus.

Other alternatives exist to explain the improvement in engraftment in the current experiments. It has been shown that noncycling donor cells may have a competitive advantage over regenerating (and cycling) host stem cells, resulting in improved engraftment during regeneration [23]. Alternatively, movement of hematopoietic progenitors between the hematopoietic microenvironment and the peripheral blood is thought to involve transendothelial migration through the cells lining the marrow sinus. Damage to the endothelial cells that form the interface between the hematopoietic system and the peripheral blood is one sequela of cyclophosphamide administration [24, 25]. This damage may facilitate access of donor cells to the hematopoietic microenvironment and account for the increased engraftment observed early in the mobilization protocol. Repair of the endothelium following cyclophosphamide may, in part, account for the absence of any increase in long-term syngeneic engraftment during the period of maximal mobilization. It is reported that the blood-bone marrow endothelial barrier is disrupted within three days of cyclophosphamide administration and that this disruption can persist for up to one week. The integrity of the blood-bone marrow endothelial barrier appears to be restored within two weeks after initial administration of the cyclophosphamide [25]. However, if this timing exists for the current protocol, the damage from cyclophosphamide would have been present during both times of enhanced and nonenhanced engraftment and suggests that this is not the only mechanism for increased engraftment in the current experiments.

It is possible that the administration of G-CSF after cyclophosphamide may impair long-term donor engraftment. This was not tested in the current experiments. The administration of G-CSF stimulates the production and activation of neutrophils. Activated neutrophils synthesize and release IL-8 [26]. In the presence of IL-8, neutrophils are reported to release a number of matrix metalloproteinases [27] which may act to release progenitors from the hematopoietic microenvironment by enzymatic degradation of components of the extracellular matrix [28, 29]. The direct administration of IL-8 i.p. in mice [30] and i.v. in primates [31] has been shown to rapidly mobilize hematopoietic progenitors from the bone marrow into the peripheral blood. This mechanism is thought to involve the ß2-integrin leukocyte function-associated antigen-1 and to require mature functional granulocytes [32, 33]. The potential role of IL-8 in the mechanism of G-CSF-induced progenitor cell mobilization in man has also been investigated. The administration of G-CSF to healthy volunteers has been shown to be associated with a sharp increase in serum IL-8 levels [34]. The elevation in serum IL-8 was shown to correlate with the period of CD34+ mobilization into the peripheral blood. Finally, G-CSF may facilitate progenitor mobilization by directly, or indirectly, reducing the expression of adhesion molecules that bind hematopoietic progenitors within the hematopoietic microenvironment [21, 35-37], and thus, might promote mobilization or alternatively aid engraftment depending perhaps on timing of administration. In the current experiments, decreased engraftment was seen only during the last day of G-CSF administration, suggesting that if G-CSF has a negative effect on engraftment, the effect is delayed by several days from initial administration.

In conclusion, the current experiments suggest that engraftment is not enhanced solely by a reduction in the number of progenitor and stem cells within the marrow. Increased engraftment, however, is seen during hematopoietic regeneration. This may be a consequence of the release of cytokines and/or changes in the cell surface characteristics of the marrow stromal cells. Alternatively, the cycling of marrow progenitor cells during hematopoietic regeneration may put them at a competitive disadvantage to noncycling donor cells allowing enhanced engraftment. Further understanding of the mechanisms of engraftment using minimal conditioning regimens may allow improved stable donor chimerism without the toxicity seen with myeloablative regimens, and may provide a strategy for achieving long-term engraftment of transduced marrow into recipients with genetic disease.


   Acknowledgments
 
Supported by NIH CA R01-10491.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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  6. Ploemacher R, van Os R, van Beurden C et al. Murine haemopoietic stem cells with long-term engraftment and marrow repopulating ability are more resistant to gamma-radiation than are spleen colony forming cells. Int J Radiat Biol 1992;61:489-499.[Medline]

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accepted for publication December 21, 1999.




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