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Stem Cells, Vol. 17, No. 4, 210-218, July 1999
© 1999 AlphaMed Press

Engraftment of Primates with G-CSF Mobilized Peripheral Blood CD34+ Progenitor Cells Expanded in G-CSF, SCF and MGDF Decreases the Duration and Severity of Neutropenia

Robert G. Andrewsa,b, Robert A. Briddellc, Robert Hillc, Mike Goughd, Ian K. McNiecec,e

a Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA;
b Department of Pediatrics, University of Washington School of Medicine, Seattle, Washington, USA;
c Amgen, Inc., Thousand Oaks, California, USA;
d University of Washington Regional Primate Research Center, Seattle, Washington, USA, and
e Bone Marrow Transplant Program, University of Colorado Health Science Center, Denver, Colorado, USA

Key Words. CD34 • Expansion • Hematopoietic progenitors • MGDF • SCF • Transplantation

Dr. Robert G. Andrews, Pediatric Oncology Program, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N., P.O. Box 19024, Seattle, Washington 98109-1024, USA.


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We used a primate model of autologous peripheral blood progenitor cell (PBPC) transplantation to study the effect of in vitro expansion on committed progenitor cell engraftment and marrow recovery after transplantation. Four groups of baboons were transplanted with enriched autologous CD34+ PBPC collected by apheresis after five days of G-CSF administration (100 µg/kg/day). Groups I and III were transplanted with cryopreserved CD34+ PBPC and Groups II and IV were transplanted with CD34+ PBPC that had been cultured for 10 days in Amgen-defined (serum free) medium and stimulated with G-CSF, megakaryocyte growth and development factor (MGDF), and stem cell factor each at 100 {eta}g/ml. Group III and IV animals were administered G-CSF (100 µg/kg/day) and MGDF (25 µg/kg/day) after transplant, while animals in Groups I and II were not. For the cultured CD34+ PBPC from groups II and IV, the total cell numbers expanded 14.4 ± 8.3 and 4.0 ± 0.7-fold, respectively, and CFU-GM expanded 7.2 ± 0.3 and 8.0 ± 0.4-fold, respectively. All animals engrafted. If no growth factor support was given after transplant (Groups II and I), the recovery of WBC and platelet production after transplant was prolonged if cells had been cultured prior to transplant (Group II). Administration of post-transplant G-CSF and MGDF shortened the period of neutropenia (ANC < 500/µL) from 13 ± 4 (Group I) to 10 ± 4 (Group III) days for animals transplanted with non-expanded CD34+ PBPC. For animals transplanted with ex vivo-expanded CD34+ PBPC, post-transplant administration of G-CSF and MGDF shortened the duration of neutropenia from 14 ± 2 (Group II) to 3 ± 4 (Group IV) days. Recovery of platelet production was slower in all animals transplanted with expanded CD34+ PBPC regardless of post-transplant growth factor administration. Progenitor cells generated in vitro can contribute to early engraftment and mitigate neutropenia when growth factor support is administered post-transplant. Thrombocytopenia was not decreased despite evidence of expansion of megakaryocytes in cultured CD34+ populations. Stem Cells 1999;17:210-218


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Committed hematopoietic progenitor cells (HPC) are thought to contribute to early engraftment after bone marrow or peripheral blood stem cell transplantation. Clinical studies have demonstrated a correlation between the number of HPC measured in vitro and engraftment measured by the rate of neutrophil and platelet recovery. This has led to the hypothesis that if the number of HPC transplanted can be sufficiently increased, then it may be possible to abrogate the period of neutropenia and thrombocytopenia that is obligate even with transplants of autologous and allogeneic mobilized peripheral blood progenitor cells (PBPC).

In vitro expansion or generation of HPC from CD34+ bone marrow, PBPC, and umbilical cord blood cell populations using various culture conditions has been documented [1-7]. The culture conditions and growth factors used to stimulate proliferation influence the types of progenitors and mature cells produced [8-12]. Murine models have demonstrated that specific growth factors modulate the ability of cultured cells to engraft despite evidence for expansion of progenitors [13-19]. The effect of culture conditions on the subsequent engraftment of cells in humans is less clear. Several studies have recently been reported in which expanded cells were transplanted for the purpose of reducing post-transplant cytopenia [20-24]. Transplanting cultured cells along with non-cultured cells and the use of non-myeloablative therapies have obscured analysis of both the short- and long-term marrow repopulating potentials of the cultured cells. The study by Holyoake et al. [21] suggests caution in assuming that expansion in vitro will predict engraftment in vivo.

We utilized a primate model of autologous PBPC transplantation to study the role of in vitro expanded CD34+ PBPC in engraftment after transplantation. Recombinant human G-CSF (G-CSF, filgrastim®) can mobilize marrow repopulating cells in baboons; however, the engraftment of G-CSF mobilized PBPC is not as rapid as in humans [25, 26]. Preliminary in vitro studies demonstrated that both human and baboon CD34+ progenitor cells expanded in a similar fashion when cultured in serum-free medium in the presence of the combination of recombinant human stem cell factor (SCF), G-CSF, and recombinant human megakaryocyte growth and development factor (MGDF). We therefore asked whether G-CSF-mobilized CD34+ baboon PBPC cultured in vitro maintained the ability to engraft lethally irradiated recipients, if the kinetics of engraftment are altered and if administration of post-transplant growth factors is needed to maintain or enhance the engraftment potential of expanded CD34+ PBPC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Healthy juvenile baboons (Papio cynocephalus) were housed at the University of Washington Regional Primate Research Center, under American Association for Accreditation of Laboratory Animal Care approved conditions, as previously described [25]. Studies were conducted under Institutional Review Board and Animal Care and Use Committee approved protocols. All animals were provided with water, biscuits, and fruit ad libitum throughout the study. Immediately prior to transplantation, the animals were given a single dose of 1,020 cGy total body irradiation (TBI) from two opposing 60Cobalt sources at a rate of 7 cGy per minute. This TBI dose is myeloablative, and in the absence of an appropriate source of marrow repopulating cells, animals die of complications of marrow aplasia two to six weeks after transplant [25]. Animals were transfused with irradiated (2,000-3,000 cGy) fresh whole blood for treatment of thrombocytopenia and anemia and administered broad spectrum antibiotic prophylaxis until stable engraftment had occurred. A central venous catheter associated with a tether system was placed at the time of transplantation for the administrations of i.v. fluids, prophylactic antibiotics, transfusions, and for daily blood draws. This catheter was removed after evidence of engraftment, ability to maintain adequate oral intake, and minimal transfusion requirements. Six animals received daily s.c. injections of G-CSF (100 µg/kg/day) and MGDF (25 µg/kg/day) starting on the day that cryopreserved or cultured cells were infused and then continued for 28 days. Complete blood counts were followed daily until the catheters were removed, after which time they were obtained every other day to weekly as indicated.

Growth Factors and Schedules for In Vivo Administration
Pegylated MGDF (0.5 mg/ml; Amgen, Inc.; Thousand Oaks, CA), SCF (1.5 mg/ml; Amgen) and G-CSF (filgrastim®, 1.0 mg/ml; Amgen) were produced in E. coli, as previously described and purified to >99% purity [25, 27] and were used as described. Purified material was stored at 4°C until used. Growth factors were administered once daily by s.c. injection for each factor. The injections were administered in the lateral aspect of the thigh or midline of the back, which had been shaved prior to the start of each study.

Apheresis
Apheresis was performed on all animals under general anesthesia the morning after the fifth dose of G-CSF (100 µg/kg/day). To provide vascular access for apheresis the right or left femoral artery was visualized by cut-down and cannulated with an 18-gauge catheter and the saphenous or brachiocephalic vein on the opposite side was catheterized with an 18-gauge intracath. Apheresis was performed using a Cobe Spectra (Cobe; Lakewood, CO). Prior to each apheresis the instrument was primed with 240 to 300 ml of blood from adult baboon donors that had been irradiated (3,000 cGy in a 137Cesium source) and passed through a Pall RC100 leukocyte removal filter (Pall Biomedical; East Hills, NY). The total period of apheresis was 2 h for each animal. During apheresis the flow rate was 40 ml per minute, the centrifuge speed was 908 rpm, and the Spectra was run in manual mode with a collection rate of 1.5 ml per minute. Animals were anti-coagulated using preservative-free heparin (Elkins-Sinn, Inc.; Cherry Hill, NJ). One thousand units were administered immediately prior to apheresis and 10,000 units in 500 ml of saline were administered at 0.8 ml per minute during the procedure. Immediately after apheresis animals were given an appropriate dose of protamine sulfate. Cells were collected into a bag containing 20 ml of ACD-A. The apheresis product was shipped at controlled ambient temperature by overnight courier to Amgen, Inc. for cell separation, cryopreservation, and in vitro culture.

Isolation of CD34+ Cells from Apheresis Products
Apheresis products were washed once with an equal volume of 0.1% human serum albumin in D-PBS containing 1 mM EDTA (separation buffer), resuspended at 1 ¥ 108 WBC/ml and labeled at a concentration of 20 µg/ml with a mouse anti-human CD34 antibody (12.8) for 30 min at 4°C. After incubation, the cells were washed once with an equal volume of separation buffer, resuspended at 2 ¥ 108 WBC/ml and stained at a ratio of 100 µl/ml with an iron-dextran conjugated rat anti-mouse IgM (Miltenyi Biotech Inc.; Woodburn, CA) for 30 min at 4°C. After incubation, the cells were washed twice with equal volumes of separation buffer and resuspended at 2 ¥ 108 WBC/ml in separation buffer followed by CD34+ cell selection utilizing the CliniMACS cell selection device (AmCell Corp.; Mountainview, CA) according to the manufacturer's protocol. CD34+ cells were collected and washed with an equal volume of Iscove's modified Dulbecco's medium (IMDM; GIBCO Corp.; Grand Island, NY) containing 10% fetal bovine serum (FBS) for experiments utilizing unexpanded cells for transplantation or washed with an equal volume of defined ex vivo expansion medium. Experiments utilizing unexpanded cells for transplantation were frozen in IMDM containing 10% FBS and 10% DMSO using a programmable cell freezer according to the manufacturer's protocol.

Ex Vivo Expansion of CD34+ Cells
CD34+ cells were seeded at 5 ¥ 104/ml in 1-liter teflon culture bags (Amgen) in 1-liter defined ex vivo expansion medium (Amgen) supplemented with 100 ng/ml of SCF, G-CSF and MGDF (Amgen). Cultures were incubated at 37°C in 5% CO2 in 100% humidified air for 10 days. After incubation, cells were harvested, washed once with an equal volume of IMDM containing 10% FBS and transported on wet ice for same-day transplantation.

Progenitor Cell Assays
Apheresis product, CD34-, and CD34+ cells were cultured for GM-CFC-derived colony formation utilizing an alpha-media based two-layer agarose assay containing 30% serum and optimal concentrations of SCF, rHuIL-3, IL-6, G-CSF and GM-CSF [25]. Assays were incubated at 37°C in 5% CO2 in 100% humidified air for 14 days and scored at 20¥ under a dissecting microscope. A GM-CFC-derived colony was identified as a cluster of more than 50 granulocyte-macrophage cells.

FACS Analysis
In addition, these cells were analyzed for CD34+ cell content utilizing FACS analysis. Cells were labeled at a concentration of 25 µg/ml with a mouse anti-human CD34 antibody (clone 12.8) or a control mouse IgM antibody for 30 min at 4°C. After washing with separation buffer, cells were incubated with FITC-conjugated goat anti-mouse IgM antibody, at a concentration of 2 µg/ml, for 30 min at 4°C. After washing with separation buffer, cells were incubated with 1¥ FACS lysing solution (FACS-LS; Becton Dickinson Immunocytometry Systems [BDIS]; Sunnyvale, CA) for 15 min, washed and resuspended in 0.5 ml FACS-LS. We collected 50,000 events for each sample on a FACS Calibur (BDIS) and subtracted control values from our determinations.

Statistical Analysis
The analyses of the nadirs were performed using two-way analysis of variance (ANOVA) on log-transformed data, the transformation being used to stabilize the variance between groups. The time-to-recovery analysis used a Cox proportional hazards model applied to time-to-event data.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Engraftment of Cultured and Non-Cultured CD34+ PBPC
Four groups of baboons transplanted with enriched autologous G-CSF mobilized CD34+ PBPC (Table 1) were studied. We designed the experiments such that animals would engraft with the enriched CD34+ PBPC but the kinetics of engraftment would be slow enough to observe any potential benefit or deleterious effect from in vitro expansion of cells. Administration of G-CSF resulted in similar leukocytosis ( Table 2) and mobilization of CD34+ in all four groups. Two groups (I and III) were transplanted with cryopreserved, enriched CD34+ PBPC that had not been cultured. Group I animals received no growth factor support post-transplant, while those in Group III were administered G-CSF and MGDF. For Group I animals, transplanted with CD34-enriched G-CSF mobilized PBPC, the time to reach a sustained absolute neutrophil count (ANC) >500/µl and total WBC >1,000/µl was 18 ± 6 and 17 ± 4 days, respectively ( Table 3). This is comparable to our previously published data on animals transplanted with unfractionated G-CSF-mobilized PBPC where the time to ANC >500/µl and WBC >1,000/µl was 24 ± 7 and 16 ± 1 days, respectively [26]. The administration of post-transplant G-CSF and MGDF to animals in Group III, transplanted with non-cultured CD34-enriched PBPC, hastened engraftment with the time to ANC >500/µl and WBC >1,000/µl decreased to 14 ± 5 and 15 ± 7 days, respectively. This decreased the duration of time with ANC <500/µl to 10 ± 4 days for Group III animals and from 13 ± 4 days for Group I animals ( Table 3), but did not eliminate neutropenia.


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  		Table 1. Transplantation of autologous CD34+, G-CSF-mobilized peripheral blood progenitor cells
 

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  		Table 2. Effects of administration of G-CSF and SCF on peripheral blood counts in different transplant groups
 

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  		Table 3. Nadir and recovery of total WBC, ANC, and platelet counts after transplant
 
The animals in Groups II and IV were transplanted with autologous enriched CD34+ PBPC that had been cultured in vitro to expand colony-forming progenitors and megakaryocytes ( Table 4). Group IV animals received post-transplant G-CSF and MGDF while those in Group II did not. The in vitro-expanded CD34+ PBPC were cultured for 10 days in serum-free medium supplemented with 100 ng/ml of SCF, G-CSF and MGDF. This resulted in a seven- to eightfold expansion of CFU-GM detectable in colony-forming assays (Table 4). In addition, megakaryocytes were clearly produced by progenitors in these cultures ( Fig. 1). Morphologically identifiable megakaryocytes ( Fig. 1), which were absent prior to culture, represented 12% ± 2% and 14% ± 1% of cells harvested from cultures of CD34+ PBPC for a mean of 1.42 and 0.98 ¥ 108 megakaryocytes infused into Group II and IV animals, respectively.


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  		Table 4. Isolation and culture of CD34+ PBPC
 


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  		Figure 1. Photomicrographs of cytospin preparations from one animal transplanted with cultured CD34+ PBPC and administered G-CSF and MGDF after transplant (Group IV). Panel A: Cells transplanted, harvested after 10 days of culture in SCF, G-CSF and MGDF. Panel B: CD34 enriched cells prior to culture. Panel C: CD34 depleted cells. Panel D: Unfractionated apheresis product.

 
One of three animals in Group IV, transplanted with in vitro-expanded CD34+ PBPC and administered post-transplant G-CSF and MGDF, never became neutropenic as defined by neutrophil counts less than 500/µl (Fig. 2, Table 3). The nadir for both the total WBC (p < 0.001) and neutrophil (p < 0.002) counts were significantly higher for Group IV animals when compared to those transplanted with either non-expanded CD34+ PBPC (Groups I and III) (p < 0.05 for both measures), or to animals transplanted with expanded CD34+ PBPC but received no growth factor support after transplant (Group II).



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  		Figure 2. Recovery of total neutrophil counts after transplantation in the four transplant groups. The data for individual animals in each group are represented by the open symbols connected with the dashed lines. The mean value is shown by the solid symbols connected with the heavy solid lines. Panel A: Group I, transplanted with non-cultured, enriched CD34+ PBPC, with no post-transplant growth factor support. Panel B: Group II, transplanted with in vitro-cultured, enriched CD34+ PBPC, with no post-transplant growth factor support. Panel C: Group III, transplanted with enriched CD34+ PBPC and administered G-CSF (100 µg/kg/day) and MGDF (25 µg/kg/day) after transplant. Panel D: Group IV, transplanted with in vitro-cultured, enriched CD34+ PBPC and administered G-CSF (100 µg/kg/day) and MGDF (25 µg/kg/day) after transplant. The nadir for both the total WBC (p < 0.001) and neutrophil (p < 0.002) counts were significantly higher for Group IV animals (Panel D) when compared to those transplanted with either non-expanded CD34+ PBPC (Groups I and III, Panels A and C) (p < 0.05 for both measures), or to animals transplanted with expanded CD34+ PBPC but received no growth factor support after transplant (Group II, Panel B).

 
In contrast to neutrophil and total WBC recovery, platelet production and the nadir of platelet counts was not significantly different among the four groups of animals, despite the generation of significant numbers of megakaryocytes in cultures of the CD34+ PBPC transplanted in Groups II and IV ( Fig. 1, Table 4). If anything, the time to transfusion independence for platelets was prolonged in those animals transplanted with cultured CD34+ PBPC (Fig. 3, Table 3).



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  		Figure 3. Recovery of platelet counts after transplantation in the four transplant groups. The data for individual animals in each group are represented by the open symbols connected with the dashed lines. The mean value is shown by the solid symbols connected with the heavy solid lines. Panel A: Group I. Panel B: Group II. Panel C: Group III. Panel D: Group IV. See Figure 2 legend for details.

 
All Group I animals recovered normal blood counts, however one was euthanized at day 120 due to chronic diarrhea and weight loss. At necropsy this animal's marrow was 30%-50% normal cellularity with normal numbers of megakaryocytes. All Group III animals recovered normal WBC and ANC values, but one animal remained persistently thrombocytopenic and was euthanized at day 88 with hemolytic anemia and thrombocytopenia refractory to transfusion. All Group II animals recovered normal WBC and ANC values, although one died with transfusion unresponsive thrombocytopenia at day 83. Another animal in this group also developed chronic diarrhea and weight loss and needed to be euthanized on day 60. All Group IV animals recovered normal counts. One animal developed an interstitial pneumonia and was euthanized at day 133 with normal marrow function and morphology. All other surviving animals were followed for at least one year after transplant regardless of study group and had normal blood counts at that time.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The contribution of committed progenitor cells to reconstitution of hematopoiesis after bone marrow and peripheral blood stem cell transplantation has been thought to be limited to early stages of engraftment. Studies have been reported supporting the concept of a correlation between progenitor cell content of marrow and apheresis collections, measured by CD34+ analysis and in vitro colony-forming cell assays, and the time to reach a neutrophil count of 500 after transplantation [28]. Transplantation of bone marrow, mobilized peripheral blood, and cord blood cells results in periods of neutropenia and thrombocytopenia of varying lengths of time. The period of pancytopenia is shortest following transplantation of peripheral blood-derived stem cells and longest when cells from cord blood cells are used [29]. It has been hypothesized if sufficient numbers of committed progenitor cells can be infused at the time of transplant it may be possible to decrease or eliminate the period of post-transplant pancytopenia. Therefore, it is important to determine whether progenitors produced in culture will engraft and contribute to the reconstitution of hematopoiesis in vivo. We studied the ability of cultured, autologous, CD34+ peripheral blood progenitor cells to engraft primates who had received a marrow ablative dose of TBI [25].

In the present studies, CD34+ PBPC cultured in serum-free medium in the presence of SCF, G-CSF and MGDF produced increased numbers of in vitro colony-forming cells and megakaryocytes. Similar results have been obtained in studies of human CD34+ PBPC using the same culture conditions and growth factors (Briddell and McNiece, unpublished studies) and have been described also by Sawai et al. [30]. Cells in the cultured CD34+ PBPC populations clearly contributed to the initial engraftment of these otherwise lethally irradiated animals [25]. The length of post-transplant neutropenia was significantly decreased only when G-CSF and MGDF were administered to animals transplanted with cultured cells. In the absence of growth factor administration, cultured cells engrafted but no more rapidly than uncultured cells. It is of interest that only neutrophil recovery was more rapid. The recovery of platelet production was slower in animals transplanted with cultured cells despite evidence of large numbers of megakaryocytes being generated in these cultures and the post-transplant administration of MGDF. As none of the animals were treated with MGDF alone it is not possible to determine if the administration of both G-CSF and MGDF resulted in preferential stimulation of granulocyte progenitors in vivo. Despite the presence of large numbers of mature megakaryocytes in the cell populations at the time of transplant, it is also possible that the culture conditions may not have been optimal for expansion and maintenance of megakaryocytic progenitors. Further, although the administration of TPO and MGDF has stimulated more rapid recovery of platelet production in animals after low- to moderate-dose chemotherapy and radiation therapy [31, 32], after more myelosuppressive therapy there has been significantly less of an effect on platelet recovery [27].

The present study demonstrates that it is possible to significantly decrease post-transplant neutropenia by transplantation of cultured CD34+ PBPC and administration of growth factors. The fact that most animals transplanted with cultured cells survived for more than six months after transplant with adequate blood counts suggests that stem cells may have been maintained in these cultures. However, these studies did not include the introduction of a marker that would distinguish transplanted from remaining endogenous host cells. Therefore, it is not possible to definitively state that long-term engraftment arose from cells that divided during culture and was not a result of maintained quiescent stem cells or recovery of endogenous stem cells. It would be surprising if true stem cells were maintained at equivalent or greater numbers than were present at the initiation of culture, based on studies of Lansdorp and others [33]. However, this model should allow us to study the effects of such culture conditions on the engraftment of CD34+ stem cells from different sources. Sawai et al. [30] have recently demonstrated that the combination of SCF plus thrombopoietin can lead to sustained expansion of progenitors of multiple types in serum-free culture conditions. It is also possible that CD34+ PBPC collected using different mobilization paradigms may respond differently under in vitro culture and express different engraftment potentials. By the use of competitive repopulation studies that include differential marking strategies using retrovirus vectors [34], it may be possible to determine the contribution to engraftment by cell populations that divide at different times in culture. This should allow a more direct study of culture conditions that maintain stem cells for purposes of transplantation and gene transfer.

In summary, these data support further clinical studies with ex vivo-expanded cells plus post-transplant growth factor treatment as an approach to eliminate post-transplant neutropenia.


   ACKNOWLEDGMENT
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We wish to thank Ray Colby, Gary Millen, and the staff of the University of Washington Regional Primate Research Center and also the staff of the Clinical Hematology Laboratory at the Fred Hutchinson Cancer Research Center for their excellent support. We also acknowledge the technical assistance of Greg Stoney, Bret Kern, and Patrick Kerzic at Amgen, Inc.

Supported in part by grants and contracts NIHRR00166 and Amgen, Inc.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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accepted for publication June 15, 1999.




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