Stem Cells, Vol. 16, No. 4, 288-293,
July 1998
© 1998 AlphaMed Press
The Ex Vivo Expansion of Feline Marrow Cells Leads to Increased Numbers of BFU-E and CFU-GM but a Loss of Reconstituting Ability
Janis L. Abkowitz,
Monica R. Taboada,
Kathleen M. Sabo,
Grady H. Shelton
Division of Hematology, Department of Medicine, University of Washington, Seattle, Washington and Pacific Northwest Research Foundation, Seattle, Washington, USA
Key Words. Ex vivo expansion • Feline hematopoietic stem cells • Enrichment of HSC
Correspondence:
Dr. Janis L. Abkowitz, Division of Hematology, University of Washington, Box 357710, Seattle, Washington 98195-7710, USA.
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Abstract
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Some studies in mice suggest that hematopoietic stem cells can be maintained and possibly expanded ex vivo. As there is a paucity of data from larger animals, we have studied hematologic reconstitution following autologous marrow transplantation in cats. Transplantation of very low density marrow cells (<1.050 g/ml), termed "1050 cells," at 2 x 105 cells/kg leads to rapid hematopoietic recovery (granulocytes >200/µl by day 20 ± 2 and platelets >50 x 103/µl by day 21 ± 3). Recovery rates are comparable when 1-2 x 107 nucleated marrow cells/kg are infused, suggesting that reconstituting cells are enriched 50- to 100-fold in the 1050 cell preparation. To explore if the numbers of reconstituting cells could be expanded ex vivo, 1050 cells were cultured in the presence of 5 ng/ml recombinant human interleukin 1ß, 10 ng/ml recombinant canine (rc)G-CSF, 2 U/ml rHu erythropoietin, and 5 ng/ml rc stem cell factor. Maximum numbers of BFU-E and colony-forming units-granulocyte/macrophage (CFU-GM) were generated at day 6. However, when 106 1050 cells/kg (5x that needed for hematologic recovery) were cultured for six days and all resulting cells infused into irradiated donor animals, two of nine (22%) engrafted. Even when flt3 ligand (100 ng/ml) was added to cultures, only two of five animals (40%) engrafted (p = NS versus studies without flt3 ligand). These data confirm that BFU-E and CFU-GM provide inaccurate estimates of reconstituting cells and demonstrate that the number or function of feline reconstituting cells is impaired by in vitro culture with cytokines.
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Introduction
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When marrow cells are cultured in vitro in the presence of cytokines, the numbers of BFU-E, colony-forming units-granulocyte/macrophage (CFU-GM), and long-term culture-initiating cells (LTC-IC) increase. Whether hematopoietic stem cells (HSC), cells able to contribute to the long-term maintenance of lymphoid and myeloid hematopoiesis, increase in number is controversial. It is also unclear if cells functionally defined as short-term repopulating cells (STRC) expand [1-14]. Whether STRC, i.e., cells which reconstitute hematopoiesis immediately after transplantation, form a distinct population from HSC is also uncertain [15-19].
These issues are important because of their clinical implications. In many circumstances cells with a proliferative capacity of HSC are not required. For example, most chemotherapy regimens induce life-threatening, but transient, cytopenias. As the role of peripheral blood stem cell or bone marrow infusion is to provide quicker marrow recovery and thus avoid the morbidity (and mortality) of bleeding and infection, it is only important that the infused cells function as STRC. Also, several potential strategies for the gene therapy of hemoglobinopathies are premised on the concept that STRC and/or later progenitors (i.e., BFU-E) engraft. Theoretically, one could correct the genetic defect in these cells, easier than in quiescent HSC, and after repeated infusions generate sufficient numbers of corrected red blood cells to dominate the red cell compartment [20].
We have studied these issues in cats. Since the number of red cells, white cells and platelets that a mouse makes in a two-year lifetime is approximately that generated by a cat in eight days (or a man in one day) [21], the biology or kinetics of HSC in a large animal could differ from that in a mouse. Cells with the proliferative capacity of a murine STRC (or HSC) even if expandable in culture, may be unable to reconstitute feline hematopoiesis. In this manuscript we describe a simple method to enrich feline cells capable of hematopoietic reconstitution by 50- to 100-fold and consider whether the number of reconstituting cells is amplified during in vitro culture with stem cell factor (SCF) and flt3 ligand (flt3L).
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Materials and Methods
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Cell Culture Methods
Marrow cells were aspirated from the proximal femur or humerus of cats after ketamine and acepromazine anesthesia (0.11 mg/kg; 22 mg/kg). Heparinized cells were layered over Percoll (density 1.070 g/ml; Sigma Chemical Company; St. Louis, MO), centrifuged at 400 g for 20 min and interface cells collected, then washed x2 in Hank's calcium and magnesium-free media (GIBCO Laboratories; Grand Island, NY). Colony-forming units-erythroid (CFU-E), BFU-E, and CFU-GM numbers were determined by methylcellulose culture. Briefly, cultures were established in Iscove's modified Dulbecco's medium ([IMDM]; GIBCO) with 20% heat-inactivated fetal calf serum (FCS) (Summit Biotech; Fort Collins, CO), 10% heat-inactivated pooled normal cat serum, 1% bovine serum albumin ([BSA]; Intergen Co.; Purchase, NY), 10–4 M betamercaptoethanol (Sigma), 1% penicillin/streptomycin/fungizone ([PSF]; GIBCO), 1 U/ml recombinant human erythropoietin ([rHuEpo]; Amgen; Thousand Oaks, CA), 2 ng/ml recombinant canine stem cell factor ([rcSCF]; Amgen) and 1.2% methylcellulose (Kodak; Rochester, NY). After incubation at 37°C in 5% CO2/95% air for three days, erythroid colonies (from CFU-E) containing 8-50 hemoglobinized cells were enumerated with indirect microscopy. After 10-12 days, erythroid bursts (from BFU-E), containing >250 hemoglobinized cells, and GM colonies (from CFU-GM), containing >50 cells, were counted. Cultures were performed in triplicate at several cell densities (e.g., 103 - 105/ml) to assure an accurate assessment of progenitor frequency and results were expressed as the mean ± the standard error.
Preparation of 1050 Cells and Fluorescence-Activated Cell Sorter (FACS) Analysis
To prepare 1050 cells, marrow mononuclear cells (density <1.070 g/ml) were subjected to a second Percoll density gradient centrifugation (at 1.050 g/ml) then washed x2 and an aliquot was counted with trypan blue exclusion. For FACS analyses monoclonal antibodies to feline CD5 (FE1.1B11; T cells), canine/feline CD21 (CA2.1D6; B cells), and canine/feline CD11b (CA16.3E10; granulocyte/monocytes) were obtained from Peter Moore (University of California; Davis, CA). As all are IgG1, monoclonal antibody SR1 (which recognizes human but not feline c-kit, obtained from Virginia Broudy (University of Washington; Seattle, WA) was used as an isotype control. 106 cells (in 50 µl phosphate-buffered saline [PBS]) were incubated on ice for 15 min with the monoclonal antibodies (20 µl tissue culture supernatant). After washing with 2 ml of cold PBS/1% BSA, phycoerythrin-conjugated goat antimouse immunoglobulin (Biosource International; Camarillo, CA) was added (final concentration of 50 µg/ml). The mixture was then incubated for 30 min on ice in the dark, washed, and subjected to FACS analysis. In some studies, cells were fixed with 300 µl cold PBS/formaldehyde (1%) added drop-wise while vortexing, and stored at 4°C, covered with foil, until FACS analysis.
Autologous Transplantation Methods
The methods for autologous transplantation are in [21]. Briefly, marrow was aspirated from both femurs and humeri of four to eight-month-old cats. Nucleated marrow cells were prepared by centrifugation of bone marrow at 1,600 rpm x20 min, the collection and washing of interface cells, and hypotonic lysis of erythrocytes. 1050 cells were prepared as described above. Prior to transplantation, the cats then received 920 cGy of total body irradiation (7cGy/min from opposing cobalt-60 sources). To compute the number of transplanted nucleated marrow cells, the number of white blood cells (WBC) present in a comparable volume of blood was subtracted (# transplanted nucleated marrow cells/ml = # nucleated marrow cells/ml - # peripheral WBC/ml). There was no numerical compensation for potential blood cell contamination in studies with 1050 marrow cells.
Infection prophylaxis included neomycin (5 mg/kg) and polymyxin (5000 units/kg) that was administered orally every eight h beginning five days prior to transplantation and continuing until hematologic recovery. After transplantation the animals also received broad spectrum antibiotics s.c., including ampicillin (6.6 mg/kg) every 12 h, and gentamycin (4 mg/kg) every 12 h for one day and then daily. Transfusions of irradiated blood were given as needed for platelet support. Hematocrit, WBC count, WBC differential, and platelet count were measured to document engraftment. Animals were sacrificed if clinical symptoms, i.e., lethargy, fever, or bleeding, failed to respond to transfusions or antibiotic therapies. The days at which granulocytes exceeded 200/µl and 500/µl in differential counts, and platelets exceeded 50 x 103/µl were recorded.
In Vitro Expansion
In initial studies to determine the concentrations of reagents which yielded an optimal in vitro expansion of progenitor cells, 1050 cells were cultured in the presence or absence of rHuEpo (2 U/ml), rcSCF (5 and 10 ng/ml), rcG-CSF (10 ng/ml; Amgen), recombinant human interleukin 1ß ([rHuIL-1ß]; 5 and 20 ng/ml; Collaborative Biomedical Products; Bedford, MA), and rHuIL-6 (20 ng/ml; Collaborative Biomedical Products). The numbers of BFU-E and CFU-GM were determined at days 4, 6, and 8 of culture. Different percentages of FCS and cat serum were included. All cultures contained IMDM, 1% BSA, 10-4 M betamercaptoethanol, and 1% PSF. In some studies, flt3L (100 ng/ml; Immunex; Seattle, WA) was added to cultures. Cultures were fed daily as needed to maintain a cell concentration between 0.5 x 106 and 1 x 106 and facilitate logarithmic growth.
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Results
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Very Low Density (<1.050 g/ml) Feline Marrow Cells Contain High Numbers of Hematopoietic Progenitors
Percoll (1.070 g/ml) density gradient centrifugation, and not Ficoll-hypaque or LSM (1.077 g/ml), allows for the optimal separation of feline lymphocytes from granulocytes in blood and of feline hematopoietic progenitor and precursor cells from mature granulocytic cells in marrow [22]. To determine if progenitors could be further enriched by density criteria, we used discontinuous density centrifugation to assess the distribution of cells with lower density values (1.050, 1.055, 1.060 g/ml). Cells with densities under 1.050 g/ml were present in low frequency and comprised 1%-7% of the marrow mononuclear (1.070 g/ml) cell population. Mean BFU-E and CFU-GM frequencies were 853 ± 221 and 204 ± 39 per 105 cells, respectively (n = 3), representing 10 ± 2 and 16 ± 3-fold mean enrichments (mean yield = 60%). CFU-E frequency was relatively low (209 ± 84/105 cells) (range 79-365/105 cells) and thus their frequency was less than that seen among mononuclear cells. It therefore appeared that 1.050 g/ml density gradient centrifugation allowed for the enrichment of earlier (e.g., less differentiated) but not later progenitor cells. On Wright-Giemsa stain these cells, termed "1050 cells," were a heterogeneous population (47% blast cells, 17% lymphocytes, 4% monocytes, 21% myelocytes, metamyelocytes, or band, and 11% erythroid precursors). By FACS analysis (n = 2), 9% of cells were CD5-positive (T cells), 4% CD21-positive (B cells), and 26% CD11b-positive (myeloid cells), confirming that granulocyte and/or monocyte precursors were the predominant contaminating cell population. Antibodies to human and canine CD34 failed to crossreact with feline cells (data not shown) so that CD34 expression could not be assayed.
Low Density (1.050 g/dl) Centrifugation Significantly Enriches for Cells Able to Reconstitute Hematopoiesis in Cats
Autologous marrow transplantation studies were then done using nucleated marrow cell and 1050 cell preparations. As shown in Table 1, granulocyte and platelet numbers reconstituted quickly (days 15 and 14, respectively) when 2 x 108 nucleated marrow cells per kilogram were transplanted. This cell dose is generally used in human transplantation. When 1-2 x 107 nucleated marrow cells/kg were transplanted, reconstitution occurred on days 20 and 24, respectively. When 5 x 106 nucleated marrow cells/kg were transplanted, only two of three animals survived. Granulocytes reconstituted in the surviving animals on days 30 and 26 and platelets on days 31 and 26. Similar cell-dose and time-to-engraftment data have been reported in canine marrow transplantation studies [23, 24].
In contrast to these data with nucleated marrow cells, the infusion of 2 x 105 1050 cells/kg resulted in rapid engraftment and all cats receiving this cell preparation survived. Thus, the cell enabling reconstitution was retained with low density (1.050 g/dl) centrifugation. Because the time to engraftment is comparable to that observed after the transplantation of 1-2 x 107 nucleated marrow cells/kg, it appears that reconstituting cells were enriched by approximately 50- to 100-fold.
Feline Progenitor Cells Can Be Expanded In Vitro
Various combinations of cytokines were tested for their capacity to expand progenitor cells in vitro. For these studies 1050 cells were cultured in the presence of 20% heat-inactivated FCS and 10% heat-inactivated pooled cat serum. The cultures were fed every day to maintain a cell concentration between 0.5 x 106 and 1 x 106/ml and to facilitate logarithmic growth. Optimal conditions were determined to be 5 ng/ml rcSCF, 10 ng/ml rcG-CSF, 5 ng/ml rHuIL-1ß, and 2 U/ml rHuEpo. rHuIL-6 did not improve results. At day 6 maximal numbers of cells (17- to 225-fold expansion), and numbers of BFU-E and CFU-GM (4- to 26- and 2- to 11-fold expansions, respectively) were seen (n = 10).
Progenitor Cells Generated In Vitro Fail to Engraft in Irradiated Cats
For these studies, the engraftment potential of 2 x 105 (or 1 x 106) 1050 cells/kg was directly compared to the engraftment potential of the cells generated from six days of expansion culture after an input of 1 x 106 1050 cells/kg. The results are shown in Table 2. All animals quickly engrafted when transplanted with unmanipulated 1050 cells while in concurrent experiments; seven of nine animals failed to engraft after receiving expanded cells. Engraftment occurred extremely late in one of the two engrafting animals. Granulocytes greater than 200/µl occurred at day 37 and platelets greater than 50,000/µl at 40 days. Thus, one could not exclude the possibility that reconstitution in this animal resulted from the recovery from radiation damage of host marrow cells and not the engraftment and differentiation of infused cells. As fivefold more cells were input to suspension culture than were needed for rapid engraftment, the data suggest that the number of engrafting cells significantly declined during the six days of culture. Since BFU-E and CFU-GM numbers increased within each suspension culture, these data demonstrate that committed progenitor cells are not reconstituting cells in larger animals. Alternatively, cat progenitor cells may develop functional defects during in vitro culture [12, 14].
The Addition of flt3L to Suspension Cultures Does Not Permit the Expansion of Reconstituting Cells
flt3L (also called flk2 ligand) expands numbers of progenitor cells and possibly HSC [6, 25]. rHuflt3L is equally active on cells of all species, including mouse and cat, in assays evaluating the size and number of progenitor cell-derived colonies (data not shown). When added to feline suspension culture (at plateau concentration, 100 ng/ml) the kinetics of expansion were unchanged. Although the numbers of BFU-E generated were not different than in our standard culture conditions, there was a small increase in the number of CFU-GM progenitors generated at day 6 (range 2- to 20-fold over input, mean = 6.5 [versus 3.8 with rHuSCF alone, p = NS, in eight direct comparative experiments]). Five cats were transplanted with the cells that were generated from six days of suspension culture of 1 x 106 1050 cells/kg in the presence of the previous cytokines and 100 ng/ml rHuflt3L. Three animals failed to engraft and were sacrificed because of pancytopenia, sepsis, and/or bleeding. Two had late engraftment, and granulocytes increased above 200/µl at days 37 and 28 while platelets increased above 50 x 103/µl at days 30 and 28. It appears that the inclusion of flt3L did not allow for the expansion of repopulating cells.
Interestingly, the quantity of cell expansion in the two engrafting animals was less than that in the three animals that failed to engraft (17 ± 10-fold expansion after six days compared to 205 ± 148-fold expansion). However, fold expansions of BFU-E and CFU-GM were similar. Thus, when the ratio of fold expansion of total cells to fold expansion of progenitors was calculated in each animal, this ratio was significantly lower in the engrafting animals (mean = 1.3) than in cats that failed to engraft (mean = 47.8). We do not think that this difference reflects excessive numbers of differentiated cells and the lack of availability of nutrients, excessive metabolic intermediates, or other alterations in cell culture condition, as cultures were maintained at 0.5-1.0 x 106/ml by daily cell counts throughout the incubation time. Similar results were seen when data from the animals that received cells expanded in the presence of IL-1ß, Epo, G-CSF, and SCF, were analyzed (cumulative data in Table 3), so that this observation is not a property of flt3L.
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Discussion
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By suspension culture, one can vastly increase the number of cells with an immature phenotype. Similarly, the number of progenitor cells and earlier cells defined by more complex in vitro assays (e.g., LTC-IC, cobblestone-forming cells) increase in number. Some murine and human transplantation studies are compatible with the hypothesis that expanded cell populations retain their potential to engraft [1-9]. In human studies, however, limiting dilution analyses cannot be performed. If 1,000 reconstituting cells were present prior to culture and only 100 remain after "in vitro expansion," reconstitution may still be observed, yet the conclusion that reconstituting cells were maintained or expanded in number would be incorrect. In our studies we compared the engraftment capacity of 2 x 105 low density marrow mononuclear cells (1050 cells)/kg to the output of suspension cultures inoculated with 5x that number of cells. All animals receiving uncultured cells rapidly engrafted, while 10 of 14 animals receiving cells cultured for six days in the presence of IL-1ß, Epo, G-CSF, and SCF, with or without flt3L failed to engraft. These animals were persistently pancytopenic and died with sepsis and/or bleeding. Since BFU-E and CFU-GM expanded in number during the six-day suspension culture, our data suggest that progenitor cells are not reconstituting cells and that too few reconstituting cells persisted after in vitro culture to permit hematopoietic recovery. Alternatively, engraftment defects may develop during suspension culture, as has been reported in murine studies [12, 14]. Thus, the absolute number or fold increase in BFU-E and CFU-GM are inaccurate estimators of the number and/or function of reconstituting cells after in vitro culture with growth factors. We suggest that clinical trials with constant numbers of harvested cells with or without "ex vivo expansion" are needed to justify the routine application of in vitro culture protocols in patients.
The four animals that survived after receiving cultured marrow cells provide some additional insights. In three of these animals, recovery was late (at 28-40 days), so we cannot formally exclude the possibility that it was mediated by the recovery of endogenous hematopoiesis, and not the contributions of cultured cells. It is intriguing, however, that in these cultures fewer mature cells were generated while there were comparable increments of BFU-E and CFU-GM ( Table 3). Perhaps the ratio of fold increase in cell number to fold increase in progenitor number will predict conditions that allow for the maintenance of earlier cells. Calculation of this ratio could guide studies using different concentrations or combinations of cytokines, bioreactors, alternative cell sources (e.g., fetal liver or mobilized peripheral blood progenitor cells) in outbred and heterogenous populations such as large animals or man.
As a higher percentage of cats survived when transplanted with cells cultured in the presence rather than the absence of flt3L, flt3L may support the maintenance of some cells able to reconstitute hematopoiesis in vivo. The difference, however, was not significant with chi square analysis (p > 0.5). It is possible that alternate concentrations or combinations of early active cytokines may support the persistence of such cells. For example, the recent paper of Miller and Eaves [9] suggests that the addition of IL-11 to SCF and flt-3L facilitates the in vitro expansion of murine HSC.
Our studies further demonstrate that 2 x 105 (unexpanded) 1050 cells/kg can quickly reconstitute an irradiated cat. After recovery, normal hematopoiesis is maintained. The data therefore suggest that this cell population contains a 50- to 100-fold enrichment of feline reconstituting cells. Because these studies involve autologous transplantation, we cannot formally exclude the possibility that endogenous hematopoiesis recovered late after transplantation. Thus we cannot assert that cells capable of maintaining long-term multilineage hematopoiesis (i.e., HSC) were similarly enriched, although this conclusion is likely [26]. If so, 1050 cells should be an excellent source of cells for gene transfer. Cats weigh three to four kg. As retroviral supernatants contain approximately 106 infectious units per ml, multiplicities of infection of over 5:1, necessary to assure virion:cell contact, are easily achievable. Perhaps with in vivo amplification [27] before marrow harvest and low density centrifugation, there will be additional enrichment of both reconstituting cells and HSC which would facilitate the preclinical investigation of gene therapy. Importantly, our studies suggest that one should use any in vitro incubation step with caution. It is possible even during the two- to four-day cultivation time generally employed for retroviral gene transfer that numbers of reconstituting cells will decline [28, 29].
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Acknowledgments
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The authors thank Allan Dimaunahan for help in preparation of the manuscript.
These studies were supported by R01s HL31823, HL46598, and DK49652 from the National Institutes of Health. Dr. Abkowitz is the recipient of a Faculty Research Award from the American Cancer Society.
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accepted for publication on May 28, 1998.
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Abstract
Introduction
