Stem Cells, Vol. 15, No. 5, 347-352,
September 1997
© 1997 AlphaMed Press
Evaluation of a Cytokine Combination Including Thrombopoietin for Improved Transduction of a Retroviral Gene into G-CSF-Mobilized CD34+ Human Blood Cells
Hyeoung Joon Kima,b,
Yasuhiro Okamotoa,
Michinori Itoa,
Yoichi Takauea,
Yoshifumi Kawanoa,
Tsutomu Watanabea,
Takashi Yamauea,
Yoshihiro Tohdaa,
Takeshi Ogosea,
Takashi Shimadac,
Akihiro Shimosakad,
Yasuhiro Kurodaa
a Department of Pediatrics, University of Tokushima, Tokushima, Japan;
b Department of Medicine, Chonnam University School of Medicine, Kwangju, Korea;
c Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan;
d Kirin Brewery Co., Tokyo, Japan
Key Words. NeoR gene transduction • CD34+ cells • Thrombopoietin • Cytokines • Cell culture
Dr. Yoichi Takaue, Department of Pediatrics, University of Tokushima, Kuramoto-cho 3, Tokushima 770, Japan.
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Abstract
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We examined cell culture conditions with various combinations of cytokines including thrombopoietin (TPO) to obtain the most efficient transduction of recombinant retrovirus vectors into G-CSF-mobilized blood CD34+ cells which were obtained from children and purified with an Isolex 50 system (Baxter; Deerfield, IL). Three different 4-day culture conditions for the stimulation of CD34+ cells were compared in terms of a cell-cycle analysis by fluorometry and gene transduction efficiency as determined by resistance to G418 and NeoR polymerase chain reaction (PCR) for individual colony-forming unit-granulocyte/macrophage (CFU-GM) grown in a methylcellulose culture system. The cytokines tested were: A) interleukin (IL)-6 + stem cell factor (SCF); B) IL-3 + IL-6 + SCF, and C) IL-3 + IL-6 + SCF + TPO. Without a cell culture, the percentage of CD34+ cells in the cell cycle (the percentage of cells in phases S and G2/M) was 4.6%. After a four-day culture (n = 5), this value increased with the addition of IL-3 (22%) or IL-3 + TPO (27%, p < 0.05) as compared to that with the baseline cocktail of IL-6 + SCF (15%). The cell number uniformly increased approximately 10-fold in each culture condition. The average efficiency of gene transfer into incubated CD34+ cells with the corresponding combinations of cytokines was, respectively, 57%, 47%, and 30% for G418-screened CFU-GM and 72%, 68%, and 51% for polymerase chain reaction-positive CFU-GM. A statistically significant difference (p < 0.01) was found for G418/CFU-GM with IL-3 + IL-6 + SCF (57%) versus IL-3 + IL-6 + SCF + TPO (30%). Hence, it is likely that the increased cell proliferation produced by the addition of TPO was not necessarily translated into an increased rate of retroviral-mediated gene transduction, possibly because TPO preferentially induced the differentiation of stem cells into mature progenitors in these culture systems.
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Introduction
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Since the biology of hematopoiesis has been characterized and clinical hematopoietic stem cell transplantation procedures are well established, these cells may be an ideal target for the stable transduction of new genetic material [1, 2]. Peripheral blood stem cells (PBSC) are widely used as a source of hematopoietic stem cells. The advantage of PBSC is that multiple collection procedures can be performed without anesthesia or invasive surgery; this may be an important consideration in practical gene therapy, since the target patients could be very small children. To improve the transduction and/or long-term expression of marker genes, purification of CD34+ cells is necessary to reduce both the amount of retrovirus supernatant and the risk of insertion mutagenesis [3-5]. In previous studies, we suggested that mobilized PBSC from small children were a realistic target for clinical gene therapy [6] and reported that transplants with purified blood CD34+ cells were successful with no difference in the engraftment speed between autografts with unmanipulated PBSC versus PB CD34+ cells [7].
Since the transduction of retroviral-vector-mediated genes occurs only in actively cycling cells [8], the activation of target cells by a combination of growth factors is essential for facilitating the transduction process and for making the procedure practical. In addition, ex vivo exposure of cells to growth factors may alter the expression of receptors for amphotropic retroviruses and thereby enhance the transduction procedure. Consequently, the culture conditions and the combination of growth factors used are considered to be the major determinants of transduction efficiency and the stable expression of the inserted gene [9]. Although most current culture protocols for this purpose use a standard combination of interleukin (IL)-3, IL-6, and stem cell factor (SCF) [10-17], the optimal combination for supporting maximum gene transduction remains to be established. In this study, we examined four-day cell culture conditions with various combinations of cytokines including newly introduced thrombopoietin (TPO) for their potential to induce the transduction of recombinant retrovirus vectors into G-CSF-mobilized blood CD34+ cells. Unexpectedly, we observed that the enhancement of cell proliferation by itself does not necessarily result in facilitated gene transduction.
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Materials and Methods
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Patients and Cell Collection
In five children (5 to 12 years old) with active cancer, PBSC were harvested during the recovery phase of chemotherapy with G-CSF by a Fenwal CS3000 Plus cell separator (Fenwal Lab; Deerfield, IL) and cells were further purified to CD34+ cells for autografts as described elsewhere [7, 18]. Some leftover samples were used for this study with the written informed consent of the children's parents. This study was approved by the Institutional Review Board.
Purification of CD34+ Cells
Blood mononuclear cells (MNC) were separated on 40%/60% Percoll gradients by centrifugation at 400 g for 30 min and washed three times with phosphate-buffered saline (PBS) solution without Ca2+ or Mg2+. MNC were stained with an anti-CD34 monoclonal antibody (9C5; Baxter; Deerfield, IL) at 0.5 µg/106 cells for 30 min at 4°C under slow end-over-end rotation using an Isolex 50 system (Baxter). Cells were washed three times with PBS/human serum albumin ([HSA]; Kaketsuken; Kumamoto, Japan). Paramagnetic microspheres coated with sheep antimouse antibodies (Isolex anti-CD34 monoclonal antibody; Baxter) were washed twice with PBS/HSA. Sensitized MNC and washed dynabeads (Dynal AS; Oslo, Norway) were mixed at 0.5 beads/cell for 30 min at 4°C with rotation. Beads and rosetted target cells were collected by exposure to magnets (two min) and washed three times with PBS/HSA. Rosetted CD34+ cells were released from the beads by incubation with 200 U/ml chymopapain (Boots Pharmaceuticals; Nottingham, UK) for 15 min at 37°C according to the method of Ishizawa et al. [19]. The cells were then cryopreserved and stored until use in a –135°C deep freezer. After thawing, CD34+ cells were placed in an AIS Micro CELLector TM T-25 Cell I Culture Flasks (Applied Immune Science; Santa Clara, CA) for one h to improve their purity, which was analyzed by side-scatter fluorescence and by the percentage of cells stained with anti-CD34 antibody (HPCA-2; Becton Dickinson Labware; Lincoln Park, NJ) using 2 to 5 x 105 cells.
Retrovirus Production
The details of this method have been previously reported [6]. Briefly, the plasmid form of the retroviral vector (XM5) that contained the NeoR gene was transfected into the 
2 ecotropic packaging cell line using calcium phosphate precipitation from which a high titer clone was obtained. After 48 h, filtered supernatants from this clone were used to transduce the PA317 amphotropic packaging cell line. Infected cells were selected in 1.0 mg/ml G418 (0.7 mg/ml active) to obtain a monoclonal cell line that produced XM5/PA317. This virus was titered at 3 to 5 x 106 colony-forming units (CFU)/ml on NIH 3T3 cells. Freshly prepared virus supernatant was filtered through a 0.45 µm filter and used for the transduction experiments.
Retroviral Transduction Procedure
CD34+ cells were suspended in Dulbecco's modified Eagle's medium (GIBCO; Grand Island, NY) and cultured for 96 h at 37°C in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2 at a density of 1 to 2 x 105/ml in supernatant containing the vector (XM5/PA317). Cultures were supplemented with 10% fetal bovine serum ([FBS]; GIBCO), 8 µg/ml polybrene, and the designated combination of cytokines as follows: A) IL-6 + SCF; B) IL-3 + IL-6 + SCF, and C) IL-3 + IL-6 + SCF + TPO. All of the cytokines were from Kirin Brewery Co. (Tokyo, Japan), and the concentrations used were 20 ng/ml for IL-3, 50 ng/mL for IL-6, 100 ng/ml for SCF, and 10 ng/ml for TPO. Every 24 h, the old supernatants were removed after the cells were centrifuged, and freshly prepared virus supernatants supplemented with polybrene and growth factors were added. On day 5, the cells were harvested for short-term clonogenic assay in methylcellulose. As a control (mock), some of the cells were maintained under the same conditions as the experimental cultures, except that no vector supernatant was added.
Flow Cytometric Analysis
The details of the method used to count CD34+ cells have been previously reported [20]. Briefly, aliquots (0.3 to 0.5 ml) of cell suspension (3 x 106 cells/ml were mixed with 1.5 ml RPMI-1640 supplemented with 10% FBS and stored at 4°C for flow-cytometry analysis performed within 48 h. One hundred µl of cell suspension were then dispensed into test tubes (Falcon 2052) for staining and for a control. Staining was performed by adding fluorescein isothiocyanate-conjugated CD33 antibody and phycoerythrin-conjugated CD34 antibody (IOM34; Immunotech; Marseilles, France) for 30 min at 4°C in the dark. Antimouse IgG1 conjugated with fluorescein isothiocyanate or phycoerythrin was used as a control. Samples were analyzed by a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems; San Jose, CA), which was calibrated using caliBRITE beads and compensated using the CD33/CD34 fluorescence information from noncoexpressing controls stained with Becton-Dickinson reagents. After function was verified, the samples were drawn into the flow cytometer using forward scatter and side scatter as gating parameters, and each group of 2 x 104 cells was analyzed. The flow cytometric data were analyzed using a gated analysis via a set of side scatter-fluorescence-2 parameters for the CD34+ cells to calculate the percentage of positive cells. Cell populations gated with an FL1-FL2 parameter were used in the analysis of lymphocyte subsets. If the CD34+ population in the detection of 2 x 104 cells fell short of 1 x 103 cells, the count was extended to a maximum of 5 x 104 cells to collect a sufficient number of CD34+ cells for evaluation. For cell-cycle analysis, the cells were pelleted in plastic tubes and 1 ml of 70% ethanol was added at 4°C for 20 min. After washing twice, 200 µl of PBS containing RNase (500 µg/ml, DNase-free) was added at 37°C for 30 min. Cells were then pelleted and 500 µl of propidium iodide (50 µg/ml, Sigma Chemical Co.; St. Louis, MO) in 0.1% sodium citrate buffer were added. After 15 min of incubation at 4°C in the dark, cells were analyzed on a FACScan cytometer. For each sample, DNA histograms were analyzed using CellFIT software (Becton Dickinson) to examine the distribution of cells in phases G0/G1, S and G2/M of the cell cycle.
CFU-GM Assay in Methylcellulose
The detailed procedure has been reported elsewhere [21]. Briefly, cells were cultured at 250 cells/well in Iscove's modified Dulbecco's medium (Flow Laboratories; Irvine, Scotland, UK) containing 0.8% methylcellulose, 20% FBS (GIBCO), 450 µg/ml human transferrin, 1% deionized bovine serum albumin, with or without 1.0 mg/ml G418 (0.7 mg/ml active). Recombinant human cytokines were added to the cultures at various prescreened concentrations (200 ng/ml for SCF, 200 ng/ml for IL-3, 2 U/ml for erythropoietin and 200 ng/ml for G-CSF). Quadruplicate cultures were plated in a volume of 0.4 ml in 24-well tissue culture plates (Corning, NY) as previously reported [21]. The plates were incubated at 37°C in a humidified atmosphere of 5% O2, 5% CO2, and 90% nitrogen for 14 days. Colonies were scored for CFU-granulocyte/macrophage (CFU-GM) at 14 days of culture using an inverted microscope.
Polymerase Chain Reaction (PCR) Amplification
Well-isolated CFU-GM from plates without G418 were picked up using a finely drawn Pasteur pipette and subjected to PCR amplification of the NeoR gene. The sequences of the primers were 5' ATC ATG GCT GAT GCA ATG CG 3' and 5' AGA TCA TCC TGA TCG ACA AG 3'. The conditions for amplification were as follows: 94°C for 5 min, then 35 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min, followed by extension at 72°C for 7 min. PCR products were separated by 3% agarose gel electrophoresis.
Statistical Analysis
The efficiency of gene transfer was assessed by: A) the ability of transduced CFU-GM to grow in the presence of G418 and B) PCR analysis of individual CFU-GM. The ratio of the number of CFU-GM colonies that grew in the presence of G418 to those which grew in the absence of G418 was determined. The results of transduction are expressed as the mean ± standard error (SE). Significance was evaluated by a two-sided paired Student's t-test.
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Results
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The mean recovery rate of CD34+ cells after purification was 18% and the purity of the final CD34+-enriched fraction was 88 ± 14% (mean ± SE). Five experiments were performed and the results are summarized in Table 1. Without the cell culture step, the percentage of CD34+ cells in the cell cycle (the percentage of cells in phases S and G2/M) was 4.6% ( Fig. 1). After a four-day culture, the cell number uniformly increased approximately 10-fold in each culture condition and the percentage of cells in the cell cycle increased to 15% with the combination of IL-6 + SCF, 22% with IL-3 + IL-6 + SCF, and 27% with IL-3 + IL-6 + SCF + TPO; thus, significantly more cells (p < 0.05) were brought into cycling status by the full combination of IL-3 + IL-6 + SCF + TPO compared to the other combinations without TPO ( Fig. 2). On the other hand, the transduction efficiency evaluated by both G418-screened CFU-GM and PCR-positive CFU-GM with the same cytokine combinations was 57% and 72%, 47% and 68%, and 30% and 51%, respectively, with a statistically significant difference found only for CFU-GM with IL-3 + IL-6 + SCF versus IL-3 + IL-6 + SCF + TPO (p < 0.01). No colonies grew from mock-transduced PB CD34+ cells after G418 selection.
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Figure 1. Cell-cycle analysis of isolated CD34+ cells. Significantly more cells (p < 0.05) were brought into cycling status by the full combination of IL-3 + IL-6 + SCF + TPO, compared to the other combinations without TPO.
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Discussion
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Retrovirus-mediated transduction of target genes occurs only when cells are in an actively cycling status. Therefore, it is widely recognized that efficient cell activation by a combination of hematopoietic growth factors is required for a practical procedure in hematopoietic stem cells. Optimum combinations of cytokines for this purpose have been evaluated for their cell-growth potential as an endpoint, and in this study, we introduced TPO. The biological effect of TPO, which is also referred to as megakaryocyte growth and development factor or the c-Mpl ligand, is not restricted to cells of megakaryocytic lineage, and its ability to support the general expansion of very immature progenitor cells, possibly including stem cells, has been reported [22]. In addition to its role as a hematopoietic growth factor, there is a possibility that TPO behaves as a survival factor for human hematopoietic cells by suppressing apoptosis [23]. Indeed, in a separate experiment, we observed that TPO-treated cells showed a lower population of apoptotic cells than standard cytokine-treated cells (70% versus 30%; Kim, unpublished data). Therefore, the addition of TPO was expected to play a critical role in establishing an improved gene transduction procedure.
In this study, we observed that our three different cell culture conditions resulted in a similar expansion of the absolute number of cells, and hence, of transduced cells. However, when the transduction efficiencies were compared, we unexpectedly found a significant difference between the culture conditions only for CFU-GM, but not by PCR evaluation, despite a significant increase in the percentage of cycling cells produced by the addition of TPO. The possibility that our culture condition was inadequate to fully support the proliferation of immature progenitor cells needs to be clarified. Nevertheless, one possible explanation for our results is that the increased rate of cell proliferation produced by the addition of TPO preferentially induced the differentiation of stem cells into mature progenitors; at this stage of cell differentiation, any meaningful increase in retroviral-mediated gene transduction would not be seen. Similar data have been reported by van Beusechem et al.; i.e., retroviral-vector mediated transduction of long-term repopulating CD34+ cells in IL-3-supplemented cultures is as efficient as that supplemented with SCF or with combinations of SCF + IL-3 or IL-6 + SCF [15]. Although other combinations including Flt3/Flk-2 ligand still need to be evaluated [16], these findings imply that factors other than the cell cycle, such as retrovirus receptor transduction, also significantly influence gene transduction of human hemopoietic stem cells, as suggested by Knaan-Shanzer et al. [24].
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Acknowledgments
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This work was supported by grants from the Second-term Comprehensive 10-Year Strategy for Cancer Control, the National Center of Neurology and Psychiatry (NCNP) of the Ministry of Health and Welfare (5A-6), the Ministry of Science, Education, and Culture of Japan (07457180), and the Chonnam University Research Institute of Medical Sciences.
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accepted for publication June 9, 1997.
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Abstract
Introduction
