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The Impact of Progenitor Enrichment, Serum, and Cytokines on the Ex Vivo Expansion of Mobilized Peripheral Blood Stem Cells: A Controlled Trial

^a Department of Oncology and Hemato-Oncology, PF Calvi Hospital, Noale (VE), Italy;
^b Cytofluorimetric Unit, PF Calvi Hospital, Noale (VE), Italy;
^c Department of Biomedical Sciences, University of Padova, Padova, Italy;
^d Blood Transfusion Service, Civic Hospital, Mirano (VE), Italy

Key Words. Stem cell • Expansion • CD34 selection • Serum-free media • Cytokine

Giuseppe Azzarello, M.D., Dept. of Oncology and Hemato-Oncology, PF Calvi Hospital, Largo S. Giorgio 3, 30033 Noale (VE), Italy. Telephone: 041 5896221; Fax: 041 5896259; e-mail:
oncnoale{at}inwind.it

	ABSTRACT

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The aim of this study was to verify, and possibly improve, culture conditions to expand human mobilized peripheral blood stem cells (PBSCs). We investigated the role of three parameters: A) the culture medium (serum-free versus serum-dependent); B) the initial cell population (Ficoll-separated mononucleated cells versus CD34⁺-selected cells), and C) the low concentration of recombinant cytokines, flt3 ligand, and thrombopoietin in association with a basic cocktail of stem cell factor, interleukin (IL)-6, IL-3, GM-CSF, and erythropoietin. Eighteen leukapheresis samples were monitored in static culture for 15 days. The expansion potential was assessed at day 10 and 15 by total nuclear cells, colony-forming-units (CFUs) (burst-forming units-erythroid [BFU-E], colony-forming units-granulocyte-macrophage [CFU-GM], and colony-forming units-granulocyte-erythroid-macrophage-megakaryocyte [CFU-GEMM]), and flow cytometry immunophenotyping (CD34⁺/CD38^-, CD38⁺, CD33⁺, CD41⁺, GlyA⁺ progenitor cells). The results, evaluated by multivariate analysis of variance, emphasize that some variables affected the outcome of stem and progenitor cell expansion. CD34⁺ enrichment increased expansion of total nuclear cells, number of CD38⁺ and CD33⁺ late precursors, and number of the CFU-GM compartment. Interestingly, however, quantitative expansion of GlyA⁺ and the early progenitor cells (CD34⁺/CD38^-, CFU-GEMM, BFU-E) are favored by the use of unselected mononucleated cells. Regarding the role of serum, no significant difference was observed except for expansion of total nuclear cells, CFU-GM, and BFU-E. Cytokine combinations, in particular the use of flt3 ligand, stimulated expansion of almost all the cellular subsets, reaching a statistical significance for total nuclear cells and CFU-GM. Our study indicates that progenitor and late precursor multilineage cell compartments of mobilized PBSCs may be significantly expanded in short-term cultures by well-defined experimental conditions. Furthermore, these data might be useful when evaluating ex vivo expansion of hematopoietic cells for clinical purposes.

	INTRODUCTION

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Hematopoiesis is maintained by the activity of pluripotent stem cells, which have the dual capacity to self-renew and to differentiate into all of the blood cell lineages. One of the major challenges in stem cell research is to stimulate self-renewal divisions of hematopoietic stem cells (HSCs) ex vivo to expand the primitive compartment and the progenitor and precursor cells and to increase transplantable HSC numbers.

Peripheral blood stem cells (PBSCs) mobilized by chemotherapy and cytokines are widely used to provide hematologic rescue following myeloablative therapy. Nevertheless, we still face some problems such as the period of absolute or severe neutropenia and thrombocytopenia (9 and 14 days, respectively) [1–3], the side effects of collection procedures, and the contamination of the grafts with malignant cells [4–6]. As a consequence, over the past years, several attempts have been made to increase the ex vivo expansion of HSCs, with the aim of shortening the time to engraftment, for tumor purging, and for new graft engineering strategies [7–9].

Regarding the use of cytokines, it has been well established that optimal ex vivo expansion requires different cocktails, including early-acting (stem cell factor [SCF], interleukin [IL]-6) and proliferative (IL-3) cytokines [10]. However, the specific combinations of growth factors that are able to balance the self-renewal divisions with minimal apoptosis and that can promote the differentiation program of different HSC subsets, remains to be elucidated [11].

Recently, it was found that a combination of thrombopoietin, flt3 ligand, and SCF in absence of stroma preserves the purity of the CD34⁺ cell population from mobilized peripheral blood [12]. In particular, association of thrombopoietin and flt3 ligand might be sufficient to stimulate long-term culture-initiating cells from cord blood CD34⁺ cells. However, additional cytokines, such as GM-CSF, SCF, IL-3, and IL-6, are required for optimal expansion of adult PBSCs [13,14].

Finally, the use of serum free-media, which provides biochemically defined culture conditions [15], and the selection of highly purified CD34⁺ cells [16], are currently being tested for improving ex vivo expansion and for testing cytokine effects in a clear-cut manner.

In our study, aiming at looking for an efficient culture system for clinical application, we investigated the role of some experimental parameters on the ex vivo expansion and maturation of PBSCs in short-term cultures. In particular, we considered three different parameters: A) the use of serum-free versus serum-dependent medium; B) different initial cell populations (Ficoll-separated monucleated cells versus positively selected CD34⁺ cells), and C) different combinations of cytokines at low concentrations (thrombopoietin and flt3 ligand) in association with a basic cocktail of SCF, IL-3, IL-6, GM-CSF, and erythropoietin (EPO).

	MATERIALS AND METHODS

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Cell Preparation
PBSCs were obtained from 18 patients with cancer in complete or partial remission mobilized with chemotherapy plus filgrastim (5 µg/kg/day) and planned for high-dose chemotherapy and autologous PBSC rescue (multiple myeloma, 6; non-Hodgkin’s lymphoma, 3; breast cancer, 7; ovarian cancer, 1; small-cell lung cancer, 1). Aliquots of peripheral blood mononuclear cells were obtained from leukapheresis samples using an AS-104 blood cell separator (Fresenius AG; St. Wendel, Germany; http://www.fresenius-ag.com).

All valuable samples were randomly assigned for testing the medium (serum-free:serum-dependent medium = 2:1), cell population type (mononucleated cells:CD34⁺-selected cells = 1:1), and cytokine combinations (basic cocktail [BC], BC/flt3 ligand, BC/thrombopoietin, BC/thrombopoietin/flt3 ligand = 1:1:1:1).

Mononucleated cells were separated over a Ficoll density gradient (Neyergaard; Oslo, Norway), and the interface was collected, washed twice, and counted. The CD34⁺ fraction was isolated using colloidal superparamagnetic microbeads in a strong magnetic field and run over a Minimacs separation column (Miltenyi Biotech; Gladback, Germany; http://www.miltenyibiotec.com). The purity and recovery rate of the CD34⁺ cells were assessed with anti-CD34 (fluorescein isothiocyanate [FITC]; HLDA-6 IL-SPA) antibody.

Cytokines
Recombinant human IL-3, IL-6, GM-CSF, SCF, flt3 ligand, and thrombopoietin were provided by BIOSPA (StemCell Technologies, Inc.; Vancouver, Canada; http://www.stemcell.com); recombinant human EPO was purchased from Boehringer (Mannheim, Germany; http://www.boehringer-ingelheim.com/corporate/home/home.asp). The growth factors were used at the following concentrations: 10 ng/ml (IL-3, IL-6, GM-CSF, SCF, and flt3 ligand), 5 ng/ml (thrombopoietin), and 2 U/ml EPO.

Expansion in Short-Term Cell Culture
Mononucleated cells and the CD34⁺ population were grown in a static, short-term, stroma-free culture system. Cells were plated at 3 x 10⁵ cells/dish, seeded in triplicate in 1.5 ml of assigned medium, and maintained at 37°C in 5% CO₂ for 15 days. Media were: serum-dependent medium (Iscove’s modified Dulbecco’s medium [Seromed; Berlin, Germany; http://biochrom.de], 10% fetal calf serum [Poyesis; Trieste, Italy], 2 mM L-glutamine and 1% penicillin/streptomycin solution [GIBCO/BRL; S. Giuliano Milanese, Italy; http://www.invitrogen.com]) and serum-free medium (Stem Pro-34^TM plus StemPro-34^TM nutrient [Life Technologies; S. Giuliano Milanese, Italy; http://www.lifetech.com], 2 mM L-glutamine, and 1% penicillin/streptomycin solution [GIBCO/ BRL]). Four cytokine combinations were employed: a basic cocktail (IL-3, IL-6, SCF, GM-CSF, EPO) and a basic cocktail supplemented either with thrombopoietin or flt3 ligand, or both. Medium containing the different cytokines was replaced every 5 days. Aliquots of cultured cells were harvested on days 10 and 15 and analyzed for total nuclear cell number and viability (trypan-blue exclusion test), progenitor assay number, and flow cytometry immunophenotyping. Fold expansion on days 10 and 15 are evaluated from the number of viable cells divided by the cell number on day 0. The absolute number of cells positive for cluster differentiation markers was calculated as the percentage of positive cells.

Progenitor Cell Assay
The colony-forming unit (CFU) assay was performed in a single-layer methylcellulose culture according to Eaves and Eaves [17]. Enriched CD34⁺ cells and mononucleated cells were stimulated with 20 ng/ml GM-CSF, 20 ng/ml G-CSF, 10 ng/ml IL-3, 3 U/ml EPO, and 50 ng/ml SCF (BIOSPA; StemCell Technologies, Inc.) and seeded in duplicate to a final concentration of 2 x 10³ cells/ml and 2 x 10⁵ cells/ml, respectively. The numbers of BFU-E, CFU-granulocyte-macrophage (CFU-GM), and CFU-granulocyte-erythroid-macrophage-megakaryocyte (CFU-GEMM) were scored at day 14 of culture using an inverted microscope (Nikon; Sesto Fiorentino, Italy).

Flow Cytometry Immunophenotyping
Initial cells and subsequent cultures were labeled with the following monoclonal antibodies purchased from IL-SPA (Milan, Italy): FITC-CD34⁺ (clone581), FITC-glycophorin-A (clone 11E4B-7-6 KC16), FITC-CD38 (clone T-16), FITC-CD33, FITC-CD41 (clone P2), and FITC-CD45. Isotype-matched, FITC-conjugated antibodies were used as controls. Cells were counted with an EPICS-2 flow cytometer and 10,000 events per sample were analyzed using the EPICS-2 software (Coulter; Milan, Italy; http://www.coulter.com).

Statistical Analysis
Statistical analysis was performed using a multivariate analysis of variance with repeated measures and grouping factors. The Greenhouse-Geisser and Huynh-Feldt adjustments were adopted as a protection in case of violation of homogeneity of covariance [18].

	RESULTS

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Ficoll-separated mononucleated cells or CD34⁺-selected cells from PBSCs were maintained in stroma-free, short-term culture in three replicates using either serum-free or serum-dependent medium and various combinations of cytokines. Figures 1 and 2 summarize our data on the time course of expansion as mean fold increase at 10 and 15 days of culture. Histograms in Figure 1 refer to total nuclear cells and precursor cells, and histograms in Figure 2 refer to the progenitor cell compartment. One hundred forty-four and 72 experiments were performed with serum-free and serum-containing medium, respectively; 108 experiments had mononucleated cells and 108 experiments had CD34⁺-selected cells; 108 experiments were performed with flt3-based cytokine combinations, and 108 experiments were performed with cytokine combinations without flt3 ligand. As reported in Table 1, all experimental conditions induced an expansion of the hematopoietic compartments. Flow cytometry analysis showed that CD34⁺/38^- cells were maintained only to a limited degree, even if satisfactory results were recorded for the progenitor cell compartment, such as CFU-GEMM and CFU-GM. Otherwise, cell populations committed to myeloid (CD33⁺ and CD38⁺), megakaryocytic (CD41⁺), and erythroid (GlyA⁺) lineages predominated. A multivariate analysis was performed to define the statistical significance of these results.

View larger version (41K): [in this window] [in a new window]   Figure 1. Time course expansion data (mean fold increase at 10 and 15 days of culture) for total nuclear cells and precursor cells. A = total nuclear cells; B = CD34+/CD38+; C = CD33+; D = CD41+; E = GlyA+.

View larger version (34K): [in this window] [in a new window]   Figure 2. Time course expansion data (mean fold increase at 10 and 15 days of culture) for progenitor cells. A = CD34+/38-; B = CFU-GM; C = BFU-E; D = CFU-GEMM.

Factors Affecting Late Precursor Cell Compartment Expansion
Three factors were statistically significant for the expansion of the late precursor cell compartment: A) CD34⁺ enrichment, B) serum-dependent medium, and C) addition of flt3 ligand to the cytokine combinations (Table 2). CD34⁺ enrichment increased expansion of total nuclear cells (p < 0. 000) and of the CD38⁺ (p = 0.001) and CD33⁺ (p = 0.008) subsets. Only the GlyA⁺ cells expanded much better in mononucleated cell culture (p 0.000). Ex vivo expansion of total nuclear cells worked better with serum-dependent instead of serum-free medium (p = 0.054); no significant fold increase was observed for other phenotypes of the late precursor cell compartment. Fold-increase values showed that only the flt3-based cytokine combinations had a positive effect, reaching a borderline statistical significance for the ex vivo expansion of total nuclear cells (p = 0.055).

	DISCUSSION

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The ex vivo expansion of hematopoietic stem cells is a very promising approach for different clinical applications, ranging from rescue after myeloablative therapy to purging of contaminating tumor cells or other graft engineering techniques [19–24]. The major goals of this study were to define the optimal culture conditions for the ex vivo expansion of mobilized PBSCs in a short-term, stroma-free, static culture and to provide new insights into the procedures that should be used in a clinical setting. Although our results are in line with previous reports [8, 25, 26], they provide new data on the conditions that promote ex vivo expansion of hematopoietic stem cells and clarify previous reports, thanks to the application of a multiparametric analysis of variance.

First, we found that CD34⁺ enrichment is a prerequisite for efficient ex vivo expansion, but with some important exceptions. In particular, the overall results for the early progenitor subsets, such as CD34⁺/CD38^-, CFU-GEMM, and BFU-E favor the use of nonselected mononucleated cells. The same results were observed in only one subset of the late precursor compartment, namely the GlyA⁺ cells. It has been reported that accessory cells might play a role in the expansion of bone marrow/cord blood-derived stem cells [27–28], and this might also be important for the expansion of early PBSCs. Moreover, the existence of CD34^- long-term repopulating cells has to be evaluated when CD34⁺ enrichment is planned for expansion procedures [29]. Undoubtedly, cell selection is important for large clinical scale expansion and for potential purging of minimal contaminating disease. Second, regarding the performance of serum-free in comparison with serum-dependent medium, we did not observe any significant differences in almost all cell populations. However, by multivariate analysis, we detected a better performance in serum-dependent medium, particularly for total nuclear cells and CFU-GM. Our data are in contrast with previous studies reporting better expansion by serum-free media in different experimental models [11, 26, 30, 31). However, they are in agreement with recent data by Shadduck et al. [32], which showed that human cord blood hematopoietic stem cells expand better when initially cultivated in serum-dependent medium for 1-2 weeks and then shifted to a serum-free medium. Our data stress that serum contains unknown growth factors that are required for ex vivo expansion, at least for some cellular subsets, but confirm that serum is dispensable for an efficient expansion of almost all progenitor and precursor cells. In addition, the use of serum-free medium is recommended for clinical applications because the absence of bovine-derived components potentially prevents allergens or prion-type contamination. Furthermore, fetal calf serum may also contain inhibitors or stimulating factors of hematopoiesis, which might affect stem cell populations [30].

Finally, the use of flt3 ligand, but not of thrombopoietin, in combination with a cocktail of cytokines, gave the best expansion values of all the cellular subsets, with the exception of CD34⁺/CD38^-. This trend reached statistical significance for total nuclear cells and the CFU-GM population. Optimal consistence of flt3 combined with different growth factor cocktails on peripheral blood or cord blood stem cells was evaluated in detail in previous works [12, 31]. In spite of some negative interactions between factors that can modify the overall expansion, our results favor, whenever possible, the use of a flt3-based cytokine combination to expand simultaneously the late precursor and progenitor cell compartments. The expansion rates reported in our experiments are of particular interest for large-scale clinical application, since they were obtained with low doses of cytokines. Flt3 ligand enhances human progenitor cell growth [10, 33]; moreover, in combination with thrombopoietin and SCF, flt3 ligand suppresses apoptosis of human mobilized CD34⁺ cells and recruits primitive CD34⁺Thy-1⁺ cells into rapid division [12]. Our results with early progenitor CD34⁺/CD38^- cells confirm previous data on other ex vivo expansion procedures with peripheral blood. In fact, hematopoietic PBSCs showed limited potential to expand in vitro as compared with cord blood [31]. This may be due to differences among stem cells from various sources or to the lack of particular ancillary cells [34, 35]. It is noteworthy, in this respect, that conditioned medium from cord blood cultures transiently stimulates the proliferation of CD34⁺ cells from peripheral blood [31]. Recently, another human stem cell antigen (AC133) has been identified, which might improve the purity and the potential expansion of the stem cell population [36].

In conclusion, the results of our study clearly show that the ex vivo expansion of progenitor and precursor cells from mobilized peripheral blood is dependent upon controlled experimental conditions, which might be useful when evaluating ex vivo expansion of hematopoietic cells for clinical purposes. Defining the best-controlled conditions of culture might allow optimization of expansion [37] and, possibly, engineering of some cellular functions.

	ACKNOWLEDGMENT

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This work was partially funded by a grant from the Veneto Region and from the Cassa di Risparmio di Venezia (CARIVE). We are grateful to Abigail Johnson for editorial assistance.

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