Stem Cells, Vol. 14, No. 4, 430-438,
July 1996
© 1996 AlphaMed Press
Functional and Morphological Characterization of Immunomagnetically Selected CD34+ Hematopoietic Progenitor Cells
Federica Servidaa,
Davide Soligob,
Lorenza Canevaa,
Francesco Bertolinid,
Etienne de Harvena,
Simona Campiglioa,
Chiara Corsinic,
Giorgio Lambertenghi Deliliersb
a Fondazione Matarelli,
b Centro Trapianti di Midollo and
c Centro Trasfusionale e di Immunologia dei Trapianti, Ospedale Maggiore, IRCCS, Milan, Italy;
d Fondazione Maugeri, Pavia, Italy
Key Words. CD34 antigen • Cell separation • Ex vivo expansion • Growth factors • Hematopoietic stem cells • Long-term culture-initiating cells • Ultrastructure
Dr. Davide Soligo, Centro Trapianti di Midollo, Ospedale Maggiore, IRCCS, Via F. Sforza, 35 - 20122 Milan, Italy.
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Abstract
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We evaluated the potential of immunomagnetically selected (miniMACS) progenitor cells to give rise to colony-forming cells and their precursors, detected as long-term culture-initiating cells (LTC-IC), as well as their capacity to expand in liquid cultures. A 90% mean purity, a 43.2% yield and a 55.8-fold enrichment were achieved from normal bone marrow. When corrected for enrichment, the mean number of committed progenitor cells and the frequency of LTC-IC (evaluated by means of limiting dilution assay [LDA]) were not statistically different in low density mononuclear cells or in the CD34-enriched fractions. In five cases CD34+ selected cells grown in a stroma-free long-term bone marrow culture system with the addition of stem cell factor, interleukin 3, interleukin 6 and GM-CSF every 48 h, showed a 15 (±15) and 31 (±21) mean colony forming unit-granulocyte/macrophage fold increase on cultures at days 7 and 14. However, when corrected for enrichment, the expansion capability of these cells was significantly lower than that of the unseparated fraction, particularly after the first week. Immediately after separation, electron microscopy revealed that the CD34+ selected fraction contained more than 45% of well-differentiated myeloid cells (MPO+ ), with iron beads preferentially clustered at one pole of the cell surface and sometimes already endocytosed in pinocytic vesicles. After 24 h and 48 h incubation at 37°C, the majority of the cells showed no iron particles, but about 30% of the cells were iron-labeled phagocytic cells. The percentage of apoptotic cells with internalized iron was negligible. These data show that immunomagnetically separated CD34+ cells may have a slightly impaired short-term expansion capability, but give rise to both committed and more primitive progenitor cells. During the separation, the iron beads are internalized, rapidly processed in the cytoplasm and do not seem to interfere with in vitro growth.
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Introduction
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A small population of immature he-matopoietic cells can be characterized in the bone marrow by means of their expression of the CD34 antigen surface marker [1]. CD34 expression has been found in stem cells, precursor cells and progenitor cells capable of proliferating in short- and long-term cultures [1–3]. The CD34+ cells are very infrequently encountered in bone marrow (frequency of 1%-4% in normal bone marrow), and suitable methods for their isolation and enrichment are therefore warranted.
Fluorescence activated cell sorting (FACS), the classical method for collecting CD34+ cells, is extremely time-consuming and involves the sacrifice of too many cells in the final enriched fraction.
Berenson et al. [4 ] have developed a method for CD34+ cell separation, based on the affinity between biotin and avidin, in which low density mononuclear cells (LD-MNC) stained with a biotinylated antibody directed against CD34 are passed through an avidin column, retained within the column and then released by agitation. An automated version has been developed for clinical use (CeprateTM SC System, CellPro, Inc.; Bothell, WA). However, especially in the case of low frequency cells, after enrichment in avidin-biotin columns one cannot avoid the presence of high levels of contaminating cells.
A panning method for CD34 purification (CELLector® System) [5] has also been developed in which red blood cells, B and T lymphocytes, monocytes and stromal cells are removed by attachment to a soybean agglutinin (SBA) coated flask. Nonadherent cells are then passed into a flask covalently coated with the CD34 antibody, and the adherent CD34+ cells are removed by agitation. Such panning methods are relatively inexpensive but time-consuming; they provide a level of high purity, but the recovery rate (or yield) is generally poor [6].
With a recently introduced technique one can obtain CD34+ immature hematopoietic cells rapidly and efficiently with a high degree of purity and an excellent recovery rate for further in vitro cell culture studies [7,8]. This separation method is based on the immunolabeling of target cells by means of very small paramagnetic particles and the isolation of labeled cells in a high gradient magnetic field.
The purpose of the present study was to investigate the ultrastructural morphology and the in vitro proliferation capacity of immunomagnetically separated normal CD34+ cells in short- and long-term cultures.
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Materials and Methods
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Sample Preparation
Human bone marrow aspirates from 17 healthy adults undergoing bone marrow harvest, G-CSF mobilized peripheral blood stem cells (PBSC) from six normal related donors and umbilical cord blood (CB) samples from six full-term newborns were layered on a Ficoll-Paque gradient (specific gravity 1.077 g/ml; Nycomed Pharma AS; Oslo, Norway). The LD-MNC were washed twice in Hanks' balanced salt solution (HBSS) and resuspended prior to CD34+ cell separation in phosphate buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) and 5 mM EDTA.
CD34+Cell Separation
The LD-MNC were incubated for 15 min at 4°C with a chemically modified QBEND10 monoclonal antibody directed against the CD34 antigen (hapten conjugated) [8]. The cells were then washed and incubated for 15 min at 4°C with immunomagnetic beads (Miltenyi Biotec; Bergisch Gladbach, Germany) directed against the modified QBEND10. For flow cytometry, 10 µl of CD34-phycoerythrin (CD34-PE) conjugated antibody (HPCA-2; Becton Dickinson; Mountain View, CA) were added to the cells for 5 min at 4°C. The sample was then filtered through a 50 µm nylon mesh in order to remove clumps and placed on a column in the miniMACS cell separator (Miltenyi Biotec). The labeled cells, separated by a high gradient magnetic field, were eluted from the column after removal from the magnet. The positive fraction was then placed on a new column and the magnetic separation step repeated. At the end of the separation, the cells were counted, assessed for viability by means of trypan blue dye exclusion and their purity determined by means of flow cytometry analysis. All of these steps were performed under aseptic conditions.
Flow Cytometry Analysis
The LD-MNC and the CD34+ cells stained with CD34-PE conjugated antibody HPCA-2 prior to the separation were analyzed by means of flow cytometry (FACS Vantage; Becton Dickinson).
Transmission Electron Microscopy(TEM) Preparations
About 50,000 CD34+ cells were fixed either immediately after separation (three experiments), or after a 24 h and 48 h incubation in Iscove's modified Dulbecco's medium (IMDM) and 10% fetal bovine serum (FBS) at 37°C. Fixation of washed cells took place overnight using 2% glutaraldehyde in PBS at 4°C. The cells were rinsed in PBS, postfixed for 1 h in 1% OsO4 in PBS at 4°C, and then washed in distilled water. In one experiment, the cells were also incubated immediately after fixation with a mixture of 5 mg 3,3'-diaminobenzidinetetrahydrochloride dihydrate (DAB), 10 ml TRIS/HCL buffer 0.05 M, 0.1 ml H2O2 1% (pH 7.6), washed first in TRIS/HCL buffer and then in PBS [9]. Dehydration was carried out using a graded ethanol series from 70% up to 100% and propylene oxide. After embedding in Araldite, ultrathin sections (silver or very pale gold) were obtained using a Reichert Jung ultramicrotome, counterstained with uranyl acetate and lead citrate and observed with a Philips CM10 transmission electron microscope at 80 kV. Two hundred cells were counted in at least five different sections.
Clonogenic Assays
The colony forming unit-granulo-cyte/macrophage (CFU-GM) and the BFU-E assays were carried out by plating 1 x 105 LD-MNC and 5 x 103 CD34+ cells from 13 bone marrow samples in 35 mm Petri dishes in 1 ml of methylcellulose culture medium (MethoCultTM H4230; Stem Cell Technologies; Vancouver, Canada) containing 0.9% methylcellulose, 30% pretested FBS, 1% pretested BSA, 10–4 M 2-mercaptoethanol, additioned with 15% supernatant of the 5637 cell line and 3 U/ml human urinary erythropoietin. Triplicate dishes were incubated at 37°C with 5% CO2, in a fully humidified atmosphere for 14 days. The aggregates of 
40 and of <40 cells were respectively scored as colonies and clusters, and counted after 14 days of culture.
Colony forming unit culture (CFU-C) data were presented both in absolute values and after the correction for the enrichment (Tables 2-3
). This correction allowed the comparison between data obtained from LD-MNC and CD34+ cells after the separation and a check for a possible loss of progenitor cells during the separation.
Stroma-Free Liquid Cultures
Stroma-free liquid cultures were established as previously described [10]. In particular, CD34+ bone marrow cells from five normal donors were seeded at 1 x 105 cells/ml in 25 cm2 tissue culture flasks in IMDM supplemented with 30% FBS and then incubated at 37°C in a fully humidified atmosphere with 5% CO2 in air for two weeks. On day 0, and after every 48 h, the cultures were fed with a combination of cytokines, including 50 ng/ml of recombinant human stem cell factor (rHuSCF) (Amgen; Thousand Oaks, CA), 100 ng/ml of recombinant human GM-CSF (rHuGM-CSF) (Sandoz; Basel, Switzerland), 100 ng/ml of recombinant human interleukin 3 (rHuIL-3) (Sandoz) and 10 ng/ml of rHuIL-6 (Pharmacia; Milan, Italy). At weekly intervals, the cultures were demi-populated by removing half of the culture volume, which was replaced with fresh medium and cytokines. The cells collected from the refeed were counted, assessed for viability by means of trypan blue dye exclusion and tested for progenitor cells by clonogenic assays. The CFU-GM fold increase was calculated by dividing the absolute number of CFU-GM (CFU-GM incidence multiplied by the increase in the number of living nucleated cells), at each time point during the culture, by their absolute number at time 0.
Long Term Culture Initiating Cells Limiting Dilution Assay (LTC-IC LDA)
In order to determine the frequencies of LTC-IC [11], a limiting dilution assay (LDA) [12] was performed on LD-MNC and CD34+ cells from five bone marrow samples. The M2-10B4 murine cell line was used as a feeder layer for the LTC-IC by seeding 9 x 103 M2-10B4 irradiated (80 Gy) cells in 96-well microtiter plates. The dilution steps (16 replicates for each dilution) were 60, 120, 180, 240 and 300 cells/well for the CD34+ cells, and 3,000, 6,000, 9,000, 12,000 and 15,000 cells/well for the LD-MNC in 200 µl/well of long-term bone marrow culture (LTBMC) medium. After five weeks of culture at 37°C with 5% CO2, the adherent and nonadherent cells were harvested from each well by removing the medium and adding 50 µl of trypsin per well for 10 min at 37°C. The trypsinized cells were washed from the wells with 50 µl of medium and pooled with the nonadherent cells. In order to evaluate colony formation, the cells from each well were plated in 0.25 ml of semisolid medium for colony assay (MethoCultTM H4230; Stem Cell Technologies) containing 0.9% methylcellulose, 30% pretested FBS, 1% pretested BSA, 10–4 M 2-mercaptoethanol, additioned with 15% supernatant of the 5637 cell line and 3 U/ml human urinary erythropoietin, in 24-well plates and incubated at 37°C with 5% CO2, in a fully humidified atmosphere for 14 days. The incidence of negative wells was then determined and the frequency of LTC-IC evaluated by means of Poisson's statistics [13].
Statistics
The Student's t-test was used to evaluate CFU-GM and BFU-E differences between LD-MNC and CD34+ cells. Differences in CFU-C expansion in liquid cultures and LTC-IC frequencies were evaluated by means of the Mann Whitney U-test. Values of p < 0.05 were considered as statistically significant.
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Results
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Immunomagnetic separation of CD34+ cells was performed with the miniMACS system on 17 human bone marrow samples (Table 1). The mean number of bone marrow mononuclear cells after Ficoll-Paque gradient was 90 x 106 (±21). The mean percentage of CD34+ cells in the LD-MNC fraction was 2.1% (±1.3) positive cells. The mean number of CD34+ cells after separation was 0.8 x 106 (±0.3). The mean purity of the separated cells, evaluated by means of flow cytometry analysis using a CD34-PE conjugated antibody, was 89.9% (±5.1). The yield of the separations, calculated as the ratio between the number of "positive" cells in the final fraction multiplied by the percentage purity of the CD34+ cells and the initial number of CD34+ cells, showed an average of 43.2% (±26.4). The mean enrichment, defined as the ratio between the percentage of "positive" cells in the isolated fraction and the percentage of positive cells in the unseparated fraction, was 55.8-fold (±25.5). CD34+ separation was also evaluated in six cases of mobilized PBSC and in six cases of CB: the detailed results are given in Table 1. In particular, mean purity, CD34+ cell yields and enrichment were 83.6 (±5.9), 41.6 (±5.8) and 59.3 (±35.7) for PBSC, and 83.5 (±7.7), 46.1 (±7.2) and 70.6 (±22.8) for CB.
Thirteen of the 17 bone marrow cell samples were tested in clonogenic assays: the mean CFU-GM number (calculated on 5 x 103 seeded cells) was 4 (±2.8) for LD-MNC and 129 (±50.1) for CD34+ cells, while the mean BFU-E number was 5.3 (±6.4) for LD-MNC and 156.8 (±135.4) for CD34+ cells (Table 2A
).
The CD34+ cells separated from five bone marrow samples were also cultivated in stroma-free liquid cultures supplemented with the above-mentioned cytokines every 48 h. As Table 3 shows, the mean CFU-GM fold increase on days 7 and 14 was respectively 15 (±15) and 31 (±21) in CD34+ separated cells. Under the same culture conditions, the LD-MNC had a mean fold increase of 4 (±2.6) and 3 (±3) on the same days. However, when the fold increase values were corrected for enrichment, the CFU-GM fold expansions of CD34+ separated cells were significantly lower (p < 0.05) than those of LD-MNC (Table 3) at day 7 but not at day 14.
In order to quantify LTC-IC in the CD34+ separated bone marrow cell population and in the LD-MNC, cells were seeded in limiting dilutions at five different cell concentrations (16 replicates for each concentration) into 96-microwell plates coated with the pre-irradiated murine stromal cell line M2-10B4. The frequency of LTC-IC was 4700 (±190) per 1 x 106 CD34+ cells and 75.2 (±33.5) per 1 x 106 LD-MNC. The frequencies of CD34+ cells and LD-MNC remained very similar even after correction for enrichment (Table 2B).
The ultrastructural morphological findings on CD34+ cells are summarized in Table 4. Immediately after separation, 53% of the cells showed iron-particle aggregates of different sizes, generally clustered at one pole of the cell surface (Fig. 1); in approximately half of the labeled cells, iron particles were also visible in small pinocytic vesicles immediately underneath the cytoplasmic membrane (Fig. 2). The majority of the cells, labeled or unlabeled, had the features of immature cells, i.e., a large nucleus with dispersed chromatin and scant cytoplasm (Fig. 1). To determine further the frequency of well-differentiated myeloid cells inside the CD34+ fraction, sections were treated for the demonstration of myeloperoxidase (MPO) activity: 45% of the cells had numerous MPO+ granules, and 12% of the cells had fewer than 3 MPO+ granules or MPO staining of the ergastoplasmic reticulum, which is consistent with very early myeloid commitment. On the other hand, 38% of the cells were completely MPO– (i.e., with the morphological characteristics of immature cells) (Table 4). The majority of separated CD34+ cells (around 70%) incubated for 24 h and 48 h in IMDM supplemented with 10% FBS at 37°C showed no iron particles either on the outer membrane or in the cytoplasm, whereas around 30% of the cells (generally with the morphological features of monocytic-macrophagic cells) were heavily loaded with iron-containing large phagocytic vacuoles (Fig. 3). After 24 h, 8% of the cells showed clear morphological features of apoptosis and only 1% of these cells contained ingested iron particles (Fig. 4).
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Figure 1. Transmission electron microscopy of an immunomagnetically separated CD34+cell showing an immature phenotype and a polar distribution of the labeling with iron particles (21,700x).
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Figure 2. Ultrastructural morphology of a CD34+cell immediately after the separation. Clusters of iron particles are visible on the cell surface (arrowheads), in an early endocytic vacuole (small arrow) and two pinocytic vesicles (big arrows) (29,400x).
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Figure 3. Immunomagnetically separated cells after 24 h in vitro incubation. A monocytic cell shows a large number of iron-laden phagosomes (12,500x).
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Figure 4. Immunomagnetically separated cells after 24 h in vitro incubation. An apoptotic cell shows a single phagocytic vesicle (arrow) containing iron particles (12,500x).
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Discussion
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A number of methods for the purification of CD34+ cells has been developed. FACS technology allowed the isolation of a potentially pure population of CD34+ cells, but was prohibitively time-consuming and expensive [6]. Berenson et al. [4] developed a method for isolating CD34+ cells that takes advantage of the affinity between biotin and avidin. This system has become the standard reference for small-scale purifications and has also been upgraded for clinical purifications [14,15].
The large number of experiments of the present study demonstrates that the immunomagnetic miniMACS system, initially developed by Miltenyi et al. [8], is an extremely efficient means for separating CD34+ cells. Comparing our data with those obtained using immuno-affinity methods [16], the miniMACS system, which involves a two-step immunomagnetic selection of Ficoll separated bone marrow cells, appears to produce comparable values of enrichment at higher yields (43.2% versus 32%). Furthermore, although in a limited number of experiments, even greater efficiency was obtained when CD34+ cells were immunomagnetically separated from PBSC and CB. Similar results were recently obtained by de Wynter et al. [17] who showed, comparing five different separation systems, higher purities and enrichments in CD34+ cells with the miniMACS system.
However, the principal aim of the present study was to investigate the behavior of human bone marrow CD34+ cells after immunomagnetic separation. Our findings seem to indicate that the cells start to internalize the iron beads, immediately after the separation, probably by means of a pinocytic process. No morphological alterations were observed after the separation, which suggests that the passage through a strong magnetic field does not damage the cells. The polarized labeling of CD34+ cells suggests that capping of the antigen takes place on the cell side facing the magnetic field, probably explaining the low percentage of cells with immunomagnetic particles in TEM ultrathin sections. Our ultrastructural and cytochemical data confirm a previous report [18] indicating the prevalence of differentiated myeloid cells in the CD34+ fraction, but also suggest that separated cells contain a percentage of immature cells. Furthermore, it seems that the iron particles are completely cleared by more primitive cells, already after 24 h of culture, whereas well-differentiated end-stage cells with phagocytic capacity are responsible for the clearance of the particles, at least in vitro. Of particular interest is the finding that the majority of apoptotic cells (generally observed at the onset of bone marrow cultures) did not contain any iron particles, thus indicating that the particles do not interfere with hemopoietic cell survival in vitro.
The in vitro data generated by our study suggest that immunomagnetically selected CD34+ cells retain unaltered growth and differentiation capabilities. The differences in clonogenic growth between LD-MNC and CD34+ cells in semisolid media were not statistically significant, thus indicating that the separation procedure was not associated with either the loss of precursor cells, or with any interference with the in vitro growth, as previously suggested by other authors [17,19]. Furthermore, the frequency of LTC-IC, which represents the more immature hematopoietic precursor cells [11,12], was not statistically different in both unseparated and separated bone marrow, thus demonstrating that primitive progenitor cells in the immunomagnetically separated fraction are both maintained and capable of proliferating in a long-term culture system.
Recent studies [20] have attempted to expand CD34+ cells in liquid cultures by replacing the normal hematopoietic microenvironment with an appropriate combination of growth factors. Using this system, a large number of cells and CFU-C has been generated after up to eight weeks of culture [21]. However, the number of both the CD34+ cells and of the LTC-IC rapidly declined during the culture, suggesting the preferential expansion of committed progenitor cells [21]. When grown in our cytokine-driven stroma-free liquid culture conditions [10], immunomagnetically separated cells were capable of expanding, but at a rate that was significantly less than that of the unseparated cells, as indicated by our expansion data after correction for enrichment. This result, which is in sharp contrast with that observed in the long-term cultures, may have multiple explanations. On the basis of our morphological findings, we can speculate that the iron-loaded monocytic-macrophagic accessory cells, present in the separated fraction and possibly releasing inhibitory factors, may be responsible for this effect in liquid cultures. On the other hand, in long-term bone marrow assays this effect may be diluted over the time of the culture (5 + 2 weeks) or could be somehow "neutralized" by the stromal cells of the feeder layer.
Our data demonstrate that immunomagnetic separation is a highly efficient method of isolating bone marrow CD34+ cells, which allows both the maintenance and the functional integrity of committed and early progenitor cells. A large number of clinical trials has already been conducted using immuno-affinity columns to enrich CD34+ cells for both autologous [15,22] and allogeneic bone marrow transplantation [23]. Our data indicate that immunomagnetic CD34+ separation using the MACS system may also be feasible on a large clinical scale and could eventually improve the results obtained with the other systems.
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Acknowledgments
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We are grateful to Mario Azzini (Future Image, Milan, Italy) for the photographic artwork.
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References
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Received December 6, 1995;
accepted for publication February 27, 1996.
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Abstract
Introduction
