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support myeloid progenitors in long-term cultures of CD34c cord blood and bone marrow ... The mac- rophage nature of PB-derived adherent cells was con-.
Ann Hematol (2000) 79 : 59–65

Q Springer-Verlag 2000

ORIGINAL ARTICLE J. Clausen 7 M. Stockschläder 7 N. Fehse H.T. Hassan 7 C. Gabl 7 A.R. Zander

Blood-derived macrophage layers in the presence of hydrocortisone support myeloid progenitors in long-term cultures of CD34 c cord blood and bone marrow cells

Received: February 16, 1999 / Accepted: June 21, 1999

Abstract Monocytes/macrophages secrete various cytokines that induce proliferation of colony-forming unit granulocyte-macrophage (CFU-GM) in short-term assays. To determine whether macrophages also support proliferation of more primitive progenitors, i.e., cells that give rise to colony forming cells in a 5-week longterm culture (LTC), we established plastic-adherent macrophage layers from human peripheral blood (PB) and filgrastim (G-CSF)-mobilized progenitor cell collections in the presence of hydrocortisone, and compared these layers with bone marrow (BM) stroma regarding their suitability to support proliferation and differentiation of CD34 c BM and cord blood (CB) cells in 5-week LTCs. CD34 c cells were seeded onto irradiated macrophage and BM stromal layers, as well as without any preformed layer. After 5 weeks, colony formation (CFU-GM, BFU-E/CFU-E) and cell expansion were determined. CD34 c cells from BM and CB yielded more CFU-GM and total nucleated cells at 5 weeks in the presence of both types of adherent layer compared with cultures without a layer (p~0.05). For CD34 c BM cells, macrophage layers were superior to BM stroma in enhancing CFU-GM and CFU-E/BFU-E J. Clausen 1 7 M. Stockschläder 2 7 N. Fehse 7 H.T. Hassan A.R. Zander (Y) Bone Marrow Transplantation Center, University Hospital Eppendorf, Hamburg, Germany C. Gabl Department of Pathology, University Hospital Innsbruck, A-6020 Innsbruck, Austria 1

Present Address: Department of Hematology and Oncology, University Hospital Innsbruck, A-6020 Innsbruck, Austria 2 Present Address: Department of Hematology and Oncology, Klinik Innere Medizin C, EMAU-Greifswald, D-17487 Greifswald, Germany Address for Correspondence: Prof. Dr. A.R. Zander, University Hospital Eppendorf, Knochenmarktransplantation, Martinistrasse 52, D-20251 Hamburg, Germany e-mail: zander6uke.uni-hamburg.de Tel.: 0049-40-4717-4850 Fax: 0049-40-4717-3795

output (p~0.05). In contrast, BM stroma was favorable compared with macrophages concerning nucleated cell expansion from CD34 c CB cells (pp0.027). The macrophage nature of PB-derived adherent cells was confirmed immunocytochemically by positive staining for CD68, Ki-M1p, CD31, CD54, inconstant staining for CD14, and negative staining for CD1a, CD3, CD15, CD34, and CD62E. Cytochemical reactions were positive for a-naphthyl acetate esterase and negative for peroxidase and periodic acid-Schiff, consistent with the immunophenotype. In conclusion, the results show that blood-derived macrophages support CFU-GM generation from CD34 c CB and BM progenitors for 5 weeks in vitro. Differential effects on proliferation and maturation of BM versus CB progenitors are discussed. Key words Bone marrow 7 Cord blood 7 Long-term culture 7 Macrophage 7 Peripheral blood 7 Stroma 7 CD34

Introduction In order to investigate the development of hematopoietic cells and to establish quality control for hematopoietic progenitor cell (HPC) preparations used for transplantation, various in vitro assays have been established. The long-term culture initiating cell (LTC-IC) assay, widely accepted as one of the most suitable in vitro assays for detection and measurement of immature progenitors, was originally performed using preestablished bone marrow (BM) stromal layers for 5week cultures with the hematopoietic cells [1, 2]. Human and murine fibroblast cell lines, purified endothelial cells, and even defined media containing multiple cytokines may substitute for the native BM stroma in hematopoietic long-term cultures [3–11]. However, despite the fact that macrophages are known to produce and secrete a variety of cytokines which influence hematopoiesis [12–15], their capacity to induce proliferation of HPCs has been shown only for the late stages of hematopoiesis, i.e., generation of mature blood cell col-

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onies [16, 17]. A recent study revealed that CD14 c-depleted stromal layers support proliferation of primitive HPC as effectively as unseparated stroma [18]. On the other hand, passaged marrow fibroblasts were shown to support granulopoiesis for less time than whole stromal layers [19], indicating the requirement of other cell types in addition to fibroblasts for a functionally intact stroma. To elucidate the role of macrophages in this respect, we have examined their ability to support proliferation of CD34 c BM and cord blood (CB) cells for 5 weeks by assessing end-point CFC numbers and total nucleated cell expansion. Peripheral blood (PB)-derived macrophage layers might be of interest for the investigation of regulative mechanisms in hematopoietic LTCs for two reasons: (a) The composition of PB macrophage layers is less heterogeneous than that of BM stromal layers, as the latter contain various cell types, including fibroblasts, endothelial cells, and adipocytes, in addition to macrophages. The less heterogeneous composition might further facilitate identification of cellular interactions in the LTC. (b) The comparison of macrophage layers with BM stroma regarding the interaction with HPC of different sources and determination of the underlying mechanisms will improve our understanding of the HPC-stroma interaction.

Materials and methods Isolation of CD34 c bone marrow cells BM cells were obtained from nine healthy adult BM donors with their informed consent. Freshly harvested BM cells were separated by density centrifugation on Ficoll (1077 g/l; Biochrom, Berlin, Germany). The light-density mononuclear cell (MNC) fraction was used for immunomagnetic selection of CD34 c cells, as described elsewhere [20]. Briefly, cells were labeled with antiCD34 conjugated microbeads and separated magnetically using the miniMacs system (Miltenyi Biotec, Bergisch Gladbach, Germany). Freshly selected CD34 c cells were then used as hematopoietic cells in the various LTCs. The CFU-GM and BFU-E/ CFU-E content of each starting cell population was determined in a semisolid CFU assay (see below) at the initiation of the LTC. Isolation of CD34 c umbilical cord blood cells Six separate samples of umbilical CB were kindly provided by the Department of Gynecology and Obstetrics at the University Hospital Hamburg. After delivery, clamping, and cutting of the umbilical cord, blood was aspirated into heparinized syringes. Samples were stored at 4 7C and were processed within 24 h and selected for CD34 c cells according to the BM samples. Establishment of adherent cell layers For preparation of macrophage layers, MNCs were isolated by Ficoll (1077 g/l) from peripheral blood of healthy adult volunteers and from G-CSF-mobilized PB progenitor cell (G-PBPC) collections obtained by leukapheresis from adult patients with solid tumors who were designated for high-dose chemotherapy and stem cell rescue and who received standard doses of G-CSF subcutaneously for progenitor cell mobilization. BM stromal layers were prepared from BM samples obtained from 11 healthy adult BM donors and four patients with solid tumors. Preliminary cultures of PB, G-PBPC, and BM MNCs were set up in 75-cm 2 tissue-culture plastic flasks (Falcon Primaria, Hei-

delberg, Germany) at 3!10 6/ml bone marrow long-term culture (BMLTC) medium consisting of a-MEM (Gibco BRL, Paisley, Scotland), with 12.5% fetal calf serum (Gibco BRL), 12.5% horse serum (Gibco BRL), 2 mmol/l l-glutamine (Gibco BRL), and 10 –6 M hydrocortisone (Sigma, Deisenhofen, Germany). Cultures were maintained at 37 7C and 5% CO2 in humidified air with twice-weekly changes of half the medium. PB/G-PBPC- and BMderived MNCs were cultured for a median of 23 days (range 10–32) and 24 days (14–38), respectively, to obtain semiconfluent or confluent layers. Next, all nonadherent cells were removed, and the adherent cells were detached with trypsin-EDTA solution (Biochrom), washed, resuspended in BMLTC medium, and replated at 30,000/cm 2 in 35-mm tissue-culture dishes (Falcon Primaria). On the following day, the cell layers were irradiated with 15 Gy (2 Gy/min) using a cesium-137 radiation source and were then incubated with fresh BMLTC medium at 37 7C for an additional 1–3 days before they were recharged with freshly isolated CD34 c BM or CB cells. Long-term cultures CD34 c BM cells (50,000–100,000 from nine donors) and CD34 c CB cells (30,000–45,000 from six donors) were used for initiation of LTCs. Different starting cell densities were chosen for CB and BM in order to compensate for the higher proliferative potential of CB cells. CD34 c cells were plated onto the pre-established macrophage and BM stromal layers and into dishes without any preformed layer in 3 ml BMLTC medium. For LTCs with CD34 c BM cells, macrophage layers were derived exclusively from GPBPC products, while CD34 c CB cultures were additionally set up with normal PB-derived macrophages as another experimental group. Co-cultures were then incubated at 33 7C with 5% CO2 in air. Twice weekly, half of the medium was changed carefully in order to avoid aspiration of nonadherent cells. Five weeks after initiation of the LTCs, all cells (nonadherent and adherent) were removed from the dishes, and viable MNCs were counted by trypan blue exclusion. The content of granulocyte/macrophage (CFU-GM) and erythroid (CFU-E and BFU-E) colony-forming cells was determined in a standard semisolid methylcellulose colony assay, as described below. Results were calculated per 1000 starting cells used for initiation of the LTC and were statistically compared with each other using the paired twotailed Wilcoxon test. The fold nucleated cell expansion was calculated as the number of nucleated cells present after 5 weeks divided by the input cell number. CFU assays Cells harvested from the LTCs were plated at 3!10 4 (CB) or 5!10 4 (BM) into 35 mm dishes (Greiner, Solingen, Germany) in 1.2 ml semisolid medium. Freshly selected CD34 c cells were plated at 2.5!10 3 per dish to determine the CFC count of the starting cell populations. All assays were set up in duplicate or triplicate. Semisolid medium consisted of IMDM (Gibco BRL) with 0.8% methylcellulose (Stem Cell Technologies, Remagen, Germany), 30% FCS (Gibco BRL), 5% phytohemagglutinin leukocyte-conditioned medium (Stem Cell Technologies), 5 units/ml recombinant human erythropoietin (Boehringer Mannheim), 1% bovine serum albumin (Boehringer Mannheim), 2 mmol/l l-glutamine (Gibco BRL), and 0.1 mmol/l 2-mercaptoethanol (Merck, Darmstadt, Germany). After incubation for 18 days at 37 7C with 5% CO2, granulocyte/macrophage (transparent) and erythroid (red) colonies consisting of more than 50 cells were scored with an inverted microscope at!60 magnification. Immunohisto- and cytochemistry For immunostaining normal PB- (np3) and G-PBPC-derived (np2) adherent layers were obtained from 1- to 6-week-old cultures and then were either grown for an additional week on pre-

61 viously gelatin (Sigma)-coated chamber-glass slides (Falcon) or centrifuged onto cytospin slides. After air drying, cells were fixed for 5 min with a 2c1 mixture of methanol and acetone. Primary antibodies against CD1a (clone NA1/34), CD3 (T3-4B5), CD14 (TÜK4), CD15 (C3D-1), CD31 (JC/70A), CD54 (6.5B5), CD68 (PG-M1, all from Dako, Glostrup, Denmark), and CD34 (8G12) and CD62E (H18/7, both from Becton Dickinson) were used in dilutions suggested by the producers for 60 min at room temperature. A secondary biotinylated antibody (incubation for 30 min), streptavidin-alkaline phosphatase (45 min), and naphthol-MXphosphate/fast red chromogen were used according to the producer’s instructions (Universal Immunostaining Kit, Immunotech, Marseille, France). All steps were performed at room temperature, and phosphate-buffered saline (PBS) was used for triple washing steps between the incubations. In one case, cytospin preparations of PB-derived adherent cells cultured for 3 weeks with and without hydrocortisone (HC) were stained for CD68 (clone KP1, Dako) and Ki-M1p (Prof. M. Parwaresch, Kiel, Germany) using a standard avidin-biotin peroxidase complex method and irrelevant antibodies as negative controls. Cytochemical reactions for peroxidase and a-naphthyl acetate esterase (Sigma) and with periodic acid-Schiff (PAS, Merck), were tested with cytospin preparations and culture slides, as described previously [21].

Results Long-term cultures Cord blood. Parallel LTCs of CD34 c CB cells (np6) were set up on PB macrophage layers, G-PBPC macrophage layers, BM stromal layers, and without adherent layers, respectively. After 5 weeks, CFC numbers and the expansion of nucleated cells were determined. Cultures with an adherent layer consistently yielded a higher CFU-GM output and nucleated cell expansion than control cultures without pre-established layers (pp0.028). Comparison of macrophage with BM stromal layers showed no statistical difference with regard to CFU-GM (Fig. 1A) and BFU-E/CFU-E (data not shown) output numbers. However, in co-cultures with BM stroma the nucleated cell expansion was higher than on PB macrophage layers in all six experiments and higher than on G-PBPC-derived macrophage layers in five of six experiments (p~0.05, Fig. 2A). Bone marrow. In nine paired sets of cultures with CD34 c BM cells, G-PBPC-derived macrophage layers were compared with BM stroma and with control cultures without a layer. Both macrophage and BM stromal layers resulted in a significantly higher CFU-GM output (p~0.01) and cell expansion (p~0.05), compared with cultures without pre-established layers. In eight of nine experiments CD34 c BM cells yielded a higher CFU-GM output when co-cultured with macrophage layers compared with BM stroma (pp0.02, Fig. 1B). In addition, the CFU-E/BFU-E output was higher in LTCs on macrophage layers compared with BM stroma (pp0.028; median BFU-E per 1000 starting cells: macrophages, 0.52 (0.09–1.36); BM stroma, 0.17 (0–0.55); no layer, 0.04 (0–1.77)). In contrast, the median fold cell expansion was slightly but not significantly higher in BM stroma cultures (pp0.14; Fig. 2B).

Fig. 1A,B Week-5 CFU-GM per 1000 initially plated CB (A) and BM (B) cells on the indicated layers. Data points (i) connected by lines represent one experiment initiated with the same CD34 c cell population on the different layers, and horizontal bars (—) indicate the respective median values. Mf macrophage

Comparison of the progenitor content of CB and BM CD34 c cells Umbilical CB was compared with BM regarding the CFU-GM content of freshly isolated CD34 c cells and their progeny after 5-week LTCs on BM and macrophage layers. Regarding freshly isolated CD34 c cells, the median CFU-GM number was approximately two times higher in CB than in BM. After 5 weeks, however, CB cultures contained 13.5 times more CFU-GM than BM cultures (Fig. 3). Additionally, the fold cell expansion was one log higher from CD34 c CB cells during 5 weeks than from BM cells (data not shown). Morphology, cytochemistry, and immunostaining In cultures of PB- and G-PBPC-derived MNCs semiconfluent adherent cell layers developed after approximately 1 week. These layers consisted mainly of small elongated cells and some larger cells with a typical round and flat macrophage morphology (Fig. 4a). The

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Fig. 3 CFU-GM numbers of fresh CD34 c CB and BM cells (day 0) and after 5-week cultures on feeder layers (day 35, data pooled from BM stroma and macrophage cultures). Significance levels were calculated using Kruskal-Wallis statistics

tate esterase and negative for peroxidase and with periodic acid-Schiff.

Discussion

Fig. 2A,B Fold nucleated cell expansion of CB (A) and BM (B) cells during 5-week cultures on the indicated layers. Corresponding data points are indicated by lines

delayed appearance of adherent cells and their predominantly elongated morphology was likely due to the presence of HC in the culture medium, as in HC-free media a large proportion of monocytes adhered within 1 h and developed the morphology of round and flat macrophages with no or very few elongated cells (Fig. 4b). Immunostaining of HC-cultured PB-derived adherent cells was positive for the macrophage antigens CD68 and Ki-M1p, the adhesion molecules CD31 (PECAM-1) and CD54 (ICAM-1), weakly positive for CD14, and negative for CD1a, CD3, and CD15. Importantly, no endothelial cells were detected by staining for CD62E (E-Selectin/ELAM-1) and CD34. There was no apparent difference in the staining pattern between adherent cells from PB and those from G-PBPC products. Also, despite the different morphology of PB-derived adherent cells in cultures with and without hydrocortisone, in both cases the cells stained equally with anti-CD68 and Ki-M1p (Fig. 5a,b). Cytochemical tests on cytospin and culture (chamber) slides of PBderived adherent cells were positive for a-naphthyl ace-

The results presented show that macrophages derived from PB and from G-CSF-mobilized PBPC collections support the generation of CFU-GM from CD34 c CB and BM cells over a 5-week period under standard LTC conditions. Comparison with BM stromal layers revealed that macrophage layers are equivalent in terms of supporting CD34 c cord blood progenitors and even superior to BM stroma with regard to generation and/or maintenance of CFU-GM in cultures of CD34 c BM cells. To test the validity of these findings, we compared our reference cultures on BM stroma with experimental data from other groups [1, 22–24]. To exclude deviations resulting from different conditions in the CFU assays (e.g., addition of defined growth factors), the end-point CFU-GM numbers of LTCs were correlated to the input CFU-GM counts. The reported CFU-GM numbers after 5 weeks ranged between 7% and 13% of the original input CFU-GM numbers. In the present study we found a mean proportion of 8.2% in cultures on BM stroma, suggesting that our data are comparable with those from other studies. In BM cultures on macrophage adherent layers, the mean CFUGM yield after 5 weeks was 15% of the input CFU-GM number. Two aspects of this study are of particular interest: The superiority of macrophage layers regarding support of bone marrow CFU-GM is in contrast to (a) the equal support of CB CFU-GM by BM stromal layers and (b) the higher expansion of nucleated cells induced by BM stroma in case of CD34 c CB (p~0.05) and, not significantly, BM cultures. The differential conditions

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Fig. 4a,b Phase-contrast photography of unstained PB-derived macrophage layers after 3 weeks of culture (a) with and (b) without 10 –6 M hydrocortisone in plastic tissue-culture dishes. Original magnification!400 Fig. 5a,b Immunoperoxidasestaining of PB-derived macrophages for CD68. Cells were cultured for 3 weeks (a) with or (b) without 10 –6 M hydrocortisone

promoting either the generation of CFU-GM (i.e., macrophage layers) or the expansion of nucleated cells (BM stroma) from CD34 c BM cells during 5 weeks of culture might be explained by a faster proliferation and differentiation occurring in LTCs on BM stroma, leading to a higher output of mature cells at the expense of CFU numbers. This suggests that BM stromal layers produce stronger stimuli for proliferation and differentiation than macrophage layers. To explain the fact that, unlike CD34 c BM cells, CD34 c CB cells yielded comparable CFU-GM numbers when cultured on BM stromal layers, the different proliferative potential of CD34 c cells from BM and CB should be taken into account. In fresh CD34 c fractions the median CFUGM content in CB was only two times higher, while the CFU-GM output measured after 5 weeks was more than 13 times higher in CB compared with BM. This suggests a greater proportion of immature to committed progenitors in CB compared with BM, and/or a

higher clonogenic potential of the immature HPC in CB. This observation has been reported previously using single-cell plating and limiting-dilution experiments with CB- and BM-derived HPC [25, 26], and it is consistent with the recent finding of longer telomeres in CD34 c cells from CB and fetal liver compared with adult BM and PB [27]. Therefore, if the stimulus for maturation was stronger in LTCs on BM stroma than in LTCs on macrophage layers, it is understandable that the more primitive CB progenitors profit from the stronger activity produced by BM stromal layers. On the other hand, CD34 c BM cells would benefit from a weaker differentiation stimulus, resulting in less maturation. This latter condition is observed in LTCs on macrophage layers, as indicated by a higher CFU-GM output and a lower expansion of mature cells. Though immunophenotypical determination of cultured cells may be influenced by culture conditions and may differ from that of native cell preparations, the immunocytochemical staining pattern of PB-derived adherent cells is in accordance with that of macrophages, as indicated by positive intracellular staining with antiCD68 and Ki-M1p [28]. CD31 and CD54, found on the PB-derived adherent cells, are also expressed by endothelial cells, but as the PB-adherent cells are negative for CD62E (E-Selectin) and CD34, it is unlikely that they are of endothelial origin. Negative staining for CD3 and CD15 indicates that they are not T lympho-

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cytes or granulocytes, and lack of CD1a expression at different time points of the culture argues against a dendritic cell differentiation that is observed in HPC cultured with GM-CSF and TNF-a [29, 30] or in CD14 c leukocytes stimulated with GM-CSF and IL-4 [31, 32]. Adherent cells from neither PB- nor G-CSF-mobilized PBPC collections formed colonies when plated in standard semisolid CFU assays. This finding is in line with the mature macrophage phenotype of the adherent cells. However, it has been shown that CD34 c progenitors can be found among the plastic-adherent cells in liquid cultures of mobilized PB leukocytes [33]. In these experiments, adherence was performed in the absence of steroids and required only 2 h, while a considerable portion of the adherent cells in the present study were rarely observed before the end of the first week, likely due to the presence of HC added at the beginning of the cultures. Additionally, when examined after 1 week or later, no CID34 c cells were detected within the adherent layers. As no significant difference was found between PB- and G-PBPC-derived adherent cells, either in their immunophenotype or in their function as feeder cells, it seems likely that monocytes/macrophages circulating independently of G-CSF mobilization are responsible for the supportive capacity in the LTC. Monocytes/macrophages have long been known to produce activity that stimulates the formation of myeloid colonies from human and murine BM cells [16, 17]. This activity can be attributed in part to the cytokines G-CSF, macrophage CSF (M-CSF), and granulocytemacrophage CSF (GM-CSF). These directly acting myeloid growth factors are secreted from monocytes/ macrophages following stimulation by other cytokines, including interleukin 1 (IL-1), gamma interferon (IFNg), and tumor necrosis factor (TNF), or by endotoxin, and in a paracrine/autocrine mode among each other [12–15]. Adherence to plastic was also shown to be a regulatory signal for cytokine production in monocytes [34]. Furthermore, human monocytes produce fibronectin, one important component of the stromal extracellular matrix, upon macrophage differentiation in vitro [35]. The ability of monocytes/macrophages to support hematopoiesis in vitro depends on the presence of physiological concentrations of steroids in the culture, as HC was shown to induce CFU-GM-supporting activity and to diminish CFU-GM-inhibitory activity from monocytes [36]. More recently, enhancement of G-CSF and inhibition of TNF-a secretion from IL-1-stimulated monocytes have been revealed to contribute to the mechanisms underlying this effect of HC [37]. Apart from our former findings [38], PB-derived adherent layers capable of supporting myelopoiesis have been reported in another recent study [39]. These layers were generated from whole PB MNCs of G-CSFtreated pediatric patients with solid tumors. In contrast to the adherent cells in the present study, the wholeblood MNC-derived layers reached confluence and

showed a typical fibroblast-like appearance. However, fibroblast-like morphology was not observed when leukapheresis products were used, which is consistent with the recently reported inability to recover BM mesenchymal cells from PB progenitor cell collections [40]. In another recent study, PB-derived adherent cells were shown to support the maintenance of CD4 c and CD8 c T lymphocytes for up to 3 months [41]. In conclusion, we have established a 5-week LTC system using PB-derived macrophage adherent layers, which is, regarding the generation of CFU-GM from CD34 c BM and CB cells, comparable with the original two-step BM LTC. The present results directly demonstrate that macrophages have a supportive effect on immature myeloid progenitors in a 5-week LTC, while the influence of macrophages or their secretory products was previously investigated mainly with respect to the late stages of hematopoiesis, i.e., colony formation from CFCs in short-term assays. Determination of the mechanisms responsible for the differential effects on myelopoiesis compared with BM stromal layers might help to further elucidate the complex relationship between stem/progenitor and accessory cells. In addition, the ability to produce fibronectin might render cultured macrophages an interesting tool for retroviral gene transfer. Acknowledgements We thank A. Uhde and S. Hennings for their technical support, the Delivery Team at the Department of Gynecology and Obstetrics for collecting the cord blood samples, and the Department of Transfusion Medicine for providing the leukapheresis samples.

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