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1991 78: 3192-3199

Myeloid differentiation of purified CD34+ cells after stimulation with recombinant human granulocyte-monocyte colony-stimulating factor (CSF), granulocyte-CSF, monocyte-CSF, and interleukin-3 T Egeland, R Steen, H Quarsten, G Gaudernack, YC Yang and E Thorsby

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Myeloid Differentiation of Purified CD34' Cells After Stimulation With Recombinant Human Granulocyte-Monocyte Colony-Stimulating Factor (CSF), Granulocyte-CSF, Monocyte-CSF, and Interleukin-3 By Torstein Egeland, Rita Steen, Hanne Quarsten, Gustav Gaudernack, Yu-Chung Yang, and Erik Thorsby CD34+ cells isolated from bone marrow or umbilical cord blood from healthy donors were studied for proliferation and differentiation in liquid cultures in the presence of recombinant human granulocyte-monocyte colony-stimulating factor (GM-CSF), granulocyte CSF (G-CSF), monocyte CSF (M-CSF), and interleukin-3 (IL-3). followed by immunophenotyping for myeloid and myeloid-associated cell surface markers. IL-3, either alone or together with GM-CSF, G-CSF, or M-CSF, induced, on average, 50-fold cell multiplication, GM-CSF five fold t o 10-fold, and G-CSF and M-CSF less than fivefold. Cells from cultures stimulated with GM-CSF, G-CSF, or M-CSF alone contained cells with a "broad" myeloid profile, "broader" than observed in cultures with IL-3. However, since IL-3 induced rapid cell multiplication, high numbers of cells expressing early (CD13, CD33) and late myeloid markers (CD14, CD15) were recovered. The presence of other CSFs

together with IL-3 did not alter the IL-%induced effect on the cells. When 5,000 CD34' cells were cultured with IL-3 alone, the cultures still contained 2,000 t o 5,000 CD34' cells after 14 days of culture, while cells cultured with GM-CSF, G-CSF, or M-CSF contained less than 1,000 CD34+ cells. Furthermore, 1,000 t o 3,000 cells were positive for the megakaryocytic lineage marker CD4lb after cultures with GM-CSF or IL-3, while cultures with G-CSF or M-CSF did not contain detectable numbers of CD4lb+ cells. Finally, erythroid cells could also be generated from purified CD34' cells. The results show that IL-3 and GM-CSF can induce rapid proliferation of purified CD34' cells in vitro with differentiation t o multiple myeloid lineages, while certain subsets maintain expression of CD34. o 1991by The American Society of Hematology.

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cal cord blood CD34' cells from healthy donors by means of an immunomagnetic technique, and immunophenotyped the cells before and after incubation for 14 days in the presence of recombinant human GM-CSF, G-CSF, or M-CSF, alone or in combination with IL-3. For phenotyping, a panel of monoclonal antibodies (MoAbs) against more than 20 different myeloid and lymphoid cell surface markers was used.

ROM 0.5% T O 2.5% of gradient centrifugated mononulear cells (MNC) from bone marrow or umbilical cord blood are CD34' cells. The CD34 antigen is expressed on myeloid precursors of the granulocytic, monocytic, erythroid, and megakaryocytic lineages.'.' Furthermore, purified CD34' cells can be kept in long-term bone marrow cultures for up to 10 weeks and still contain myeloid precursors.' Enriched CD34' cells can also engraft after infusion in lethally irradiated baboons4or humans.' Because CD34' cells also exhibit pluripotent hematopoietic stem cell characteristics, transplantation with purified CD34' cells can possibly be used as an alternative to conventional bone marrow transplantation. However, to perform a successful stem cell transplantation, some manipulation of the cells to support rapid multilineage engraftment would be an advantage. Among substances that might be used to stimulate CD34' cells ex vivo before transplantation are one or more of the following colony-stimulating factors (CSFs): granulocyte-monocyte CSF (GM-CSF), granulocyte CSF (G-CSF), monocyte CSF (M-CSF), or interleukin-3 (IL-3). To study myeloid differentiation induced by CSFs on purified CD34+ cells, we isolated bone marrow and umbiliFrom the Institute of Transplantation Immunology and Department of Gynecology and Obstetrics, Rikshospitalet University Hospital, Oslo, Norway; and the Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN. Submitted June 21,1991; acceptedAugust 27,1991. Supported by a postdoctoral fellowship fiom the Norwegian Royal Councilfor Industrial and Scientific Research. Address reprint requests to Torstein Egeland, MD, Associate Professor, Institute of Transplantation Immunology, Rikshospitalet University Hospital, N-0027 Oslo 1, Norway. The publication costs of this article were defiayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.section 1734 solely to indicate this fact. 0 1991 by The American Society of Hematology. 0006-4971I91 I7812-00O9$3.00/0

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MATERIALS AND METHODS

Mononuclear Cells MNC were isolated by gradient centrifugation on Lymphoprep (Nyegaard, Oslo, Norway) of heparinized bone marrow or umbilical cord blood samples obtained after informed consent. The samples were taken from healthy bone marrow donors or umbilical cord veins after normal delively. MNC were washed in Iscove's modified Dulbecco's medium (Flow Laboratories, Irvine, Scotland) without any supplements.

Isolation of CD34' Cells First, the relative number of CD34' cells in MNC was assessed with immunomagnetic phenotyping; using 12.8, a murine monoclonal IgM antibody against CD34,'.' and screening of approximately 1,000 cells. For isolation of CD34' cells, 12.8 or another murine antLCD34 MoAb, MylO (IgGl),' were used. MNC were either sensitized with MylO, followed by washing and rosette formation with paramagnetic M450 beads coupled with sheep antibodies against murine IgGl (Dynabeads; Dynal, Oslo, Norway), or cells were made to rosette directly with M450 beads that had previously been coupled with saturating amounts of 12.8. The rosette formation took place during cold (4°C) centrifugation of beads and MNC at 100 x g for 3 minutes. Afterwards, beads and rosette forming cells were separated from unbound cells by washing seven times using a magnet.' Finally, rosette-forming cells were detached from the beads by treatment with chymopapain (Chymodiactin; Boots, Lincolnshire, IL) according to Civin et a1,9 and free cells were washed twice in Iscove's medium supplemented with 10% fetal calf serum (Flow) and glutamine (Flow). The viability was always more than 95% as measured by staining with acridine orange and ethidium bromide.

Blood, Vol78, No 12 (December 15). 1991: pp 3192-3199

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MYELOID DIFFERENTIATION OF CD34’ CELLS

Table 1. MoAbs Used for Phenotyping

Cultures of CD34‘ Cells All cell cultures were performed in Iscove’s modified Dulbecco’s medium enriched with 10% fetal calf serum and 2 mmol/L glutamine. Isolated CD34’ cells were cultured in the presence of recombinant human GM-CSF (80 U/mL), G-CSF (1:750), M-CSF (320 U/mL), alone or in combination with IL-3 (80 U/mL). The CSFs were all produced at the Genetics Institute’s Research Laboratory, Cambridge, MA. The concentrations that gave the highest cell proliferation in titration experiments were used. The cultures were kept in 96-well tissue culture plates (U-shaped wells) (Costar, Cambridge, MA) with 1,OOO CD34+cells per well and five wells per series. Parallel culture series with CD34+ cells and a supernatant from the carcinoma cell line 5637 or PHA-stimulated normal peripheral blood MNC, PHA (Welcome, Dartford, England), alone or in combination with recombinant human IL-2 (Genzyme, Cambridge, MA), 10 U/mL, served as controls. After 14 days of culture, viable cells from individual series were counted and phenotyped.

Phenotyping of Cells Newly isolated CD34+ cells, as well as cells that had been cultures for 14 days, were phenotyped. The newly isolated CD34+ cells were phenotyped after a short preincubation to allow the cell surface antigens to recover from the chymopapain treatment used for detachment from the beads. The phenotyping was performed as previously described.6Briefly, M450 beads coupled with more than 20 different MoAbs against myeloid and lymphoid markers were used (Table 1).Approximately 100 cells were counted. To demonstrate the purity of the isolated cells, CD34+ cells were also analyzed in flow cytometry on Facscan (Becton Dickinson, Mountain View, CA) with 12.8 and HKB-1 (Table l), using fluorochromeconjugated isotype-specific antibodies (Southern Biotechnology, Birmingham, AL) as second layer. Before flow cytometry, the beads were detached from the isolated cells by incubation for 45 minutes at rmm temperature with a goat antiserum against murine Fab fragments (DetachAbead; Dynal).

Assays for CFU-GMand BFU-E Isolated CD34’ cells were also analyzed in triplicate dishes for colony-formingunit-granulocyte-monocyte (CFU-GM) and burstforming unit-erythroid (BFU-E) as described elsewhere.” One hundred cells were seeded per dish, and the cells were stimulated with the supernatant from the cell line 5637 and erythropoietin (Polyclone, Marnes, France), 3 U/mL. Colonies were enumerated after 14 days of culture. RESULTS

Phenotyping of Isolated CD34’ Cells Isolated CD34’ cells were greater than 96% pure as judged by flow cytometry (Fig 1). A majority of the cells also expressed HLA class I1 molecules (Fig 1, Table 2). Table 2 also shows that a large subset of the isolated cells was positive for the cell surface markers CD13. CD33, CDllb, CD14, CDla, CD71, CD24, and CD15 were also expressed on smaller subpopulations. Proliferative Responses to CSFs Growth ability of isolated CD34+cells in the presence of CSFs or supernatants from the cell line 5637 or phytohemogglutinin (PHA)-stimulated MNC is shown in Fig 2. There were large variations between individual experiments.These

Cell Surface Marker

MoAb

Main Specificities*

Source of MoAb

Andrew J. McMichael NA1I34 Thymocytes BMAOlll Pan T cells Behringwerke 38.1 T cells, associated to John A. Hansen the T-cell antigen receptor Subset of T cells and Paul J. Martin CD4 66.1 (early) monocytes Paul J. Martin 10.2 Pan T cells CD5 CD6 Pan T cells Paul J. Martin 12.1 CD7 Barton F. Haynes 3A1 Pan T cells Gustav Gaudernack Subset of T cells 5C2 CD8 Patrick G. Beatty 60.1 Granulocytes and CD11b monocytes CD12 20.2 Monocytes and mac- John A. Hansen rophages CD13 3D10 Early and late myeloid Gustav Gaudernack (granulocyte and monocyte) cells CD14 Early and late mono- John A. Hansen 20.3 cytes CD15 7H1 Early and late granu- MonoCarb locytes and monocytes CD19 AB1 B cells, from early Steinar Funderud stages CD24 32D12 B cells and early my- Steinar Funderud eloid CD37 HH1 Late stage B cells Steinar Funderud CD33 Earliest myeloid cells Robert G. Andrews 67.6 and Irwin D. Bernstein CD34 12.8 Pluripotent stem cells Robert G. Andrews and lineage-reand Irwin D. Bernstricted progenitors stein CD4la ITI-PL-3 Megakaryocytic lin- Gustav Gaudernack eage CD41b ITI-PL-1 Megakaryocytic lin- Gustav Gaudernack eage CD42a ITI-PL-2 Megakaryocytic lin- Gustav Gaudernack eage HLA class I1 HKB-1 A number of cells in- Steinar Funderud molecules cluding most early myeloid cells Glycophorin A IC10 Erythroid cell lineage Steinar Funderud CD71 284 Transferrin receptor Gustav Gaudernack CDla CD2 CD3

Cells were phenotyped in the rosette-forming cells assay; 12.8 and HKB-1were also used in flow cytometry. *For references, see Knapp et aI.’O

variations were as large in cells obtained from bone marrow as from umbilical cord blood, and the results with cells from these two compartments are therefore pooled. The proliferation was pronounced in cultures stimulated with IL-3 irrespective of the presence of other CSFs, reaching up to 100-fold multiplicity after 14 days of culture. GM-CSF generated three to 10 times the original cell number during the same period, while G-CSF or M-CSF induced less than one to five times cell multiplication. Only negligible num-

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EGELAND ET A1

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Fig 1. Flow cytometry of isolated CD34+cells. (A) Forward and side scatter dot blot shows gating for cells that are analyzed in dot blot Band C. (B) Negative control (irrelevant MoAbs and fluorochrome-conjugated secondary antibodies) on isolated cells. (C) Cells stained with 12.8 (FITC; horizontal axis) and HKB-1 (phycoerythrin; vertical axis) for CD34 and HLA class II molecules, respectively. KG-1A" and lymphoblastoid cell lines were used as positive controls (data not shown).

bers of viable cells could be detected after incubation in medium, PHA, or PHA with IL-2. Expression of Myeloid-AssociatedMarkers on the Cultured Cells

Figure 3 shows individual and mean results of the relative number of cells according to expression of 13 myeloidassociated cell surface markers. The results from cells harvested from bone marrow and umbilical cord blood are treated together. Generally, the results from cultures with IL-3 were not influenced by the presence of other CSFs. Data from incubations in medium only or with PHA or PHA and IL-2 are not shown, since there were too few viable cells to allow reliable phenotyping, but several of the

myeloid-associated markers were expressed on some of the cells. Relative number of cells positive for CD34 and CD33. The relative number of CD34' cells was low ( < 1% to 10%) after 14 days of culture, and only occasionally were the figures between 10% and 20% (GM-CSF or M-CSF) (Fig 3). The relative number of cells expressing CD33 varied extensivelybetween experiments, ie, from less than 10% to as great as 70%, for the same CSF. Generally, cultures containing IL-3 induced lower relative numbers of CD34 and CD33 than the other CSFs. Relative number of cells positive for CD15, CD14, CDllb, CDI3, and HLA class 11 molecules. GM-CSF, G-CSF, and M-CSF induced high relative numbers of cells that were

Table 2. Phenotyping of Cells Isolated With MoAbs Against CD34 Cell Surface Marker

Relative No. of Cells

CD34 CD33 CD15 CD14 CD11b

96 (92-98) 10 (2-36) l(1-7) 2 (1-20) 5(