PB to BM immature hemopoietic cells appeared to be complex [lo], in part ..... more immature cells (Neelis et al., manuscript submitted for publication) be it not ...
In Vivo Expansion of Hemopoietic Stem Cells GERARD WAGEMAKER, SIMONE C.C. HARTONG, KAREN J. NEELIS, TORSTEIN EGELAND; ALBERTUS W. WOGNUM Institute of Hematology, Erasmus Universiteit Rotterdam, The Netherlands and "Institute of Transplantation Immunology, Oslo, Norway Key Words. Stem cells . In vivo expansion of stem cells . Mobilization ofstern cells Thrornbopoietin CD34' cells ' Circulating CD34' cells Rhesus monkeys Hernopoietic growth factors
ABSTRACT Under conditions of steady-state hemopoiesis, a small fraction of immature hemopoietic cells, including stem cells, circulates in peripheral blood (PB). In rhesus monkeys, a median number of 1.2 X 107/1CD34' cells was observed as opposed to a median number of 1.5 X 10y/l in aspirated bone marrow (BM). The concentration of circulating CD34' cells is therefore approximately two logs less than that in BM. Since a 4-kg rhesus monkey has an estimated number of 3 X 10" BM cells and approximately 300 ml of blood, the fraction of CD34' cells that circulates can be estimated at approximately 0.4% of the total pool of CD34' cells. During hemopoietic reconstitution following a cytotoxic insult such as results from a midlethal dose of TBI, PB CD34' cell
numbers appeared to be correlated to those of BM, suggesting that PB CD34' cells may reflect reconstitution of BM CD34' cells. Reconstitution of BM immature cells can be accelerated by treatment with pharmacological doses of growth factors, resulting in largely expanded immature cell populations within a few weeks after TBI. Growth factors observed to exert such an effect included, notably, thrombopoietin. Such an acceleration can be monitored by daily assessment of circulating CD34' cells. Expansion of immature circulating cells indicates expansion of similar cells in the bone marrow rather than growth factor-induced selective mobilization of immature cells. Stem Cells 1998;16(suppl I ):1 85-I91
INTRODUCTION It is long known that a small fraction of hemopoietic stem cells circulates [l, 21, probably as part of homeostatic mechanisms required to control blood cell production from the scattered bone marrow (BM) sites. The fraction of circulating immature hemopoietic cells characterized by the CD34 surface antigen may be expanded by administration of pharmacological doses of growth factors such as G-CSF [3] and GM-CSF [4], Kit-ligand [ 5 ] ,Flt-3 ligand [6] and interleukin 8 (IL-8) [7], indicating that either their BM numbers or their mobilization into the peripheral blood (PB), or both, are under growth factor control. More recently, advantage has been taken from circulating stem cells in that these cells provide an alternative to BM to harvest large numbers of stem cells for either autologous or allogeneic transplantation [8, 91. We have earlier advocated that these cells may also be used as an early marker for residual stem cell numbers and a monitor for immature cell reconstitution after TBI and/or hemopoietic cell transplantation [ 101, or during hemopoietic growth factor therapy. However, the relation of
Characteristics and Potentials of Blood Stem Cells STEMCELLS 1998;16(suppl 1):185-191 OAlphaMed Press. All rights reserved
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In Vivo Expansion of Hemopoietic Stem Cells
PB to BM immature hemopoietic cells appeared to be complex [lo], in part because of fluctuations and genetic influences [ 111 which determine circulating cell numbers. We approached these issues from experience in a preclinical model for radiation-induced myelosuppression in rhesus monkeys, with special reference to normal levels and expansion of immature BM and PB CD34' cells by thrombopoietin (TPO) [ 12-15] therapy. As described previously, TPO treatment accelerates the reconstitution of immature BM CD34' cells [16, 171, an effect that is augmented by coadministration of GM-CSF [18]. This feature is in accordance with the presence of the TPO receptor, c-Mpl, on immature BM cells and with stimulatory effects of TPO on immature hemopoietic cells in vitro [ 18-20].
MATERIALS AND METHODS Animals Purpose-bred male rhesus monkeys (Macaca Mulatta) weighing 2.5-4.0 kilograms and aged two to three years were used. The monkeys were housed in groups of four to six monkeys in stainless steel cages in rooms equipped with reverse-filtered air barrier, normal day light rhythm and conditioned to 20°C with a relative humidity of 70%. Animals were fed ad libitum with commercial primate chow and fresh fruits, and received acidified drinking water. All animals were free of intestinal parasites and seronegative for herpes B, simian T-lymphotropic viruses and simian immunodeficiency virus. Housing, experiments and all other conditions were approved by an ethical committee in conformity with legal regulations in the Netherlands. TBI Monkeys were irradiated with a single dose of 5 Gy TBI delivered by two opposing x-ray generators, operating at a tube voltage of 300 kV and a current of 10 mA. The half-layer thickness was 3mm'. The focus skin distance was 0.8 m and the average dose rate 0.20-0.22 Gy/min. During TBI, the animals were placed in a cylindrical polycarbonate cage which rotated slowly (three times per min) around its vertical axis. Supportive Care Two weeks before TBI, the monkeys were placed in a laminar flow cabinet and the gastrointestinal tract was selectively decontaminated by giving orally Ciprofloxacin (Bayer; Mijdrecht, The Netherlands), Nystatin (Sanofi BV; Maassluis, The Netherlands) and Polymyxin B (Pfizer; New York, NY). This regimen was supplemented with systemic antibiotics, in most cases ticarcillin (Beecham Pharma; Amstelveen, The Netherlands) and cefuroxim (Glaxo; Zeist, The Netherlands), when leukocyte counts dropped below 109/l. Guided by fecal bacteriograms, the antibiotics were continued until leukocyte counts rose to levels >109/l. Dehydration and electrolyte disturbances were treated by appropriate fluid and electrolyte administration S.C. The monkeys received irradiated (15 Gy y irradiation) platelet transfusions whenever thrombocyte counts reached values below 40 x IO'/l, packed red cells whenever hematocrits were lower than 20% and occasionally, whole blood transfusions in case of coincidence of both transfusion criteria. Test Drug Recombinant full-length human TPO produced by Chinese hamster ovary cells was supplied by Genentech Inc. (South San Francisco, CA). The daily dose of 10 p,g/kg was administered S.C. for 2 1 consecutive days after TBI. The dose was diluted to a volume of 1 ml with phosphate-buffered saline/O.Ol% Tween 20 prior to administration. The diluent was used as placebo. Each treatment group consisted of four consecutive monkeys.
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BM Aspirates BM was aspirated under neurolept anesthesia using Ketalar (Apharmo; Arnhem, The Netherlands) and Vetranquil (Sanofi; Maassluis, The Netherlands). Small BM aspirates for analytical purposes were taken from the shafts of the humeri using pediatric spinal needles and collected in bottles containing 2 ml Hank's buffered Hepes solution (HHBS) with 200 IU sodium heparin/ml (Leo Pharmaceutical Products; Weesp, The Netherlands). Low density cells were isolated using a Ficoll (density 1.077) (Nycomed Pharma AS; Oslo, Norway) separation. Hematological Examinations Complete blood cell counts were measured daily using a Sysmex F-800 hematology analyzer (Toa Medical Electronics Co., LTD.; Kobe, Japan). Measurements of Surface Antigens Once weekly, a fluorescence-activated cell sorter (FACS) scan analysis was done on PB and BM samples for a variety of surface antigens, including CD34, by use of a human CD34 monoclonal antibody (mAb 566) that had been fluoresceinated with fluorescein isothiocyanate (Sigma; St Louis, MO) according to standard procedures. For PB CD34' cells this analysis was done daily. 0.5 mL of whole blood or BM was lysed in 10 ml lysing solution (8.26 g ammonium chloride/l.0 g potassium bicarbonate and 0.037 g EDTA per 1) for 10 min at 4". After lysing the cells were washed twice with HHBS containing 2% fetal calf serum and 0.05% (wthol) sodium azide (HFN). The cells were resuspended in 100 p1 HFN containing 2% normal monkey serum to prevent a specific binding of the mAbs. mAbs were added in a volume of 5 pl and incubated for 30 min on ice. After two washes, the cells were measured on the flow cytometer. Ungated list mode data were collected for 10,000 Ioo1 events and analyzed using the Lysis I1 software (Bectonone marrow Median 1.47 x 109/1 Dickinson; San Jose, CA).
I
Statistics Standard deviations were calculated and are given in the text and in the figures on the assumption of a normal distribution. The significance of a difference was calculated by Fisher's exact test for categorical data, and for continuous data by a one-way analysis of variance followed by a nonpaired Student's t-test. RESULTS
Normal Levels of BM Aspirate and PB CD34' Cells ~i~~~~ shows the absolute numbers Of CD34' in BM aspirates and PB of 50 consecutive
0.001
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lo
Peripheral blood Median 1.I 5 x 107/1 I
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0.01 0.001
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Individual monkeys (n = 50)
Figure 1. BM aspirate and PB CD3& cells in 50 consecutive normal rhesus monkeys, representing baseline data collected just before TBI. PB is presented by circles and BM by squares. Upper panel: BM and PB data of individual monkeys dissociated a i d ranked in ascending order. Lower panel: BM and PB data of individual monkeys associated, BM data ranked in ascending order.
In Vivo Expansion of Hemopoietic Stem Cells
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Table 1. Distribution pnr;irnetrrs of BM aqiratc and PB CD3-I' A h (it' 50 nmnal rhejuh monkeys Frequency (%) Mean ? SD Median Range Absolute number (X 10y/I) Mean k SD Median Range
BM CD34' cells
PB CD34' cells
3.2 f 3.5
0.2 k 0.2
2.2
0.1 ND - 1.1
0.2 - 17.5 2.2 ? 2.8 1.5
0.02 ? 0.02 0.01
0.02 - 16.5
ND - 0.09
ND: Not detectable
Table 2. Approximation of the fraction of C D X srlls thdt circuldtr,. bawd on incJian Ie\rls Number of BM CD34' cells/monkeyd
6.6 X 10'
Number of PB CD34' cells/monkeyb
0.3 X 10'
Fraction of circulating CD34' cells
0.4%
"Calculatedon the assumption that a 4-kg monkey has 3 X 10" BM cells of which 2.2% are CD34'. hCalculatedon the assumption that a 4-kg monkey has approximately 300 ml blood containing 107/1CD34' cells.
rhesus monkeys, sampled to obtain baseline data just before TBI. In the upper panel, the individual monkey BM and PB values are dissociated and both ranked in ascending order, displaying a median value of 107ACD34' cells in PB and of 1.5 X 109/1in aspirated BM, and a variance which spans for both BM and PB approximately two logs in magnitude. In the lower panel, the PB CD34' cell numbers of individual monkeys are reassociated with those of the BM, which clearly shows that a direct relationship between BM and PB CD34' is not evident. The distribution parameters of the 0.1 BM and PB CD34' cells are given in Table 1 and the calculation of 0 the fraction of circulating CD34' X UJ cells is shown in Table 2, on which 5 0.01 0 basis it is concluded that approxi+ * mately 0.4% of the total pool of c) n 0 CD34' cells circulates.
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7 14 Time after TBI (davs)
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Figure 2. Circulating CD34' cells of TPO (closed circles) and placebotreated (open circles) rhesus monkeys, revealing acceleration of CD3# cell reconstitution by TPO and consequently more than one-log expansion of those cells compared to placebo controls two weeks after TBI. Mean values of four individual monkeys in each group. The difference at two weeks is highly significant (p
