diluted marrow was gently layered over 4.0 ml of separation fluid in a 15 ml ... by Worton, McCulloch and Till (32) for use with mouse bone marrow. A 0.8%.
Colony Formation in Culture by Bovine Granulopoietic Progenitor Cells J. M. Walker, V. E. 0. Valli and J. H. Lumsden*
Calf bone marrow cells cultured in a semisolid medium of 0.8% methyl cellulose produced colonies of granulocytic cells and macrophages by seven days. A prerequisite for colony growth was the presence of serum obtained from a calf three hours after intravenous injection of endotoxin. Three morphological types of colonies were seen but cell types within these types of colonies did not differ. Cultured cells were identified by morphological and cytochemical characteristics. Optimum growth occurred when serum from endotoxin stimulated calves and fetal calf serum were present in a volumetric ratio of 7:3. Inhibition of colony growth occurred when endotoxin-stimulated serum was present at greater than optimum concentration. Normal calf serum, fetal calf serum, mouse L-cell conditioned medium and bovine urine did not stimulate significant colony growth when 8.0 x 104 marrow cells were cultured. There was a linear relationhip between the number of marrow cells in the cultures and the number of colonies produced. Colony forming efficiency ranged from 13 to 59 colonies per 105 cells plated. The behaviour of calf colony forming units in suspension culture was similar to that reported for mouse colony forming units.
La culture de cellules de la moelle osseuse de veaux, dans un milieu semi-solide dont la teneur en methyl cellulose s'elevait a 0.8%, donna naissance 'a des colonies de granulocytes et de macrophages, en sept jours. La presence, dans ce milieu de culture, de serum preleve chez un veau, trois heures apries l'injection intra-veineuse d'endotoxine, s'avera essentielle au diveloppement des colonies. On en observa trois types morphologiques; les types de cellules composant ces colonies ne presentaient cependant aucune difference. On les identifia d'apris leurs caracteres morphologiques et cytochimiques. On obtint la croissance la plus luxuriante en enrichissant le milieu de culture avec du serum d'un veau ayant requ l'endotoxine et celui d'un foetus bovin, dans la proportion volumetrique de 7:3. La croissance des colonies se trouva inhibee lorsque la quantite de serum provenant d'un veau ayant requ l'endotoxine depassait celle de la proportion optimale. Du serum de veau normal ou de foetus bovin, un milieu de culture conditionn6 par les cellules L de souris et de l'urine bovine ne stimulerent pas de faCon appreciable la croissance des colonies, lorsqu'on ensemenqait 8.0 x 104 cellules de la moelle osseuse. On nota une relation lineaire entre le nombre de cellules ensemencees et celui des colonies ulterieures. Chaque fois qu'on ensemengait 10 cellules, on pouvait prevoir le developpement de 13 it 59 colonies. Le comportement des unites generatrices de colonies, dans des milieux de culture en suspension, en ce qui concerne le veau, ressemblait 'a celui qu'on a dej'a rapport6 relativement aux souris.
*Department of Pathology, Ontario Veterinary College, University of Guelph, Guelph, Ontario. This work was done while Dr. Walker was a Fellow of the Medical Research Council of Canada. Supported by grants from the Medical Research Council of Canada, the National Research Council of Canada, and the Ontario Ministry of Agriculture and Food. Submitted May 31, 1973.
Hemopoiesis depends upon cellular proliferation and differentiation occurring in populations of undifferentiated progenitor cells [for recent reviews see Lajtha (7) and McCulloch (10)]. These have not been identified morphologically, hence they must be studied indirectly using developmental criteria. There are two widely used methods of study based on such criteria. The first is the spleen colony technique (11, 29) which detects mouse pluripotent stem cells by their capacity to form macroscopic hemopoietic colonies in the spleens of heavily irradiated or genetically anaemic recipients. A second method detects committed granulopoietic progenitor cells from mouse (1, 19), rat (2), rabbit, (8), marmoset (20), monkey (15) and man (17, 25) by their capacity to form colonies in cell culture. The two methods have been compared extensively in the mouse and it is generally agreed that the cells giving rise to spleen colonies are progenitors of those that yield colonies in culture. The objective of the study described here was to develop a culture assay for bovine progenitor cells analogous to that available for murine cells. Comparisons were made of colony formation in culture from bovine marrow cells with the similar procedure for mouse cells in terms of culture requirements, cellular composition of colonies and behaviour in suspension cultures. We found that marrow from normal calves would form colonies in culture when stimulated by 25 -
Endotoxin Stimulated Normal Calf Serum
Percent of Total Serum In Cultures (V/V)
Fig. 1. Number of colonies formed when 8 x 104 nucleated marrow cells were cultured with varying concentrations of endotoxin-stimulated calf serum or normal calf serum made up to a constant volume with fetal calf serum. Each point represents the mean colony counts from seven calves. Four dishes were counted from each calf at the serum concentrations indicated. Vertical bars represent the standard error of the mean.
serum obtained from an endotoxin-treated calf. The procedure described herein yields a quantitative assay for a class of progenitor cells similar in their position in hemopoietic differentiation to the committed granulopoietic progenitors detected in culture assays of murine marrow cells.
MATERIALS AND METHODS SOURCE OF HEMOPOIETIC TISSUE Purebred castrated male and female Holstein-Friesian calves were used as marrow cell donors. Their ages ranged from three months to eight months and weights varied between 90 and 193 kg. Clinical, hematological and parasitological examinations were conducted regularly to ensure that all marrow donors were clinically normal. The calves were identified by letters and placed in three groups for experimental purposes. Bone marrow from calves A to G inclusive was used to determine optimum concentration of colony stimulating activity. Calves F to J inclusive served as marrow donors for establishing linearity of the assay. Calves G, H, K and L were used to study the behaviour of progenitor cells in suspension culture. COLLECTION OF MARROW CELLS FOR CULTURE The calves were restrained in lateral recumbency and marrow aspiration was carried out using local anesthesia as described by Wilde (31). The marrow cavity of the third and fourth sternebrae was penetrated, using a 16 gauge 1.5 inch Yale biopsy needle. Three to 5.0 ml of marrow was aspirated into a plastic 35 ml syringe containing 50 units of preservative free heparin.' Coarse debris was removed by passing the aspirate through a fine metal sieve (Swinney adaptor) into 4.0 ml of tissue culture medium [alpha medium (27)]. Cell clumps were dispersed by repeated aspiration into a 10 ml pipette. tSodium Heparin, 100,000 USP units, Connaught Medical Research Laboratories, Toronto, Ontario.
Can. J. comp. Med.
CELL SEPARATION Because bovine red blood cells sediment at an extremely slow rate (24) a modification of the density gradient centrifugation technique described by Thorsby (28) for the separation of granulocyte and lymphocytes from human peripheral blood was employed to obtain a concentrated suspension of nucleated cells free of red cells. The method used was as follows: Six ml of the diluted marrow was gently layered over 4.0 ml of separation fluid in a 15 ml conical polycarbonate centrifuge tube.2 The separation fluid was prepared by combining 24 parts of 9.0% Ficoll3 in distilled water with 10 parts of 33.3%;^ Isopaque.4 The diluted marrow and separation fluid were centrifuged at 900 x gravity for 40 minutes at 18°C. During centrifugation the red cells and polymorphonuclear leukocytes collect at the bottom of the conical tube. All other leukocytes and their precursors plus platelets sediment to the interface of the separation fluid and diluted marrow where they form a narrow band. This interface layer was drawn off, washed twice in alpha medium and resuspended in 5.0 ml of alpha medium. The nucleated cells were counted using a hemocytometer. This method usually yielded suspensions containing eight to 20 million nucleated cells per ml and varying numbers of red blood cells which were generally not in excess of a 10:1 ratio with nucleated cells. Cell suspensions of appropriate concentrations for culture were prepared from this pool.
SOURCE OF COLONY STIMULATING ACTIVITY
Serum collected from a calf treated with endotoxin5 provided the source of colony stimulating activity (CSA) for the culture system. This serum was produced as follows: A sterile solution of 5%c, dextrose in normal saline containing 1.0 mg Piromen/ ml was injected into the right jugular vein of a 214 kg Holstein-Hereford heifer at the rate of 25.0 ug/kg body weight. Total white blood cell counts were done on venous blood
Sorvall, Inc., Norwalk, Con-
3Ficoll, Pharmacia, Upsala, Sweden. 41sopaque-400, Winthrop Laboratories, Aurora, Ontario. 5Piromen (a trypsinized whole cell culture of Pseudomonas aeruginosa), Baxter Laboratories, Malton, Ontario.
at hourly intervals post injection and at the nadir of peripheral leukocyte counts (three hours post injection) blood was collected from the left jugular vein into 10.0 ml vacutainers6 without anticoagulant. The blood was allowed to coagulate in a water bath at 37°C for one hour, and the serum was separated after centrifugation at 630 x gravity for ten minutes. The serum was filtered through a 0.45 u Millipore filter7, collected in 10 ml lots in sterile 17 x 100 mm plastic tubes8 and stored at -20°C until just prior to use, at which time it was thawed and re-filtered through a 0.45 /i, Millipore filter. Control serum was collected from a normal Holstein calf three hours after the intravenous injection of 4.28 ml of sterile saline containing 5% dextrose. This serum was prepared and stored the same way as the endotoxin-stimulated serum. The serum was stored frozen for five months without apparent loss of activity. Other possible sources of CSA were tested. These included mouse L-cell conditioned medium9 and bovine urine prepared as described by Robinson, Stanley and Metcalf (23). Cultures employing L-cell conditioned medium as a source of CSA were prepared as described by Iscove et al
(6). PREPARATION OF CULTURES
The culture method was based on the techniques of Bradley and Metcalf (1) and Pluznik and Sachs (18) as modified by Worton, McCulloch and Till (32) for use with mouse bone marrow. A 0.8% solution (w/w) of methyl cellulose10 in alpha medium provided a viscid environment suitable for cell growth and support so that discrete clones could be easily counted. Cultures were prepared as follows: Alpha medium 1.5 ml, 0.5 ml of a suspension of cells under test in alpha medium and 1.0 ml of serum were combined in a 17 x 100 mm plastic test tube. The serum fraction was composed of 70% (v/v) endotoxin-stimulated calf serum and 30% (v/v) 6Becton-Dickinson Company, Mississauga, Ontario. 7Millipore Limited, Montreal, Quebec. 82001 Tube, Falcon Plastics, Oxnard, California. 9Mousc L-cell conditioned medium supplied by Dr. E. A. McCulloch, Ontario Cancer Institute, Toronto, Ontario.
1OMethocel, Premium grade, 4000 c.p.s. viscosity. Dow Chemical.
TABLE I. A Comparison of the Hemopoietic Colony Stimulating Activity in Serum, Urine and Culture Medium
Source of CSA
L-cell conditioned medium ...... 0, 1 . Normal calf serum ........ 1, 1 . 20, 24, 25, 21 Endotoxin-stimulated calf serum.... ... Calf urine ........................... 0, 0, 0, 0 -8 x 104 nucleated marrow cells per dish
fetal calf serum" (F.C.S.). Two ml of 2% stock methyl cellulose in alpha medium were added using a 14 gauge needle and 6 ml plastic syringe. The final concentration of the resulting 5 ml suspension was: alpha medium 40% (v/v), fetal calf serum 6% (v/v), endotoxin-stimulated calf serum 14% (v/v), 2% methyl cellulose 40% (v/v). The final concentration of methyl cellulose was 0.8% (w/v). This suspension was thoroughly mixed using a vortex mixer at low speed. One ml of this mixture was then transferred to each of four 35 x 100 mm plastic culture dishes" using an 18 gauge needle and 6.0 ml plastic syringe. The cultures were spread evenly by gentle swirling. Two dishes were placed in a 100 x 15 mm Petri dish'3 along with a 35 x 100 mm dish containing distilled water. The latter provides a humid atmosphere for culturing. The dishes were incubated for eight days in an atmosphere containing 10% C02 in air and maintained at a temperature of 370 C. Colony counts were made at eight days, using an inverted microscope with a 2.5 x objective and 10 x wide angle ocular. Colonies of 20 cells were arbitrarily chosen as the minimum size to be scored as a colony.
Colonies Per Disha Calf B 1, 1 0, 0 18, 19, 14, 14 0, 0, 0, 0
0, 0 1, 1 34, 30, 36, 29
x 106 nucleated cells in 1 ml of alpha medium supplemented with 10% F.C.S. Cultures with CSA were prepared by suspending 8.0 x 10' nucleated cells in 1 ml of alpha medium supplemented with 3% F.C.S. and 7% endotoxin-stimulated calf serum. Cultures were maintained in a humidified incubator flushed with 10% C02 in air at 37°C. At appropriate intervals the cells in suspension were harvested, washed and assayed for colony formation in vitro.
ISOLATION AND STAINING OF COLONIES
Under inverted microscopic examination individual colonies were drawn into a capillary tube and smeared onto glass slides. After drying the smears were stained with Wright's stain in an automatic slide
MARROW SUSPENSION CULTURE Marrow cells were cultivated in suspension cultures in 35 x 10 mm plastic dishes both with and without CSA. Cultures without CSA were prepared by suspending 3.0 Number of Nucleated Marrow Cells per Dish
"Fetal Calf Serum, Gibco, Grand Island, New York. "1008 Petri Dish, Falcon Plastics, Oxnard, California. 31009 Ace Petri Dish, Becton-Dickinson, Clarkson, Ont-
Fig. 2. Relationship between the number of nucleated cells plated and number of colonies formed from calf F. Each point represents the mean colony counts of four dishes. Bars indicate the standard error of the mean at each point. Correlation coefficient = 0.96.
Can. J. comp. Med.
Fig. 3. Two small, compact colonies after eight days in culture. Colonies consist of granulocytes in various stages of maturation and macrophages. X59.
Fig. 4. A single colony with compact core and 1003e periphery after eight days in culture. This colony contained granulocytes in various stages of maturation and macrophages. X150.
stainer'4. Enzyme specific stains were also used to differentiate the granulocytic series (chloroacetate esterase, peroxidase) from the monocytic series (non specific esterase) after the method of Yam, Li and Crosby (33). NOMENCLATURE
The cell in an inoculum that gives rise to colonies under the given experimental conditions is usually described in operational terms as a colony-forming unit (CFU). This is usually a single cell and when colonies are formed in a splenic environment it is designated CFU-S. Where colony formation depends on the addition of CSA to a culture it is designated CFU-C.
COLONY STIMULATING ACTIVITY
In the mouse, colony formation in culture depends on the presence of molecules with colony stimulating activity. Various culture systems derive these molecules from a number of sources including culture medium conditioned by mouse -cells (32), serum from normal and leukemic mice
'4Hema-Tek slide stainer, Ames Company, Elkhart, Indiana.
Vol. 38 - April, 1974
Fig. 5. A diffuse colony photographed after eight days in culture. This colony contained granulocytes in various stages of maturation and macrophages. X59.
(22), and serum from mice treated with endotoxin (21). For the calf, we found that serum collected from a calf three hours after treatment with endotoxin was effective in promoting colony formation in culture. Figure 1 contains pooled data from seven experiments on the effect of concentration of serum from an endotoxintreated calf. In each experiment the amount of bovine serum in the cultures was kept constant by decreasing the amount of fetal calf serum as serum from an endotoxintreated animal was increased. It is evident that colony formation was not observed in the absence of serum from endotoxintreated calves and that the number of colonies increased to a maximum at 70%°! added serum and thereafter declined. Ac-
TABLE II. Colony Forming Efficiency of Calf Marrow Cells Calf G ................ H ................ I ................ J ................
Colonies/105 Nucleated Cells24 (19 - 29) 13 (10 - 16) 23 (20 - 26) 59 (51 - 67)
-Mean number of colonies estimated from the linear regression of number of colonies on number of cells plated at 105 cells. 95%o confidence limits in parenthesis
cordingly 70% serum from an endotoxintreated calf and 30% fetal serum was used as the standard serum supplement in subsequent experiments. Human urine provides a source of CSA for growth of murine marrow cells in culture (23). The suitability of calf urine as a source of CSA was tested. The results shown in Table I indicate that calf urine did not provide significant CSA. The results presented in Table I also indicate a species specificity for the CSA in the serum of the endotoxin-treated calf when compared with the effect of CSA in L-cell conditioned medium active for mouse cells. It is evident that CSA from mouse sources was ineffective in stimulating colony formation by calf marrow cells.
COLONY MORPHOLOGY Three types of colonies were observed in cultures of calf cells. These are illustrated in Figs. 3, 4 and 5. Figure 3 shows two compact colonies. Figure 4 shows a colony with a compact central core and a more diffuse peripheral mantle of cells. Figure 5 is a diffuse colony. Using Wright's stain all three colony types were shown to contain granulocytes in different stages of maturation, and macrophages. Positive reactions were detected for chloroacetate esterase, peroxidase (neutrophils and precursors) and nonspecific esterase (macrophages) in the cytoplasm of cells from each of the three colonies (33). These histochemical reactions were similar to those found for neutrophils and monocytes normally present in calf marrow and peripheral blood.
BEHAVIOUR IN SUSPENSION CULTURE Mouse cells that form colonies in culture are shown to increase in number in suspension cultures when CSA is added (12, 19).
-with CSA ---
LINEARITY OF ASSAY In a valid assay system of progenitor cells based on colony formation it is necessary that a linear relationship be demonstrated between the number of marrow cells plated and the number of colonies formed. This criterion was achieved for cultures of calf marrow. Figure 2 contains the results of a typical experiment. The straight line relating cell number to colony formation was fitted by the method of least squares using a Wang calculator. The extrapolation of the line to the origin did not differ significantly from zero (P 0.05). Table II presents data from similar experiments on four calves. The number of colonies per 105 nucleated marrow cells plated in an expression of the colony forming efficiency in this system and was determined from the lines of best fit. It is evident that considerable animal to animal variation occurred. 150