Cell Tissue Kinet. (1974) 7 , 537-548.

QUANTITATIVE IN V I T R O STUDIES ON STIMULATION

STIMULATING FACTOR OF MURINE HAEMOPOIETIC CELLS BY COLONY

GERRIT J. V A N DEN ENCH

Department of Radiobiology of the Erasmus University, Rotterdam, and Radiobiological Institute TNO, Rijswijk (Z.H.), The Netherlands

(Received4 March 1974; revision received21 May 1974)

ABSTRACT

A new standardization method for Colony Stimulating Factor (CSF) is described and criteria are introduced which enables stimulating activity to be assessed independent of absolute colony numbers. With this method CSF preparations from different sources are compared and evidence is presented that suggests an identical mechanism of action for these substances. Data are presented on the increase in colony numbers that is induced by the addition of erythrocyte lysates to the cultures. The relationship between colony forming cells that are stimulated by CSF alone and cells stimulated by the combined action of CSF and erythrocyte lysate is discussed.

I N T R O D U C T I O N

Since the introduction of an in vitro cloning technique for myeloid progenitor cells (Pluznik & Sachs, 1965; Bradley & Metcalf, 1966), much work has been done on the physicochemical characterization of a factor which is an essential requirement for the colony formation (Colony Stimulating Factor or CSF). This factor(s) has been shown to be present in most organs and tissues of rodents and primates. Many tissue extracts, culture media conditioned by various cell lines, and urines may serve as a source of stimulating activity (Stanley & Metcalf, 1969; Bradley, Stanley &Sumner, 1971a; Austin, McCulloch & Till, 1971). In those preparations only small amounts of the active substance are present and, up to now, absolute purification of the factor has not been reported. The CSF prepared from human urine, for example, is estimated to show maximal activity at concentrations as low as lo-” M but is present in urine at much lower concentrations (Stanley, 1972). CSF from various sources varies in physicochemical properties such as electrophoretic mobility and calcium phosphate binding characteristics (Bradley et al., 1971a). In fact, most mouse tissues appear to contain a specific type of CSF (Sheridan & Stanley, 1971 ; Sheridan & Metcalf, 1973). Surprisingly enough, little information is available on possible differences in the biological activities of various kinds of CSF.

Correspondence: Dr Gerrit J. van den Engh, Department of Radiobiology, The Erasmus University, Rotterdam, The Netherlands.

537

538 Gerrit J. van den Engh The cell that in uitro gives rise to a colony in the presence of CSF is a progenitor cell of the

myeloid series and is known as colony forming unit-culture (CFU-c). This cell type has been reported to differ in density (Haskill, McNeill & Moore, 1970; Worton, McCulloch & Till, 1969), velocity sedimentation rate (Worton et al., 1969), and cell cycle characteristics (Lajtha et al., 1969; Iscove, Till & McCulloch, 1970; Metcalf, 1972) from the haemopoietic stem cell. This cell type is characterized by its ability to produce colonies in the spleens of lethally irradiated mice and is therefore called the colony forming unit-spleen (CFU-s) (Till & McCulloch, 1961). In contrast to the culture systems in which the CFU-c has been shown to be different from CFU-s, Dicke and co-workers have suggested that, in marrow cultures stimulated by a feeder layer of fibroblasts, at least some of the colony formation can be attributed to proliferation of haemopoietic stem cells (Dicke, 1969; Dicke, Platenburg & Van Bekkum, 1971). Survival of CFU-s in liquid culture in the presence of feeder layers has also been reported elsewhere (McCulloch & Till, 1971). Factors other than CSF may be present and influence the growth of haemopoietic cells in these systems.

Lysates of erythrocytes contain a factor that influences the activity of CSF. Although these lysares are unable to stimulate by themselves, they induce a substantial increase in the incidence and size of colonies when added together with CSF (Bradley, Telfer & Fry, 1971 b). Addition of such lysates also prolongs the survival of stem cells in liquid culture (Testa & Lajtha, 1972).

The present paper reports on a comparison ofthe stimulating activity from different sources. In this study, the biological activity of the CSF preparations has been assessed rather than their chemico-physical properties. It appears that, with regard to this criterion, the various types of CSF (at least those studied) form a homogeneous group. A detailed description of the enhancing effect of lysates of erythrocytes is also given.

M A T E R I A L S A N D M E T H O D S

Fibroblast cultures The trunks of 10-12-day-old mouse embryos were minced and trypsinized with Puck’s

trypsin solution (0.3% trypsin, DIFCO) for 30 min. The cell pellet was resuspended in Waymouth’s medium supplemented with 5 % calf serum. 10 ml of this suspension were pipetted onto plastic Petri dishes (Falcon, 10 cm diameter). The cultures were kept in a humidified incubator at 37°C in an atmosphere containing 10% CO,. Three days after plating, fresh medium was pipetted onto the cultures. The fibroblasts were trypsinized every week and transferred to new Petri dishes. Such cell lines could be maintained for I year with- out loss of CSF producing capacity.

CSF-containing preparations (a) Fibroblast-conditioned medium. Medium from fibroblast cultures was collected

aseptically and stored at -20°C. The medium could be used directly as a source of CSF or could be purified by dialysis for 24 h against glass distilled water followed by concentration by batch chromatography on calcium phosphate gel.

(b) Mouse placenta membranes and pregnant uterus extract ( P M U E ) . Tissue extracts were prepared according to the method described by Bradley et al. (1971 a). Whole pregnant mouse uteri were collected and stored at -20°C until used. The tissues were homogenized in a Sorval mixer in 3 volumes of cold glass distilled water for 60 sec. The homogenate was centrifuged

Stimulation of murine haemopoietic cells by CSF 539 (1 5,000 g, 30 min) and ammonium sulphate was added to the supernatant until 50 % satura- tion was reached.

Following centrifugation (20,00Og, 30 min), the sediment was discarded and the supernatant was saturated with ammonium sulphate. After another centrifugation (20,OOOg, 30 min), the supernatant was discarded and the precipitate was suspended in one quarter of the original volume of cold glass distilled water. The resulting suspension was dialysed against glass distilled water for 3 days and the precipitate was removed by centrifugation. After sterilization by millipore filtration, the material could be used directly or could be purified further by batch chromatography on calcium phosphate gel. Preparations were routinely heat inactivated at 60°C for 30 min.

(c) Human urine CSF. CSF from human urine was prepared according to the method described by Stanley et al. ( I 972). Human urine was collected from volunteers and dialysed for 3 days against glass distilled water at 4°C. Following centrjfugation, the precipitate was discarded and the supernatant was purified and concentrated by adsorption on calcium phosphate gel. The eluate in one tenth of the original urine volume of 0.1 M phosphate buffer was used after sterilization by millipore filtration.

Assay for colony stimulating activity The assay used was a slight modification of the technique described by Metcalf & Moore

(1971). The activity of the dilutions of CSF containing material was assayed by mixing 0.1 ml with 1 ml nutrient agar containing 5 x lo4 nucleated bone marrow cells obtained from 8-10-week-old CBA/Rij mice. Each dilution was tested in triplicate. The nutrient agar con- sisted of 0.3 % agar (Bacto agar, DIFCO), in Dulbecco’s medium (prepared from lox stock solution, Biocult) supplemented with 20% of a mixture of equal volumes of fetal calf serum (Flow), horse serum, and 3 7; Trypticase Soy Broth (BBL). The cultures were kept at 37°C in a humidified incubator (National) and gassed with 10% CO, in air. The cell aggregates produced were counted on day 7 with an inverted microscope (Zeiss) at a 25x magnification. Small aggregates were inspected at 63x magnification. At the time of counting, a distinction was made between clusters (aggregates containing less than fifty cells) and colonies (aggre- gates containing more than fifty cells). When the size of cell aggregates is not taken into account, they are referred to as clones. In routine titrations only colonies were scored. In experiments in which cells were harvested from cultures, CSF was mixed in a 1 ml under- layer of 0.5 % agar medium and 2 x lo5 cells in 0.5 ml of liquid medium were placed on top of this.

CFU-s assajl CFU-s were determined by the spleen colony assay of Till & McCulloch (1961).

RESULTS

CSF levels and colony formation Since the absolute concentration of CSF-containing solutions is unknown, it is impossible

to compare different types of CSF on the basis of direct dose-response studies. Therefore, as a means of normalizing titrations, the cells growth induced by varying dilutions of CSF containing preparations has been plotted against the logarithm of their relative concen- trations. When plotted in this way, the shape of the resulting curve is independent of the CSF

540 Gerrit J . oan den Etigh concentration in the undiluted material, since multiplication of all dose values by a constant factor results in a shift of the curve along the dose axis. In other words, two identical substances will give parallel curves and the distance between the curves as measured along the dose axis corresponds to the ratio of their concentrations. When substances are compared which have a similar effect but have a different mechanism of action, the probability that one will find parallel curves decreases as the dose-response relationship becomes more complex. When plotted in this manner these plots reveal a very simple relationship between the colony induction and the concentration of CSF.

x Stock concenlrotion

FIG. I . Titration of CSF from a pregnant mouse uterus extract (PMUE) against 5 x lo4 mouse bone marrow cells.The graphs are drawn by hand to fit clone (2) and colony (0) numbers counted at day 7 of culture. Each point represents an average value of three plates.

Fig. 1 shows a typical dose-response curve for the growth of mouse marrow cellsinduced by varying dilutions of a CSF containing preparation from a pregnant mouse uterus extract (PMUE). Cell aggregates werecounted after 7 days ofculture. Adistinction wasmade between clones (total cell aggregates irrespective of cell number) and colonies (aggregates containing more than fifty cells). Above a threshold level, both the clone and the colony curve increases linearly with the logarithm of the CSF concentration. The curves form a plateau when a saturation concentration is reached. Depending on general culture conditions such as quality of serum batch and stability of temperature and humidity over the culture period, the values for the plateaus may vary between 300 and 500 clones and between 70 and 140 colonies/105 cells plated. The saturation concentration for the induction of clones is approxi- mately a quarter of that for colony induction. The concentration at which the linear part of the colony curve reaches the plateau is approximately 8 times the concentration at which the first colony appears. These observations most probably coincide with a certain CSF con- centration and therefore they can be used as reference points for an arbitrary unit

Stimulation of murine haemopoietic cells by CSF 54 I system that is proportional to the CSFconcentration. The value of 1 unit of CSF/mlis assigned to the initial point of clone formation. This is the lowest concentration at which cells are stimulated in the presence of CSF. Colony formation is first observed at 5 units/ml. Cultures are maximally stimulated for clone and colony formation at approximately 10 and 40 CSF unitslml, respectively. Note that no reference is made to absolute colony numbers in this unit system.

The relationship described appears not to be dependent on the kind of CSF used. CSF produced by embryonic fibroblasts in culture and highly purified CSF isolated from human urine show similar dose-response relationships (Figs. 2 and 3). Since the shapes of these curves represent a complex distribution of CSF sensitivity over the marrow cell population, their similarity strongly suggests an identical biological activity of these preparations.

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FIG. 2. Titration of CSF from fibroblast-conditioned medium against 5 x lo4 mouse bone marrow cells. The graphs are drawn by hand to fit clone (0) and colony (0) numbers countedat day 7 of culture. Each point represents an average value of three plates.

If this is the case, the number of colonies induced by mixtures of different types of CSF should be predictable by simple summation of the individual concentrations rather than by the summation of the individual effects. Fig. 4 shows the results of an experiment in which mixtures of human urine CSF and PMUE were assayed for their stirnulatory activity. Both preparations are expressed in the CSF unit system on the basis of their titration curves. The sum of urine CSF and PMUE doses is plotted on the lower axis of the graph. The drawn line represents the result that is obtained when only one of the preparations is used. This curve fits the measured points indicating that the mixtures of CSF act as if they consist of only one kind of CSF.

These titrations also give an indication of the purity of the material used. Although prepared in an identical fashion different preparations of PMUE were found to contain

542 Gerrit J. van den Engh

i Stock concentr.31ion

FIG. 3. Titration of CSF from human urine against 5 x lo4 mouse bone marrow cells. Colonies are counted at day 7 of culture. Each point represents an average value of three plates.

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8 I0 20 40 8 0

CSF u r l n e + CSF embryo ex l roc t

Frti. 4. Colony stimulating activity of mixtures of human urine CSF and PMUE. The strength of the individual preparations is expressed in an arbitrary unit system. The dose axis represents the sum of the two doses. m, Mixtures that contain 4 units of urine CSF + varying amounts of PMUE; 0, mixtures which contain 8 units of urine CSF; -, the curve that is obtained when either pure urine CSF or PMUE is used.

Stimulation of murine haemopoietic cells by CSF 543 varying amounts of impurities. The left panel of Fig. 5 shows a PMUE preparation which inhibited colony formation at high doses. The right panel shows a preparation that induced colony numbers above normal levels and the colony number did not seem to reach a plateau (Fig. 1). Both preparations yielded a normal titration curve after additional heat treatment and dialysis. This suggests that the abnormal shapes of the curves were caused by some interfering factors that have been selectively removed by the treatment.

150

b n //

x Stock concentration

FIG. 5. Titration of two PMUE preparations before (C) and after (0) additional heat treatment and dialysis. The preparation of panel A contained impurities with an inhibiting effect on colony formation. The graph of panel B shows the presence of an enhancing factor. By heating the preparations to 60°C for 30min followed by dialysis against glass distilled water for 24 hr, the contaminants were selectively removed.

Efiects of erythrocyte lysates 011 colony formation Addition of a lysate of rat erythrocytes to cultures stimulated by CSF leads to a dramatic

increase in colony numbers (Bradleyet al., 1971 b). Theincrease has been reported to belinear with the amount of lysate added (Bradley et al., 1972). Lysates also enhance the survival of stem cells in culture (Testa & Lajtha, 1972). The additional CFU-c which are enhanced by these lysates could represent entirely different cells from the CFU-c population that is normally monitored. If so, this population can be expected to have a different CSFrequirement which would be reflected by a change in the shape of the titration curve. A titration of PMUE in the presence of a fixed concentration of erythrocyte lysate is shown in Fig. 6. The same factor of increase as compared to the standard titration curve is found at all CSF levels. No cell growth occurs with only the lysate present. The curve levels off at the saturation concentration. When the effect of the different CSF concentrations is expressed as a percentage of the effect that can be maximally induced, the two curves are identical. This indicates that the additional cell population that is expressed as colonies under the influence of the lysate factor, has the same CSF requirements as the cells that form colonies without the lysate. Therefore, if these cells represent a different cell population, these cells have transformed into normal CSF-dependent CFU-c before giving rise to colonies.

544

300

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Cerrit J . van den Engh

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x Stock concentration

FIG. 6. Titration of PMUE with and without the addition of an erythrocyte lysate. Various amounts of PMUE are assayed in the presence of a constant amount of the lysate.

CSF lei~els and CFU-c proliferation

Most authors reporting observations on cultures of CFU-c confine themselves to stating that the CSF levels used allowed optimal cell growth. This only indicates that the plateau of colony formation is reached. CSF, however, has other effects on the marrow cells besides the induction of colonies. An increase in colony size can still be observed at doses well above the saturation concentration (Metcalf & Moore, 1971). As Fig. 7 shows, very high levels of CSF also promote self-renewal of the CFU-c. Cells were recovered from cultures stimulated by PMUE at saturation concentration and at 4 times the saturation concentration the numbers of CFU-c present were determined on different days of culture. Although both concentrations are optimal with regard to colony formation, the higher CSF level appears to be more favourable for CFU-c multiplication than is the lower.

CSF lecels and stem cell proliferation

Stem cells can be recovered for up to 1 week of culture from bone marrow cultures that are stimulated by a feeder layer of fibroblasts. This has not previously been reported with medium conditioned by fibroblasts. Since conditioned medium is maximally present as 20 of the culture medium, the CSF levels can be expected to be much higher when fibroblasts are present. To determine whether prolonged stem cell survival is an effect of high doses of CSF or whether the presence of fibroblasts is essential, the stem cell recovery from cultures with 4x saturation concentration of PMUE was determined on different days. Fig. 8 shows that even at these high CSF levels, only few stem cells could be recovered after 2 days of culture while in the cultures stimulated by fibroblasts, stem cells could be recovered until day 6.

Stimulation of murine haemopoietic cclls bey CSF 545

I60

140

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" 100

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g 80 aJ m

c +

p 6 C c?

4c

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FIG. 7. Recovery of CFU-c at various times of culture. CSF is mixed in 1 ml of medium and 05% agar to a final concentration of I time (0) and 4 times (0) the saturation concentration. On top of this, 2 x lo5 marrow cells in 0.5 ml of liquid medium are put. Each point represents three plates.

DISCUSSION

It has been demonstrated in Results that the quality and the quantity of the source of stimulating activity may influence the in uitro colony formation in a number of ways. Preparations which contain varying amounts of CSF and impurities may have different effects on colony number, colony size and CFU-c replication. When statingcultureconditions, it is therefore not sufficient to describe the stimulating activity of the stimulus used as optimal with regard to the maximal colony number.

Stanley et al. (1972) described a method to estimate CSF levels. This method is based on curve-fitting by computer of the asymmetrical sygmoid curve which is obtained when CSF concentration is arithmetically plotted against colony formation. This technique seems too complicated for occasional CSF determinations. The simple graphical method presented in this paper is more suitable as a generalreference method. It has the advantage that, in addition to an estimation of the CSF content, it also yields an indication of the purity of the material used. Another advantage of the proposed unit system is that it is not dependent on the absolute colony number. The strength of a preparation is estimated by determining the points of intersection of the extrapolated linear part of the titration curve with the dose axis and the plateau. Since the ratio of the concentrations with which these points correspond

546 Gcrrit J . van den Eiigh

\

\

b - - b - - - _ _ I I I 2 3 4 5 6

Doys of culture

FIG. 8. Recovery of CFU-s at various times of culture. The same technical set up was used as in Fig. 7 . 0 , Cultures stimulated by CSF at 4 times saturation concentration; 0, cultures stimulated by embryonic fibroblasts covered by the agar layer.

has been found to be the same for all CSF preparations studied in this paper, the height of the plateau (e.g. the colony number that can be maximally induced) does not influence the outcome of such a concentration estimation. I t can be expected, therefore, that concen- tration estimates vary little with culture conditions. Furthermore, this unit system might have some biological significance since 1 unit corresponds with the onset of cluster formation, as observed at day 7 of culture.

It has been shown that semi-purified CSF from different sources stimulates mouse marrow cells in a comparable manner. The study of mixtures of different kinds of CSF determines their identity in biological activity. In many papers, the biological identity of CSF is taken to be implicit, but i t has never been proved. The problem remains as t o why human marrow cells are not stimulated by human urine CSF. Considering the identity of human and murine CSF in biological activity towards mouse cells this discrepancy must be due t o differences in the physiology of murine and human marrow cells. The human cells might need a cofactor in order to form colonies. Price, McCulloch & Till (1973) reported the existence of a unique small molecular factor that has a stimulatory effect on human marrow. I t seems possible that the presence of such a small molecular factor is essential for stimulation of human cells by human urine CSF.

It is still a matter of debate as t o what cells give rise to the additional colonies and clusters when erythrocyte lysatc is added. One possible cause of the phenomenon could be that lysate improves the efficiency at which CFU-c are detected. Metcalf (1970) demonstrated that the number of colonies increases with time. The CFU-c were still being recruited t o form colonies at day 7. Thus, some of the clones generated by cells with colony forming potential are still in the cluster stage at this time. Since the number of colonies found at a given time depends on both the number of proliferating CFU-c and their division rate, erythrocyte lysates could increase the number of CFU-c expressed as colonies by altering either of these parameters. If this is the case the additional colonies represent CFU-c from the same cell population

Stimulation of murine haemopoietic cells by CSF 547

that is expressed in cultures stimulated with CSF alone. Another possibility is that erythrocyte lysates allow the proliferation of a new population of CFU-c. It has been shown that the additional CFU-c require CSF in the same manner as CFU-c that form colonies without lysate. Thus, if the enhanced CFU-c represent a new cell population this cell population becomes sensitive to CSF under the influence of the lysate. The additional CFU-c then could represent either a precursor of the CFU-c that responds to CSF or a more mature cell type that regains its capacity to respond to CSF. There are no experimental data that favour any particular one of these possibilities.

A C K N O W L E D G M E N T S

The author is greatly indebted to Professor D. W. van Bekkum for introducing him into this area of research and for constructive advice during the course of these studies. Dr B. Lowenberg, Dr K. A. Dicke and Dr N. T. Williams have contributed many valuable thoughts during lengthy discussions on scientific and other subjects. Miss Pia van Oosterom is acknowledged for expert technical assistance. Many thanks to the collaborators of the Radiobiological Institute TNO for their stimulating activities.

R E F E R E N C E S

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ISCOVE, N.N., TILL, J.E. & MCCULLOCH, E.A. (1970) The proliferative states of mouse granulopoietic progenitor cells. Proc. SOC. exp. Biol. Med. 134, 33.

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PLUZNIK, D. H. & SACHS, L. (1965) The cloning of normal ‘mast’ cells in tissue culture. Comp. Physiol. 66,319. PRICE, G.B., MCCULLOCH, E.A. &TILL, J.E. (1973) A new human low molecular weight granulocyte colony

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548 Gerrit J. van den Engh SHERIDAN, J.W. & METCALF, D. (1973) A low molecular weight factor in lung-conditioned medium stimulating

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poietic Cells: Proceedings of a workshop symposium on in vitro culture of hemopoietic cells, p. 26. STANLEY, E.R. & METCALF, D. (1969) Partial purification and some properties of the factor in normal and

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TESTA, N.G. & LAJTHA, L.G. (1972) Some factors affecting survivalof CFU and CFU-c inculture. In: In Vitro Culture of Hemopoieric Cells: Proceedings ofa workshop symposium on in vitro culture of hemopoietic cells, p. 102.

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