IN VITRO Volume14, No. 6, 1978 Allrightsreserved9
STUDIES ON IN VITRO COLONY FORMATION BY MOUSE BONE-MARROW CELLS USING DIFFERENT SOURCES OF COLONY-STIMULATING FACTOR' J A M E S L. SHELLHAAS, 2 M E L V I N S. R H E I N S , 3 ANDJOYCE A. F I L P P I
Department of Microbiology, The Ohio State University, Columbus, Ohio 43210 SUMMARY Conditioned media of a primary mouse embryo and a mouse cell line were compared as sources of colony-stimulating factor. The incorporation of embryo cell conditioned medium into semisolid cultures of mouse bone-marrow cells induced the formation of a larger number of in vitro colonies than did the addition of equal volumes of LM cell conditioned medium. This finding did not appear to be the result of quantitative differences in the levels of C.S.F. between the sources since concentration of the LM cell conditioned preparation did not enhance effectively the number of colonies produced. Both the colonial morphology and the cellular components of the developing colonies were found to differ in accordance with the C.S.F. source employed for stimulation. Colonies that developed under the influence of embryo cell conditioned medium were typically larger and more disperse than were those produced in cultures stimulated with the LM cell conditioned source. The latter colonies were smaller, more compact and contained fewer cells. Differences also were noted in the relative proportion of granulocytes to mononuclear cells comprising the colonies. Those colonies stimulated with LM cell conditioned medium rapidly underwent a transition from primarily a granulocytic composition to one comprised principally of mononuclear cells. Cultures stimulated with embryo cell conditioned medium contained a greater number of granulocytic colonies which persisted for a protracted period during cultivation. The addition of 2-mercaptoethanol to cultures stimulated with embryo cell conditioned medium increased the number of colonies produced. Such synergy did not occur in cultures stimulated with the LM cell conditioned medium.
Key words: colony-stimulating factor; in vitro colonies; embryo cell conditioned; LM cell conditioned. INTRODUCTION
stimulated to undergo proliferation by the addition to the culture of a glycoprotein that has been assigned the functional title of colony stimulating factor (CSF). This activity is associated with a Variety of sources including conditioned medium from both established (1) and primary embryonic mouse cell monolayers (2), leukemic serum of both mouse ~3,4) and human t5) origin, and human and mouse urine concentrates (6-8). In this study, we compared LM cell conditioned medium obtained from an established LM 1Supported by United States Public Health Service cell line with that obtained from a primary embryo cell explant with regard to their utility as Research Grant CA 13752. 2Present address: Department of Microbiology and sources of CSF for mouse CFU-c. In particular, Immunology, University of Louisville, Louisville, Ken- these investigations examined the influence of tucky 40232. 3To whom requests for reprints addressed at Depart- each of the stimulator sources on the cellular comment of Microbiology, 484 W. 12th Avenue, The Ohio position of the colonies, total number of colonies State University, Columbus, Ohio 43210. produced, the rate of progressive transformation 550 The in vitro culture of hematopoietic cell populations in the presence of a suitable stimulator results in the clonal derivation of hematopoietic cell colonies. Contingent upon the nature of the stimulator, it is possible to culture erythrocytic, megakaryocytic, lymphoid or myeloid/mononuclear cell colonies. Requisite to the development of mouse granulocytic/mononuclear cell colonies, the precursor cell, CFU-c, must he
COLONY-STIMULATING FACTORS of the cells comprising the colonies from predominantly a granulocytic to a mononuclear cell composition, and differences in the developing colonies to be modified by the addition of 2-mercaptoethanol, an assumed inducer of cell division. MATERIALS AND METHODS
Mice. Two- to three-month-old female C 3 H / H e J mice purchased from Jackson Laboratories iBar Harbor, Maine) were utilized throughout this study. Animals were housed in metal cages in the Animal Facility of the College of Biological Sciences, The Ohio State University, and were provided water and Purina mouse pellets ad libitum. Marrow cell collection. Mice were sacrificed by cervical disarticulation, three to four per experiment; and bone marrow was harvested by flushing each femur with 1.0 ml bone-marrow collection fluid, prepared according to the formulation of Metcalf and Moore (9j. Single cell suspensions were prepared by gentle trituration, and cell viability was assessed by trypan blue dye exclusion. Thin layer agar technique. The semisolid medium for the establishment of bone-marrow cell cultures required for the assay of colony-stimulating factorIs) (CSF) was prepared by the admixture of equal volumes of 0.6% agar (Difco) and a double-strength highly enriched tissue culture medium prepared as described by Metcalf and Moore (9). Bone-marrow cells were added to the medium to a final concentration of 5 x 104 cells per ml, and 1-ml aliquots were plated into 35- by 10-mm plastic tissue-culture dishes (Falcon). Following solidification of the gel, aliquots of the CSF test sources ranging from 0.02 to 0.6 ml were added to individual culture dishes. In some experiments, 1 luM to 100 /~M concentrations of 2mercaptoethanol (2-ME) also were simultaneously incorporated into the semisolid culture with the CSF test source. Marrow cultures were incubated in a 37 ~ C humidified incubator with an atmosphere of 10% CO2. Total colony counts were performed after 7 days incubation, utilizing a stercomicroscope with 25 diameters total magnification. All tests were performed in triplicate and the means determined. The results were examined for significance by subjecting the data to statistical analysis employing Student's test. Sources of CSF. The two sources of CSF examined were the supernatant fluids decanted from cell culture monolayers of Ca) the L M cell line, a derivative of the 929 clone of L cells, and (b) pri-
551
mary monolayer cell cultures of C3H embryo cells. Primary embryo cell cultures were prepared by mincing, with iris scissors and forceps, 14- to 17-day mouse embryos into 1-mm 3 pieces of tissue. The tissue was transferred to a fluted trypsinization flask and washed with 30 ml Hanks' balanced salt solution (HBSS) to remove erythrocytes. After washing, the HBSS was decanted and 50 ml warm t37 ~ C) 0.25% EDTA-trypsin (GIBCO) was added to the flask and the tissue trypsinized for 20 min. The cell-rich supernatant fluid was filtered through sterile cheesecloth into 5 ml ice-cold fetal bovine serum. Following a second trypsinization, the cells were washed and plated in 75-cm 2 culture flasks (Falcon) at a concentration of 5 x l0 s cells per ml in minimum essential medium (MEM~ plus 5% fetal bovine serum. Both the LM cell line and the primary embryo cultures were maintained at 37 ~ C for 7 days in a humidified atmosphere of 5% CO2. At the termination of incubation, the supernatant fluids were decanted and centrifuged at 100 x g for 10 min. to remove any cells that might have detached from the monolayer. The fluids were sterilized by positive pressure filtration and stored at - 2 0 ~ C in 10-ml aliquots. Cellular morphology. The cellular composition of the bone-marrow colonies cultured in vitro was determined by "picking" individual colonies from the semisolid gel with a Drummond Wiretrol capillary pipette (Drummond Scientific Co.). Single colonies were picked from cultures daily. Care was exercised to isolate only those cellular aggregates that conformed to the minimum colony size requirements of 18 to 20 cells. Aggregates of suitable size for examination were observed initially after 2 days incubation. Individual colonies were deposited on alcohol-cleaned microscope slides, and a single drop of 0.6% orcein (Matheson, Coleman and BelB in a 60% acetic acid was added. An alcohol-cleaned cover slip was gently lowered onto the colony and the edges sealed to prevent drying during observation. Cells were examined and classified as to their granulocytic or mononuclear nature according to the criteria described by Metcalf, Bradley and Robinson ~10~. RESULTS Because the active principle contained within CSF has been obtained from a variety of sources, it was of interest to determine if differences existed with regard to the stimulation pattern produced by the addition of CSF to mouse bonemarrow cells.
552
SHELLHAAS, RHEINS, AND FILPPI
Colonial morphology. The first effect attributed to the different stimulatory sources was the morphological appearance of the developing colonies. The majority of the colonies produced in cultures stimulated by the addition of embryo cell conditioned medium were typically 0.4 to 0.6 mm in diameter and were characterized by large numbers of cells arranged in a relatively dispersed fashion within the colony proper. There was no
conspicuous center to the colony (Fig. laL Moreover, because the leading or advancing edge of the colony consisted of fewer cells than did the remainder of the cellular aggregate, the colony assumed a "fringe" or "veil" appearance. Whereas the morphology of each of the colonies was relatively independent of the length of incubation, the colony size generally increased during the test period.
FIG. 1. a, Typical colonial morphology of culture stimulated with embryo cell conditioned medium; b, typical colonial morphology of culture stimulated with LM cell conditioned medium, x50.
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FIG. 2. Dose-response relationship of bone marrow cultures stimulated with LM cell or embryo cell conditioned medium. The majority of the colonies produced following stimulation with L M cell conditioned medium exhibited the morphology depicted in Fig. l b . These colonies were smaller, ranging from 0.2 to 0.4 mm in diameter, and were comprised of fewer cells than were those observed in cultures stimulated with embryo cell conditioned medium. The cellular elements of each colony appeared concentrated into a small central area with no obvious "veil" or "fringe." Dose-response relationship. Dose-response studies of both the stimulation sources resulted in basic sigmoid-shaped curves, a feature considered characteristic of all colony stimulators (11). Quantities of embryo cell conditioned medium less than 0.4 ml failed to stimulate optimal colony formation, whereas additional volumes of stimulator failed to increase the number of colonies produced. Representative peak values were 38 to 40 colonies per 5 x 104 bone-marrow cells IFig. 2). The dose-response relationship for the L M cell conditioned medium, while essentially similar, differed in some respects from the curve for the embryo cell conditioned medium. Conditioned medium from L M cells stimulated the production of only 23 to 25 colonies per 5 x 104 bone-marrow cells. Quantities of this stimulator source in excess of 0.3 ml did not increase the number of resultant colonies. Cellular composition of colonies. The relative proportions of granulocytic to mononuclear cells were compared in the developing colonies of cultures stimulated with either the L M cell or the embryo cell conditioned medium (Fig. 3). In cell cultures stimulated with optimal concentrations of
L M cell conditioned medium, approximately 75% of the colonies consisted of granulocytic cells. This value rapidly declined to 40% on the 3rd day, and by the 5th day of incubation, the colonies consisted totally of cells of a mononuclear morphology. This pattern of granulocytic to mononuclear cell transformation was in contrast to that observed for marrow cell cultures stimulated with optimal concentrations of embryo cell conditioned medium. A greater percentage of these colonies consisted principally of granulocytic cells than did comparably aged parallel cultures stimulated with the L M cell conditioned medium. Further, the observed relative increase in the number of granulocytic colonies persisted longer during the incubation. Only alter the 5th day of culture did the percentage of granulocytic colonies fall below 80~ (Fig. 3). After this, the rate of the decline of granulocytic colonies roughly paralleled that observed for colonies stimulated with LM cell conditioned medium. The observed change in the pattern of cell proportionality could have been the result of a CSF concentration-dependent transformation rate; i.e. as CSF was utilized by the target cells, or the culture medium became depleted of active stimulator, an alteration in the colonial cell character was manifested. However, 5- and 10-fold concentration of the L M cell conditioned medium did not enhance colony formation. The only influence noted was a shift in the dose-response curve to the left in comparison to that observed in experiments that utilized the unconcentrated preparations. Thus it appeared that qualitative differences between the stimulating factors from the LM cell ioo 90
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FIG. 3. Percentage of granulocytic colonies observed at various periods in cultures stimulated with LM cell or embryo cell conditioned medium.
554
SHELLHAAS, RHEINS, AND FILPPI
conditioned medium and the embryo cell conditioned medium were responsible for the observed differences in colonial morphology and doseresponse characteristics. Costimulation o/cultures. Because the addition of an optimal concentration of each conditioned medium to marrow cell cultures resulted in different maximum levels of colony formation, the possibility of the existence of subpopulations of CFU-c was suggested. To investigate this possibility, bone-marrow cell cultures were stimulated with optimal concentrations of both CSF sources. The results of such costimulation experiments did not indicate an increase in colony formation. Rather the total number of colonies in costimulated cultures was similar to that observed in cultures stimulated with embryo cell conditioned medium. The addition of 2-ME alone to marrow cultures had no stimulatory effect on colony formation. However, differences in the level of colony formation were observed when 2-ME was added to marrow cell cultures containing embryo cell conditioned medium. The incorporation of 50/aM of 2-
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FIo. 4. Effect of costimulation with both embryo cell conditioned medium ~E. C. M.t and LM cell conditioned medium (LM C.M.} or 2-mercaptoethanol (2ME) and a single stimulator source [either embryo cell conditioned medium (E. C. M. ) or LM cell conditioned medium i LM C.M. i] on colony formation.
M E into cultures containing embryo cell conditioned medium significantly increased the number of resultant colonies. Such synergy was not observed in parallel cultures that were stimulated with LM cell conditioned medium. Incorporation of 2-ME concentrations of 500/~M or greater were toxic to the cells and inhibited even low levels of normally observed sporadic colony formation. DISCUSSION In the present study it was observed that two different mouse sources of CSF varied in their capabilities to stimulate mouse progenitor cells to undergo hematopoietic cell colony formation. Differences in the number of colonies and the morphology of the colonies produced were noted. Variations also were observed in the morphological nature of the cells comprising the colonies or aggregates. The smaller size and the earlier appearance of changes in the cellular character of the colonies stimulated with L M cell conditioned medium appeared not to be a function of the concentration of the active principle in this source of stimulator. Thus qualitative, rather than quantitative, factors in the two stimulators were more likely responsible for the noted differential proliferation patterns. Stimulation with the LM cell conditioned medium also initiated earlier changes in the cellular components of the colonies as evidenced by the more rapid appearance of colonies comprised of mononuclear cells when compared with marrow cell cultures stimulated with embryo cell conditioned medium, This observation may account for the smaller colony size that is characteristic of cultures stimulated with LM cell conditioned medium if it is assumed that the period of monocyte replication is more protracted than is the proliferation associated with cells of the more immature granulopoietic system. Insight into the nature of the differences between the two stimulators might be gained by considering the CSF sources. The LM cell fibroblast is an established spontaneously transformed cell line of C3H mouse origin. It has been passaged an inestimable number of times and may have undergone mutations that were reflected by alterations in metabolism from that exhibited by the original ceils. Embryo cell monolayers consist of cells from a large number of embryonic tissues. As such, each cell type would contribute various and perhaps peculiar byproducts of cellular metabolism. Additionally, in the fetus, one might anticipate a greater production of humoral molecules associated with hematopoietic stimulation and
COLONY-STIMULATING FACTORS regulation. Therefore the two sources examined, although prepared in a similar fashion, may vary as to the metabolic byproducts contained in culture supernates. Further, the possibility exists that additional factors were present. Whereas the active CSF in the two sources might have been the same, other components in the LM cell conditioned medium may have adversely affected colony formation. The reducing agent 2-ME has been shown to be influential in a variety of in vitro immunological reactions such as the response to sheep erythrocytes (13), T cell reactivity to phytohemagglutinin t14), and the mixed leukocyte reaction (15). The agent also has been employed to culture B lymphocyte colonies from lymphoid cell suspensions (16). In the present study, the enhanced colony formation in marrow cell cultures stimulated with embryo cell conditioned medium following the incorporation of 2-ME was of interest. Whereas the mechanism responsible for this observation is unknown, it may be that the reducing activity associated with this molecule split the macromolecular CSF moiety into a more stimulatory molecule, or into a number of subunits also possessing activity. Alternatively, 2-ME may serve as a scavenger molecule, removing potentially toxic free-radicals from the immediate environment. Rather than exerting an influence on the embryo cell stimulating factor itself, 2-ME may specifically act on the CSF receptor of the CFU-c, or some combination of the above. This of course assumes that the receptor sites on the CFU-c for the two stimulatory factors are different, or vary in their avidity for binding stimulator molecules. These differences could he manifested in signals of varying intensity which then could influence the rate of cell proliferation. The data also support the possibility that CSF influences differentiation of the target bonemarrow cells rather than simply sustaining the viability of the ceils until inherent genetic factors signal proliferation and maturation. This would raise a question as to whether the CSF in LM cell conditioned medium promotes a "switch" in colonial character from that of a granulocytic to that of a monnuclear cell type, or it is the consequence of unidentified modifying substances that are ineffective in completing the differentiation of the immature granulocytic cells. It may be that cells in a given population exhibit differences as to the incidence or spatial relationships of the receptor sites on their surfaces pertinent to the CSF. Whereas it is generally agreed that the primary
555
stimulator of mouse bone-marrow cells is a glyco~ protein, conformational differences in the glycoprotein moieties are also to be considered. These may be reflected in the suitability of a particular material to serve as a stimulator. Additional substances that direct or augment the primary stimulator may affect the availability of receptor sites to the principal CSF "trigger." REFERENCES 1. Austin, P. E., E. A. McCulloch, and J. E. Till. 1971. Characterization of the factor in L-cell conditioned medium capable of stimulating colony formation by mouse marrow cells in culture. J. Cell. Physiol. 77: 121-134. 2. Pluznik, D. H., and L. Sachs. 1965. The cloning of normal "mast" cells in tissue culture. J. Cell. Comp. Physiol. 66: 31%324. 3. Metcalf, D., and R. Foster. 1967. Behavior on transfer of serum stimulated bone marrow colonies. Proc. Soc. Exp. Biol. Med. 126: 758-762. 4. Robinson, W., D. Metcalf, and T. R. Bradley. 1967. Stimulation by normal and leukemic mouse sera of colony formation in vitro by mouse bone marrow cells. J. Cell. Physiol. 69: 83-92. 5. Foster, R., and D. Metcal~. 1968. Bone marrow colony stimulating activity in human sera. Results of two independent surveys in Buffalo and Melbourne. Br. J. Haematol. 15: 147-159. 6. Metcalf, D., and E. R. Stanley. 1969. Quantitative studies on the stimulation of mouse bone marrow colony growth in vitro by normal human urine. Aus. J. Exp. Biol. Med. Sci. 47: 453. 7. Robinson, W. A., E. R. Stanley, and D. Metcalf. 1969. Stimulation of bone marrow colony growth in vitro by human urine. Blood 33: 396-399. 8. Chan, S. H. 1970. Studies on colony-stimulating factor (C.S.F.): Role of the kidney in clearing serum C.S.F. Proc. Soc. Exp. Biol. Med. 134: 733-737. 9. Metcalf, D., and M. A. S. Moore. 1971. Techniques in experimental haematology and their limitations. In: A. Neuberger, and E. L. Tatum {Eds.), Hemopoietlc Cells. Vol. 24. North Holland Publishing Co., Amsterdam, p. 35. 10. Metcalf, D., T. R. Bradley, and W. Robinson. 1967. Analysis of colonies developing in vitro from mouse bone marrow cells stimulated by kidney feeder layers or leukemic serum. J. Cell. Physiol. 69: 93-108. 11. Metcalf, D., and M. A. S. Moore. 1971. Techniques in experimental haematology and their limitations. In: A. Neuberger, and E. L. Tatum {Eds.), Hemopoietic Cells. Vol. 24. North Holland Publishing Co., Amsterdam, pp. 37-39. 12. Metcalf, D. 1974. The sermn factor stimulating colony formation in vitro from mouse bone marrow cells stimulated by kidney feeder layers or leukemic serum. J. Cell. Physiol. 69: 93-108. 13. Chen, C., and J. G. Hirsch. 1972. Restoration of antibody-forming capacity in cultures of nonadherent spleen cells by mercaptoethanol. Science 176: 60-61.
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14. Fangcr, M. W., D. A. Hart, J. V. Wells, and A. Nisonoff. 1970. Enhancement by reducing agents of the transformation of human and rabbit peripheral lymphocytes. J. Immunol. 105: 1043-1045. 15. Heber-Katz, E., and R. E. Click. 1972. Immune response in vitro. V. Role of mercaptoethanol in
the mixed-leucocyte reaction. Cell. Immunol. 5: 410-418. 16. Metcalf, D., N . L . Warner, G . J . V . Nossal, J. F. A. P. Miller, K. Shortman, and E. Rabellino. 1975. Growth of B lymphocyte colonies in vitro from mouse lymphoid organs. Nature 255: 63O-632.
STUDIES ON IN VITRO COLONY FORMATION BY MOUSE BONE-MARROW CELLS USING DIFFERENT SOURCES OF COLONY-STIMULATING FACTOR' J A M E S L. SHELLHAAS, 2 M E L V I N S. R H E I N S , 3 ANDJOYCE A. F I L P P I
Department of Microbiology, The Ohio State University, Columbus, Ohio 43210 SUMMARY Conditioned media of a primary mouse embryo and a mouse cell line were compared as sources of colony-stimulating factor. The incorporation of embryo cell conditioned medium into semisolid cultures of mouse bone-marrow cells induced the formation of a larger number of in vitro colonies than did the addition of equal volumes of LM cell conditioned medium. This finding did not appear to be the result of quantitative differences in the levels of C.S.F. between the sources since concentration of the LM cell conditioned preparation did not enhance effectively the number of colonies produced. Both the colonial morphology and the cellular components of the developing colonies were found to differ in accordance with the C.S.F. source employed for stimulation. Colonies that developed under the influence of embryo cell conditioned medium were typically larger and more disperse than were those produced in cultures stimulated with the LM cell conditioned source. The latter colonies were smaller, more compact and contained fewer cells. Differences also were noted in the relative proportion of granulocytes to mononuclear cells comprising the colonies. Those colonies stimulated with LM cell conditioned medium rapidly underwent a transition from primarily a granulocytic composition to one comprised principally of mononuclear cells. Cultures stimulated with embryo cell conditioned medium contained a greater number of granulocytic colonies which persisted for a protracted period during cultivation. The addition of 2-mercaptoethanol to cultures stimulated with embryo cell conditioned medium increased the number of colonies produced. Such synergy did not occur in cultures stimulated with the LM cell conditioned medium.
Key words: colony-stimulating factor; in vitro colonies; embryo cell conditioned; LM cell conditioned. INTRODUCTION
stimulated to undergo proliferation by the addition to the culture of a glycoprotein that has been assigned the functional title of colony stimulating factor (CSF). This activity is associated with a Variety of sources including conditioned medium from both established (1) and primary embryonic mouse cell monolayers (2), leukemic serum of both mouse ~3,4) and human t5) origin, and human and mouse urine concentrates (6-8). In this study, we compared LM cell conditioned medium obtained from an established LM 1Supported by United States Public Health Service cell line with that obtained from a primary embryo cell explant with regard to their utility as Research Grant CA 13752. 2Present address: Department of Microbiology and sources of CSF for mouse CFU-c. In particular, Immunology, University of Louisville, Louisville, Ken- these investigations examined the influence of tucky 40232. 3To whom requests for reprints addressed at Depart- each of the stimulator sources on the cellular comment of Microbiology, 484 W. 12th Avenue, The Ohio position of the colonies, total number of colonies State University, Columbus, Ohio 43210. produced, the rate of progressive transformation 550 The in vitro culture of hematopoietic cell populations in the presence of a suitable stimulator results in the clonal derivation of hematopoietic cell colonies. Contingent upon the nature of the stimulator, it is possible to culture erythrocytic, megakaryocytic, lymphoid or myeloid/mononuclear cell colonies. Requisite to the development of mouse granulocytic/mononuclear cell colonies, the precursor cell, CFU-c, must he
COLONY-STIMULATING FACTORS of the cells comprising the colonies from predominantly a granulocytic to a mononuclear cell composition, and differences in the developing colonies to be modified by the addition of 2-mercaptoethanol, an assumed inducer of cell division. MATERIALS AND METHODS
Mice. Two- to three-month-old female C 3 H / H e J mice purchased from Jackson Laboratories iBar Harbor, Maine) were utilized throughout this study. Animals were housed in metal cages in the Animal Facility of the College of Biological Sciences, The Ohio State University, and were provided water and Purina mouse pellets ad libitum. Marrow cell collection. Mice were sacrificed by cervical disarticulation, three to four per experiment; and bone marrow was harvested by flushing each femur with 1.0 ml bone-marrow collection fluid, prepared according to the formulation of Metcalf and Moore (9j. Single cell suspensions were prepared by gentle trituration, and cell viability was assessed by trypan blue dye exclusion. Thin layer agar technique. The semisolid medium for the establishment of bone-marrow cell cultures required for the assay of colony-stimulating factorIs) (CSF) was prepared by the admixture of equal volumes of 0.6% agar (Difco) and a double-strength highly enriched tissue culture medium prepared as described by Metcalf and Moore (9). Bone-marrow cells were added to the medium to a final concentration of 5 x 104 cells per ml, and 1-ml aliquots were plated into 35- by 10-mm plastic tissue-culture dishes (Falcon). Following solidification of the gel, aliquots of the CSF test sources ranging from 0.02 to 0.6 ml were added to individual culture dishes. In some experiments, 1 luM to 100 /~M concentrations of 2mercaptoethanol (2-ME) also were simultaneously incorporated into the semisolid culture with the CSF test source. Marrow cultures were incubated in a 37 ~ C humidified incubator with an atmosphere of 10% CO2. Total colony counts were performed after 7 days incubation, utilizing a stercomicroscope with 25 diameters total magnification. All tests were performed in triplicate and the means determined. The results were examined for significance by subjecting the data to statistical analysis employing Student's test. Sources of CSF. The two sources of CSF examined were the supernatant fluids decanted from cell culture monolayers of Ca) the L M cell line, a derivative of the 929 clone of L cells, and (b) pri-
551
mary monolayer cell cultures of C3H embryo cells. Primary embryo cell cultures were prepared by mincing, with iris scissors and forceps, 14- to 17-day mouse embryos into 1-mm 3 pieces of tissue. The tissue was transferred to a fluted trypsinization flask and washed with 30 ml Hanks' balanced salt solution (HBSS) to remove erythrocytes. After washing, the HBSS was decanted and 50 ml warm t37 ~ C) 0.25% EDTA-trypsin (GIBCO) was added to the flask and the tissue trypsinized for 20 min. The cell-rich supernatant fluid was filtered through sterile cheesecloth into 5 ml ice-cold fetal bovine serum. Following a second trypsinization, the cells were washed and plated in 75-cm 2 culture flasks (Falcon) at a concentration of 5 x l0 s cells per ml in minimum essential medium (MEM~ plus 5% fetal bovine serum. Both the LM cell line and the primary embryo cultures were maintained at 37 ~ C for 7 days in a humidified atmosphere of 5% CO2. At the termination of incubation, the supernatant fluids were decanted and centrifuged at 100 x g for 10 min. to remove any cells that might have detached from the monolayer. The fluids were sterilized by positive pressure filtration and stored at - 2 0 ~ C in 10-ml aliquots. Cellular morphology. The cellular composition of the bone-marrow colonies cultured in vitro was determined by "picking" individual colonies from the semisolid gel with a Drummond Wiretrol capillary pipette (Drummond Scientific Co.). Single colonies were picked from cultures daily. Care was exercised to isolate only those cellular aggregates that conformed to the minimum colony size requirements of 18 to 20 cells. Aggregates of suitable size for examination were observed initially after 2 days incubation. Individual colonies were deposited on alcohol-cleaned microscope slides, and a single drop of 0.6% orcein (Matheson, Coleman and BelB in a 60% acetic acid was added. An alcohol-cleaned cover slip was gently lowered onto the colony and the edges sealed to prevent drying during observation. Cells were examined and classified as to their granulocytic or mononuclear nature according to the criteria described by Metcalf, Bradley and Robinson ~10~. RESULTS Because the active principle contained within CSF has been obtained from a variety of sources, it was of interest to determine if differences existed with regard to the stimulation pattern produced by the addition of CSF to mouse bonemarrow cells.
552
SHELLHAAS, RHEINS, AND FILPPI
Colonial morphology. The first effect attributed to the different stimulatory sources was the morphological appearance of the developing colonies. The majority of the colonies produced in cultures stimulated by the addition of embryo cell conditioned medium were typically 0.4 to 0.6 mm in diameter and were characterized by large numbers of cells arranged in a relatively dispersed fashion within the colony proper. There was no
conspicuous center to the colony (Fig. laL Moreover, because the leading or advancing edge of the colony consisted of fewer cells than did the remainder of the cellular aggregate, the colony assumed a "fringe" or "veil" appearance. Whereas the morphology of each of the colonies was relatively independent of the length of incubation, the colony size generally increased during the test period.
FIG. 1. a, Typical colonial morphology of culture stimulated with embryo cell conditioned medium; b, typical colonial morphology of culture stimulated with LM cell conditioned medium, x50.
553
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FIG. 2. Dose-response relationship of bone marrow cultures stimulated with LM cell or embryo cell conditioned medium. The majority of the colonies produced following stimulation with L M cell conditioned medium exhibited the morphology depicted in Fig. l b . These colonies were smaller, ranging from 0.2 to 0.4 mm in diameter, and were comprised of fewer cells than were those observed in cultures stimulated with embryo cell conditioned medium. The cellular elements of each colony appeared concentrated into a small central area with no obvious "veil" or "fringe." Dose-response relationship. Dose-response studies of both the stimulation sources resulted in basic sigmoid-shaped curves, a feature considered characteristic of all colony stimulators (11). Quantities of embryo cell conditioned medium less than 0.4 ml failed to stimulate optimal colony formation, whereas additional volumes of stimulator failed to increase the number of colonies produced. Representative peak values were 38 to 40 colonies per 5 x 104 bone-marrow cells IFig. 2). The dose-response relationship for the L M cell conditioned medium, while essentially similar, differed in some respects from the curve for the embryo cell conditioned medium. Conditioned medium from L M cells stimulated the production of only 23 to 25 colonies per 5 x 104 bone-marrow cells. Quantities of this stimulator source in excess of 0.3 ml did not increase the number of resultant colonies. Cellular composition of colonies. The relative proportions of granulocytic to mononuclear cells were compared in the developing colonies of cultures stimulated with either the L M cell or the embryo cell conditioned medium (Fig. 3). In cell cultures stimulated with optimal concentrations of
L M cell conditioned medium, approximately 75% of the colonies consisted of granulocytic cells. This value rapidly declined to 40% on the 3rd day, and by the 5th day of incubation, the colonies consisted totally of cells of a mononuclear morphology. This pattern of granulocytic to mononuclear cell transformation was in contrast to that observed for marrow cell cultures stimulated with optimal concentrations of embryo cell conditioned medium. A greater percentage of these colonies consisted principally of granulocytic cells than did comparably aged parallel cultures stimulated with the L M cell conditioned medium. Further, the observed relative increase in the number of granulocytic colonies persisted longer during the incubation. Only alter the 5th day of culture did the percentage of granulocytic colonies fall below 80~ (Fig. 3). After this, the rate of the decline of granulocytic colonies roughly paralleled that observed for colonies stimulated with LM cell conditioned medium. The observed change in the pattern of cell proportionality could have been the result of a CSF concentration-dependent transformation rate; i.e. as CSF was utilized by the target cells, or the culture medium became depleted of active stimulator, an alteration in the colonial cell character was manifested. However, 5- and 10-fold concentration of the L M cell conditioned medium did not enhance colony formation. The only influence noted was a shift in the dose-response curve to the left in comparison to that observed in experiments that utilized the unconcentrated preparations. Thus it appeared that qualitative differences between the stimulating factors from the LM cell ioo 90
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FIG. 3. Percentage of granulocytic colonies observed at various periods in cultures stimulated with LM cell or embryo cell conditioned medium.
554
SHELLHAAS, RHEINS, AND FILPPI
conditioned medium and the embryo cell conditioned medium were responsible for the observed differences in colonial morphology and doseresponse characteristics. Costimulation o/cultures. Because the addition of an optimal concentration of each conditioned medium to marrow cell cultures resulted in different maximum levels of colony formation, the possibility of the existence of subpopulations of CFU-c was suggested. To investigate this possibility, bone-marrow cell cultures were stimulated with optimal concentrations of both CSF sources. The results of such costimulation experiments did not indicate an increase in colony formation. Rather the total number of colonies in costimulated cultures was similar to that observed in cultures stimulated with embryo cell conditioned medium. The addition of 2-ME alone to marrow cultures had no stimulatory effect on colony formation. However, differences in the level of colony formation were observed when 2-ME was added to marrow cell cultures containing embryo cell conditioned medium. The incorporation of 50/aM of 2-
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FIo. 4. Effect of costimulation with both embryo cell conditioned medium ~E. C. M.t and LM cell conditioned medium (LM C.M.} or 2-mercaptoethanol (2ME) and a single stimulator source [either embryo cell conditioned medium (E. C. M. ) or LM cell conditioned medium i LM C.M. i] on colony formation.
M E into cultures containing embryo cell conditioned medium significantly increased the number of resultant colonies. Such synergy was not observed in parallel cultures that were stimulated with LM cell conditioned medium. Incorporation of 2-ME concentrations of 500/~M or greater were toxic to the cells and inhibited even low levels of normally observed sporadic colony formation. DISCUSSION In the present study it was observed that two different mouse sources of CSF varied in their capabilities to stimulate mouse progenitor cells to undergo hematopoietic cell colony formation. Differences in the number of colonies and the morphology of the colonies produced were noted. Variations also were observed in the morphological nature of the cells comprising the colonies or aggregates. The smaller size and the earlier appearance of changes in the cellular character of the colonies stimulated with L M cell conditioned medium appeared not to be a function of the concentration of the active principle in this source of stimulator. Thus qualitative, rather than quantitative, factors in the two stimulators were more likely responsible for the noted differential proliferation patterns. Stimulation with the LM cell conditioned medium also initiated earlier changes in the cellular components of the colonies as evidenced by the more rapid appearance of colonies comprised of mononuclear cells when compared with marrow cell cultures stimulated with embryo cell conditioned medium, This observation may account for the smaller colony size that is characteristic of cultures stimulated with LM cell conditioned medium if it is assumed that the period of monocyte replication is more protracted than is the proliferation associated with cells of the more immature granulopoietic system. Insight into the nature of the differences between the two stimulators might be gained by considering the CSF sources. The LM cell fibroblast is an established spontaneously transformed cell line of C3H mouse origin. It has been passaged an inestimable number of times and may have undergone mutations that were reflected by alterations in metabolism from that exhibited by the original ceils. Embryo cell monolayers consist of cells from a large number of embryonic tissues. As such, each cell type would contribute various and perhaps peculiar byproducts of cellular metabolism. Additionally, in the fetus, one might anticipate a greater production of humoral molecules associated with hematopoietic stimulation and
COLONY-STIMULATING FACTORS regulation. Therefore the two sources examined, although prepared in a similar fashion, may vary as to the metabolic byproducts contained in culture supernates. Further, the possibility exists that additional factors were present. Whereas the active CSF in the two sources might have been the same, other components in the LM cell conditioned medium may have adversely affected colony formation. The reducing agent 2-ME has been shown to be influential in a variety of in vitro immunological reactions such as the response to sheep erythrocytes (13), T cell reactivity to phytohemagglutinin t14), and the mixed leukocyte reaction (15). The agent also has been employed to culture B lymphocyte colonies from lymphoid cell suspensions (16). In the present study, the enhanced colony formation in marrow cell cultures stimulated with embryo cell conditioned medium following the incorporation of 2-ME was of interest. Whereas the mechanism responsible for this observation is unknown, it may be that the reducing activity associated with this molecule split the macromolecular CSF moiety into a more stimulatory molecule, or into a number of subunits also possessing activity. Alternatively, 2-ME may serve as a scavenger molecule, removing potentially toxic free-radicals from the immediate environment. Rather than exerting an influence on the embryo cell stimulating factor itself, 2-ME may specifically act on the CSF receptor of the CFU-c, or some combination of the above. This of course assumes that the receptor sites on the CFU-c for the two stimulatory factors are different, or vary in their avidity for binding stimulator molecules. These differences could he manifested in signals of varying intensity which then could influence the rate of cell proliferation. The data also support the possibility that CSF influences differentiation of the target bonemarrow cells rather than simply sustaining the viability of the ceils until inherent genetic factors signal proliferation and maturation. This would raise a question as to whether the CSF in LM cell conditioned medium promotes a "switch" in colonial character from that of a granulocytic to that of a monnuclear cell type, or it is the consequence of unidentified modifying substances that are ineffective in completing the differentiation of the immature granulocytic cells. It may be that cells in a given population exhibit differences as to the incidence or spatial relationships of the receptor sites on their surfaces pertinent to the CSF. Whereas it is generally agreed that the primary
555
stimulator of mouse bone-marrow cells is a glyco~ protein, conformational differences in the glycoprotein moieties are also to be considered. These may be reflected in the suitability of a particular material to serve as a stimulator. Additional substances that direct or augment the primary stimulator may affect the availability of receptor sites to the principal CSF "trigger." REFERENCES 1. Austin, P. E., E. A. McCulloch, and J. E. Till. 1971. Characterization of the factor in L-cell conditioned medium capable of stimulating colony formation by mouse marrow cells in culture. J. Cell. Physiol. 77: 121-134. 2. Pluznik, D. H., and L. Sachs. 1965. The cloning of normal "mast" cells in tissue culture. J. Cell. Comp. Physiol. 66: 31%324. 3. Metcalf, D., and R. Foster. 1967. Behavior on transfer of serum stimulated bone marrow colonies. Proc. Soc. Exp. Biol. Med. 126: 758-762. 4. Robinson, W., D. Metcalf, and T. R. Bradley. 1967. Stimulation by normal and leukemic mouse sera of colony formation in vitro by mouse bone marrow cells. J. Cell. Physiol. 69: 83-92. 5. Foster, R., and D. Metcal~. 1968. Bone marrow colony stimulating activity in human sera. Results of two independent surveys in Buffalo and Melbourne. Br. J. Haematol. 15: 147-159. 6. Metcalf, D., and E. R. Stanley. 1969. Quantitative studies on the stimulation of mouse bone marrow colony growth in vitro by normal human urine. Aus. J. Exp. Biol. Med. Sci. 47: 453. 7. Robinson, W. A., E. R. Stanley, and D. Metcalf. 1969. Stimulation of bone marrow colony growth in vitro by human urine. Blood 33: 396-399. 8. Chan, S. H. 1970. Studies on colony-stimulating factor (C.S.F.): Role of the kidney in clearing serum C.S.F. Proc. Soc. Exp. Biol. Med. 134: 733-737. 9. Metcalf, D., and M. A. S. Moore. 1971. Techniques in experimental haematology and their limitations. In: A. Neuberger, and E. L. Tatum {Eds.), Hemopoietlc Cells. Vol. 24. North Holland Publishing Co., Amsterdam, p. 35. 10. Metcalf, D., T. R. Bradley, and W. Robinson. 1967. Analysis of colonies developing in vitro from mouse bone marrow cells stimulated by kidney feeder layers or leukemic serum. J. Cell. Physiol. 69: 93-108. 11. Metcalf, D., and M. A. S. Moore. 1971. Techniques in experimental haematology and their limitations. In: A. Neuberger, and E. L. Tatum {Eds.), Hemopoietic Cells. Vol. 24. North Holland Publishing Co., Amsterdam, pp. 37-39. 12. Metcalf, D. 1974. The sermn factor stimulating colony formation in vitro from mouse bone marrow cells stimulated by kidney feeder layers or leukemic serum. J. Cell. Physiol. 69: 93-108. 13. Chen, C., and J. G. Hirsch. 1972. Restoration of antibody-forming capacity in cultures of nonadherent spleen cells by mercaptoethanol. Science 176: 60-61.
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14. Fangcr, M. W., D. A. Hart, J. V. Wells, and A. Nisonoff. 1970. Enhancement by reducing agents of the transformation of human and rabbit peripheral lymphocytes. J. Immunol. 105: 1043-1045. 15. Heber-Katz, E., and R. E. Click. 1972. Immune response in vitro. V. Role of mercaptoethanol in
the mixed-leucocyte reaction. Cell. Immunol. 5: 410-418. 16. Metcalf, D., N . L . Warner, G . J . V . Nossal, J. F. A. P. Miller, K. Shortman, and E. Rabellino. 1975. Growth of B lymphocyte colonies in vitro from mouse lymphoid organs. Nature 255: 63O-632.
