Colony Formation in Agar by Multipotential Hemopoietic Cells D. METCALF, G. R. JOHNSON AND T.E. MANDEL The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria, P. 0. 3050, Australia

ABSTRACT Agar cultures of CBA fetal liver, peripheral blood, yolk sac and adult marrow cells were stimulated by pokeweed mitogen-stimulated spleen conditioned medium. Two to ten percent of the colonies developing were mixed colonies, documented by light or electron microscopy to contain erythroid, neutrophil, macrophage, eosinophil and megakaryocytic cells. No lymphoid cells were detected. Mean size for 7-day mixed colonies was 1,800-7,300 cells. When 7-day mixed colonies were recloned in agar, low levels of colony-forming cells were detected in 10% of the colonies but most daughter colonies formed were small neutrophil and/or macrophage colonies. Injection of pooled 7-day mixed colony cells to irradiated CBA mice produced low numbers of spleen colonies, mainly erythroid in composition. Karyotypic analysis using the T6T6 marker chromosome showed that some of these colonies were of donor origin. With an assumed f factor of 0.2, the mean content of spleen colony-forming cells per 7-day mixed colony was calculated to vary from 0.09 to 0.76 according to the type of mixed colony assayed. The fetal and adult multipotential hemopoietic cells forming mixed colonies in agar may be hemopoietic stem cells perhaps of a special or fetal type. Using specific stimuli, committed progenitor cells of every major hemopoietic class in t h e mouse can now be induced to proliferate in agar cultures and generate colonies of differentiating progeny cells (see review by Metcalf, '77). Such colonies are clones and, with the exception of the double population in neutrophilmacrophage colonies, the cell populations in such colonies are restricted to members of one cell class. Although it was suggested that granulocyte-macrophage colonies might be generated in agar by multipotential hemopoietic stem cells (CFU-S) (Dicke et al., '71), analysis showed that the properties of most cells forming granulocyte-macrophage colonies in agar (GM-CFC)differ sharply from those of CFU-S and most GM-CFC appear to be committed progenitor cells of the granulocyte-macrophage cell class (Moore et al., '72; Moore and Williams, '73; Metcalf, '77). Less complete evidence indicates that most cells forming erythroid, eosinophil and megakaryocyte colonies in vitro are also committed progenitor cells (GreJ. CELL. PHYSIOL. (1979)98: 401-420.

gory et al., '73; Heath e t al., '76; Metcalf et al., '74, '75). Recently, it was demonstrated that medium harvested from pokeweed mitogen-stimulated cultures of mouse spleen cells was able to stimulate erythroid colony formation in agar by mouse fetal liver cells (Johnson and Metcalf, '77). Such conditioned media lacked detectable erythropoietin as assayed in polycythemic mice. Of greater interest was the recognition that approximately one-third to one-half of the erythroid colonies contained other hemopoietic cells, the most common additional cells being macrophages, neutrophils, megakaryocytes and eosinophils. Single cell transfer studies showed that these mixed colonies were clones derived from single cells (Johnson and Metcalf, '77). The present study was undertaken to analyze in more detail the cellular content of Received May 2, '78.Accepted Sept. 11, '78. ' This work was supported by the Carden Fellowship Fund of the Anti-Cancer Council of Victoria, The National Health and Medical Research Council, Canberra and the National Cancer Institute, Bethesda, Contract No. NOI-CB-74148.

401

402

D. METCALF. G. R. JOHNSON AND T. E. MANDEL

mixed hemopoietic colonies grown in agar and to determine whether such colonies contained cells capable of being recloned in the various in vitro agar culture systems or of forming spleen colonies in irradiated mice. MATERIALS AND METHODS

Preparation of cell suspensions Mice used were of the inbred strains CBAf/ CaH, Wehi CBA T6T6 and C57B1/6f/J Wehi maintained in this Institute. Fetal tissues were obtained from pregnant mice (day of vaginal plug = Day 0 of gestation). After removal from t h e uterus, intact yolk sacs containing fetuses were washed three times and the fetuses separated. Decapitated fetuses were placed in a small volume of Eisen's balanced salt solution (EBBS) to allow collection of peripheral blood. Fetal livers were dissected free with cataract knives and converted to single cell suspensions by oral pipetting. Cell suspensions were allowed to stand for five minutes to allow undispersed fragments to sediment then the supernatant cell suspension was centrifuged through fetal calf serum (1,200 g, 7 minutes) to remove cell debris. Suspensions of cells from other fetal organs or yolk sacs were prepared in a similar manner. Cell counts were performed using eosin to determine viable cell counts. Preparation of spleen conditioned medium C57B1 spleen cells were incubated for seven days a t a concentration of 2 x lo6cells per ml in RPMI-1640 containing 5%heat-inactivated human plasma and 0.05 ml of a 1:15 dilution of pokeweed mitogen per ml of culture medium (Grand Island Biological Company, New York). After incubation, the media were centrifuged for ten minutes a t 3,OOOg. The supernatant fluid was then harvested and millipore filtered. Not all batches of human plasma were able to support the production of active conditioned medium and batches were pretested before use (Metcalf and Johnson, '78). Agar cultures Cells were cultured in 35-mm plastic Petri dishes containing 1 ml agar-medium. The agar-medium was an equal volume mixture of 0.6% Bacto-agar and double strength DulbecCO'S Modified Eagle's medium. The composition of the double strength medium was: Dulbecco's Modified Eagle's medium HG16 Instant Tissue Culture Powder (Grand Island Biological Company, New York) 10 g; 390 ml

double glass distilled water; 3 ml L-asparagine (6.7 mg/ml) (final concentration in medium 20 pg/ml); 1.5 ml DEAE Dextran (50 mg/ml) (final concentration in medium 75 p g l ml) (Pharmacia, Sweden) (molecular weight = 500,000); 0.575 ml penicillin (2 x lo5 Unitdml); 0.375 ml streptomycin (200 mg/ ml); 4.9 g NaHCO,; 250 ml human plasma (heated to 56°C for 30 minutes and centrifuged at 3,000 g for 10 minutes to remove precipitate). Not all batches of human plasma were able to support the formation of prominent red-colored erythroid colonies. Satisfactory plasmas from individual patients or normal blood donors were selected by prior testing (Johnson and Metcalf, '77; Metcalf and Johnson, '78). Cultures contained from 1,000 to 20,000 nucleated cellslml. Cell counts for fetal liver excluded yolk sac erythroblasts but these were included in counts on fetal peripheral blood. One milliliter volumes of the cell suspensions in agar-medium were pipetted into culture dishes containing 0.2 ml of spleen conditioned medium. The culture dishes were mixed, allowed to gel, then incubated for up to seven days in a fully humidified atmosphere of 10% C 0 2 in air. Control cultures of 12-day CBA fetal liver cells designed to develop only neutrophil and/ or macrophage colonies were prepared using the above medium but employing as stimulus 0.1 ml of Step I1 mouse lung conditioned medium containing GM-CSF (Burgess et al., '77). Control cultures of 75,000 CBA adult marrow cells were prepared in the above medium and stimulated either by 0.2 ml of spleen conditioned medium or 0.1 ml of lung conditioned medium. Assays for B-lymphocyte colony formation were performed using the medium described previously (Metcalf et al., '75a) which i n volves the addition of 2-mercaptoethanol and 0.1 ml of 30%sheep red cells. Scoring of cultures Cultures were scored for erythroid colonies using a n Olympus dissection microscope and semi-indirect lighting a t x 35 magnifications. Red- or pink-colored aggregates containing more than 50 cells were scored as erythroid colonies (Johnson and Metcalf, '77). Neutrophi1 andlor macrophage colonies and occasional eosinophil or megakaryocyte colonies were scored separately by the usual criteria (Metcalf et al., '74, '75). As is usual in 7-day cultures of mouse cells, few or no monocyte

403

MULTIPOTENTIAL COLONY-FORMING CELLS

colonies were observed, most colony cells in this series having the morphology of mature macrophages. Details of alternative methods for scoring such cultures will be described in the text. To determine the cellular composition of 6to 7-day colonies, individual colonies were removed using a fine Pasteur pipette and the colonies spread on microscope slides. Colony cells were stained with benzidine-Giemsa (Borsook et al., '69) and differentiated cell counts performed a t x 1,000 magnifications. Electron microscopy Special cultures of 1,000-2,000 12-day CBA fetal liver cells were prepared so that each culture dish contained only one or a few colonies presumed to have a mixed composition. Individual 6-day colonies with no adjacent cells or colonies were carefully removed from the culture dish using a fine Pasteur pipette and placed in 10 ml of diluted Karnovsky fixative (Karnovsky, '65) a t room temperature. The colonies were fixed for one to two hours at room temperature, washed twice in tap water and post fixed in 2% osmium tetroxide in 0.08 M cacodylate buffer for a further two hours. The colonies were then stained en bloc in 2% uranyl acetate, dehydrated in a graded series of acetone and embedded in Spurr's low viscosity resin (Spurr, '69), each colony being embedded in a separate gelatin capsule. After polymerization, the colonies were trimmed and 1p sections were cut for light microscopic examination. These sections were stained with toluidine blue to confirm the absence of other cells from the area surrounding the colony. When these sections indicated that the central region of the colony was being sectioned, ultrathin sections were cut for electron microscopy. These sections were silver to pale gold (= 60-80 nm) and were stained sequentially with saturated alcoholic uranyl acetate (Hayat, '70) and lead citrate (Reynolds, '63). They were examined in a Philips 300 electron microscope. Recloning of colony cells Six- or seven-day colonies grown in cultures of 2,000 to 20,000 12-day CBA fetal liver cells were picked off using a fine Pasteur pipette and individual colonies added to 3 ml of agarmedium. Colony cells were gently dispersed by oral pipetting and two, 1 ml, volumes of the cell suspension in agar medium added to Petri dishes containing 0.2 ml of spleen conditioned medium (SCM). Cultures were incubated for a

further seven days before being scored for colony ( > 50 cells) and cluster (3-50 cells) formation. In other experiments, harvested colonies were pooled in 1- to 2-ml volumes of ice-cold Eisen's balanced salt solution. Colony cells were resuspended by gentle pipetting and undispersed agar fragments allowed to settle to the bottom of the tube. Supernatant colony cells were harvested and viable cell counts performed using eosin. These cell suspensions were injected i.v. to CBA or CBA T6T6 mice irradiated previously with 850 rads wholebody irradiation (from a Philips RT 250 unit operating a t 250 kv, 15 mA.HVL was 0.8 mm copper and a focal skin distance of 50 cm with full backscatter conditions, 127 rad/min). Spleen colonies were counted at seven days, the spleens fixed in Bouin's fixative, serially sectioned and stained with hematoxylin and eosin. Karyotypic analysis of individual spleen colonies To determine the origin of spleen colonies developing in mice injected with pooled colony cells, CBA or CBA T6T6 mice were injected with colony cells grown from CBA T6T6 or CBA 12-day fetal liver cells. Metaphase spreads were obtained from individual spleen colonies by two methods. Mice were given intraperitoneal injections of Colcemid (2.5 pg in normal saline) and three hours later individual spleen colonies were dissected out and dispersed by passage through needles of decreasing bore size. Metaphase spreads were then prepared by the method described by Metcalf and Moore ('71). Slides were Giemsa-stained and coded slides scored by two independent observers. The second procedure followed, was to dissect out individual spleen colonies which were then dispersed into single cell suspensions prior to a 3-hour in vitro incubation a t 37OC with Colcemid. (Spleen colony cell suspension in 0.4 ml balanced salt solution to which was added 0.8 ml balanced salt solution containing 10%fetal calf serum and 2 pg of Colcemid). Cell suspensions were then washed in balanced salt solution and metaphase spreads were prepared and scored as outlined above. RESULTS

Frequency, size and composition of mixed colonies From previous observations, some of the red-colored erythroid colonies developing in

404

D. METCALF, G. R. JOHNSON AND T. E. MANDEL TABLE 1

Relatiue frequency oferythroid and mixed colonies in cultures offetal and adult cells Percent colonies Cells cultured

Fetal liver 11 day 12 day 14 day Yolk sac llday Fetal peripheral blood 11 day 12 day Adult bone marrow

No. colonies per lo5 cells

Neutro- Neutrophil- Macrophil macrophage phage

Eosinophil

Erythroid

19

2 2

43 30

Mixed

3,100

2 6 4

18 32 18

25

920 416

58

0

10

10 11 10

500

2

2

78

2

6

10

143 69

6 12 24

12 12

44 46 46

4 2 4

28 18 8

10

150

16

6 2

All cultures prepared using 2-5,000cells from organ p a l s per dish and scored at seven days. The absolute frequency of colonies shorn represent mean data from four replicate cultures recalculated as per lo5cultured cells. Fifty individual sequential colonies smeared, stained with Giemsa-henzldine and, where possible, differential counts performed on 500 cells. Mixed colonies contained erythroid cells mixed with more than 1%of other hemopoietic cells (see table 3 for typical composition).

cultures of CBA fetal liver cells after stimulation by spleen conditioned medium (SCM) were known to contain mixed populations of hemopoietic cells (Johnson and Metcalf, ’77). To determine more exactly the distribution and frequency of cells capable of forming mixed colonies, a survey was made of cultures prepared from various fetal and adult tissues. For this purpose, care was taken to minimise the occurrence of artefacts due to coincident colonies by culturing only 2-5,000 cells per culture. After seven days of incubation, 50 sequential colonies were removed from each culture dish and the colony cells smeared, stained and typed. As shown in table 1, most colonies developing in the cultures were composed of neutrophils and/or macrophages. However 1053%were composed either wholly of erythroid cells or contained erythroid cells mixed with other hemopoietic cells. Since most of the mixed colonies were partially or wholly red in color, it can be deduced from the data in table 1 that about 20-50% of colonies suspected on gross appearance as containing erythroid cells, in fact were mixed colonies. Multiple (burst) erythroid colonies were always composed of apparently pure populations of erythroid cells as were the numerous small red-colored colonies of 100-300 uniform cells often beginning to break down by day 7. Where red-colored colonies were large, tightly packed single colonies, it was not possible to predict whether these colonies would be com-

posed entirely of erythroid cells or be mixed colonies. However to undertake any biological studies on mixed colony cells, living cells are required and individual colonies in these cultures were not large enough to permit part of the colony to be removed for typing prior t o the use of the remainder. As a compromise, a crude method of classifying colonies was devised, based on the gross appearance of the colonies. As mentioned above, most colonies in these cultures were readily identifiable in unstained cultures as typical neutrophil and/or macrophage colonies. The remaining colonies could be grouped into three categories: (a) “Red colonies,” usually large single colonies wholly red in color or with patchy white areas. Often such colonies had a loose outer mantle of larger white-colored cells. Multiple (burst) red colonies were excluded from this category as were the numerous small 100-300 cell colonies, since these could clearly be seen to be composed of a highly uniform population of small erythroid cells. (b) “Green colonies,” very large single, green-brown colored colonies of densely packed cells usually with patchy red areas and often with an outer mantle of cells of widely varying size. The green color of these colonies appeared to be due to a combination of their very large size, their content of neutrophils and a significant content of erythroid cells. (c) “White colonies,” usually single dense colonies with or without an outer mantle of

405

MULTIPOTENTIAL COLONY-FORMING CELLS TABLE 2

Average sue of mixed colonies grown from 12-day CBA fetal liuer compared with control colonies grown from adult CBA bone marrow cells

Cells cultured

Stimulus

12-dayCBA fetal liver

Spleen conditioned medium Spleen conditioned medium Lung conditioned medium

Adult CBA bone marrow

Type of colony

Total colonies counted

Mean number of cells per colony

Red Green White Unselected

273 144 295 74

3,070 7,300 1,830 1,400

Unselected

96

2,030

Cultures contained 2,000 12-day CBA fetal liver cells or 75,000 CBA marrow cells stimulated by 0.2 ml C57B1 spleen conditioned medium or 0.1 ml C57B1 lung conditioned medium. ’ Calculated mean values from groups of 20-50 redispersed 7-day colony cells from six separate experiments. Figures indicate number of viable nucleated cells. TABLE 3

Pooled data from differential cell counts on individual mixed colonies grown from 12-day CBA fetal liver cells 5 p e of colony

Red Green White

Number of colonies typed

Erythroid

Neutrophi1

Macrophage

Percentage of cells Eosinophi1

Blast

9 7 9

96 68 1

1 8 20

3 6 58

0 17 20

0 1 1

Megakaryocyte

0 0

0

Cultures of 2,000 12-day CBA fetal liver cells stimulated by 0.2 ml of spleen conditioned medium. Individual 7-day colonies smeared and stained with Giemsa-benzidine. Where possible 500 cells typed per colony.

irregular-sized cells. These colonies were distinguishable from neutrophil colonies by the grossly irregular size of component cells. Colonies classified as “red,” “green” or “white” were provisionally regarded as “mixed” colonies for further studies. As shown in table 2, the “red” and “green” mixed colonies had a very large average size after 7 days of incubation, containing an average of 37,300 nucleated cells. These total cell counts do not include a considerable proportion of non-nucleated erythroid cells ( a mean number of 3,200 cells for red colonies and 2,800 for green colonies) so t h e total size of some colonies approximated 10,000 cells. This should be contrasted with the much smaller size of the white mixed colonies and pooled unselected 7-day colonies grown from bone marrow cells in the same culture medium stimulated either by the same dose of spleen conditioned medium or mouse lung conditioned medium (1,400-2,000cells per colony). Morphological analysis of the cells from individual colonies classified provisionally on

gross morphology as red, green or white mixed colonies is shown in table 3. The data confirmed that most such colonies were in fact composed of mixed populations, although some red colonies were composed wholly of erythroid cells and some white colonies were in fact only composed of neutrophil andlor macrophages. It can be seen that the red colonies contained a higher average proportion of nucleated erythroid cells (approximately 90%) than did the green colonies (approximately 70%).As found previously, approximately 20% of mixed colonies contained low numbers of megakaryocytes by orcein staining of intact colonies (Johnson and Metcalf, ’77) but these were damaged during the smearing process and are not seen in the data in table 3. Some white mixed colonies did contain low numbers of erythroid cells but others appeared to lack such cells at least at day 7 of colony development. The above categories of “red,” “green” and “white” mixed colonies fall far short of an optimal system for identifying every living

406

D. METCALF, G. R. JOHNSON AND T. E. MANDEL

at various stages of differentiation were present. Granulocytes were also present and showed varying degrees of differentiation. Neutrophilic polymorphs were the commonest granulocytic cells seen and ranged in differentiaElectron microscopy of mixed tion from myeloblasts, through myelocytes colonies and metamyelocytes to apparently fully maElectron microscopic studies were re- ture cells (figs. 2c,d, 3a). In addition to neutrostricted to colonies likely to contain mixed phils, granulocytic cells with large electronhemopoietic populations. Colonies chosen for dense granules and a segmented nucleus were examination contained red areas mixed with also present though not in all colonies exameither white or green areas and were well sep- ined. These cells were possibly basophils and arated from any other colonies in the culture close examination in the light microscope of 1-p thick toluidine blue-stained sections dish. Colonies processed for electron microscopy taken adjacent to the thin sections showed were first examined by light microscopy in 1-p some metachromatic staining of their granthick sections to confirm that they contained ules. In the limited number of colonies exama heterogeneous cell population and that no ined recognizable eosinophils were not idencells or clusters were present in the region tified in the electron microscope since no cells adjacent to the colony. Figure l a shows a low having the characteristic bar-shaped granules power light micrograph of a 1-pthick section of murine eosinophils were seen. Among the mononuclear cells, only macroof a typical colony. The survey light micrograph shows the relatively dense packing of phages and monocyte-like cells were identhe cells and part of the rim of cell-free resin tified and in these colonies no lymphocytes around the periphery of the colony. Even a t or plasma cells were seen. The macrophages the low magnification of this figure ( x 150) it formed a structurally diverse group and were is apparent that the cells are quite het- usually identified by their content of ingested erogeneous in both size and appearance. cell debris, phagosomes and lysosomes. Also Electron microscopy confirmed the wide present and very conspicuous in some colonies range of hemopoietic cells present within a were very large cells with huge and apparentsingle colony. Figure l b is an electron micro- ly multiple nuclei (figs. 3c,d). These cells had graph of the colony shown in figure l a and extensive cytoplasm which contained numershows the range of cells present even in a ous small granules particularly towards the single field. The field illustrated includes typi- cell margin. In addition, the peripheral cytocal nucleated red cells ranging from erythro- plasm contained a complex canalicular system blasts to relatively mature, apparently anu- and the free edge of the cell was highly irregucleate, red cells mostly a t the reticulocyte lar in outline (figs. 3c,d). These cells were stage of differentiation. Cells resembling mac- identified, on the basis of their size and charrophages are also present as well as cell debris acteristic ultrastructure, as megakaryocytes. However no free platelets were seen in the secand other evidence of degenerating cells. Figures 2 and 3 illustrate, a t higher mag- tions examined. nification, the types of cell seen in a single colThe degree of platelet budding seen in colony. These electron micrographs show a wide ony megakaryocytes appears to vary with the range of hemopoietic cells of different types batch of human plasma used in the culture and a t differing stages of maturity. medium. As seen best with pure megakaryoThe majority of the cells examined ultra- cyte colonies, budding was occasionally exstructurally showed features of incomplete treme, the individual cells being surrounded differentiation, particularly in the case of the by halos of platelets visible in unstained culred blood cell series where the most common tures. cells were erythroblasts, while reticulocytes Recloning of colony cells in agar and fully mature erythrocytes were less freIndividual 7-day colonies grown from 12quent (figs. 2a-c). The degree of differentiation between different erythroid colonies day CBA fetal liver cells were transferred to 3 varied widely and in some 7-day colonies no ml of agar-medium, the colony cells dispersed mature RBC were seen and only erythroblasts by oral pipetting and the cell suspension in

mixed colony in a culture dish. However in studies to follow, only obvious examples of each category were chosen for assay and checks showed that such obvious colonies were in fact mixed.

,

407

MULTIPOTENTIAL COLONY-FORMING CELLS TABLE 4

Colony- and cluster-formingcell content of 7-daymixed colonies grown from CBA fetal liver cells Colony-forming cell assays Type of colony

Red Red-white Multiple red (Burst) Green mixed White mixed

Number positive colonies1 number tested

3/65 4/48 0123 6/34 6/23

Percent positive

Cluster-forming cell assays

Mean number per colony

Mean posi~ tive coloniesl number tested

Percent positive

Mean number per colony

22% 39% 5%

162 16 16% 16 6

41% 47%

929 lot9

0%

-

12/54 15/38 1/20

18% 26%

824 18k 18

13/52 7/15

5% 8%

'

725 3'9

' Primary cultures were 2-20.000 12-day CBA fetal liver cells stimulated by pokeweed mitogen-stimulated spleen conditioned medium. 'Mean total content of colony-forming and cluster-forming cells inpositive colonies, calculated from colony and cluster numbers in secondary cultures. All secondary cultures stimulated by spleen conditioned medium. agar medium recultured using spleen conditioned medium as the stimulus for colony formation. All culture dishes were checked before incubation to exclude cultures containing undispersed colony fragments. Secondary cultures were scored for colony formation after a further seven days of incubation. The results have been classified according to whether the colony used for recloning was red, green or white in color. Red colonies were classified in these experiments according to whether t h e colony was a single uniformly red colony, a single colony containing red and white areas or a multiple (burst) red colony. Green-colored colonies included colonies in which only part of t h e colony was greenish and t h e remainder red-colored. From table 4 it can be seen t h a t all types of colony with t h e exception of burst erythroid colonies contained some cells capable of forming colonies on recloning in agar and, overall, 10% of the colonies were positive. The numbers of colonyforming cells in positive colonies were low with the mean content varying from 7 to 18 cells per colony. This represents approximately one colony forming cell per 1,000 colony cells. Previous studies (Johnson and Metcalf, '78) showed that in spleen conditioned medium stimulated cultures of 12-day CBA fetal liver cells there were approximately 5/1,000 neutrophil-macrophage colony-forming cells and approximately 2/1,000 erythroid colonyforming cells. In cultures of 19-day (newborn) liver corresponding to the total in vivo plus in vitro age of the present colony cells (12 7 days) the frequency of colony-forming cells was low (1/103cells forming a neutrophil-macrophage colony and 0.02/103 cells forming a n erythroid colony).

+

As shown in figure 4, t h e size of these recloned colonies tended to be small. Although some colonies of up to 1,500 cells were obtained the majority contained fewer than 200 cells. This contrasted sharply with t h e size of up to 10,000 cells for the donor colonies. Analysis of the composition of these colonies (fig. 4, table 5 ) emphasised the difference between the recloned colonies and t h e original colonies. The majority of recloned colonies were loosely dispersed collections of neutrophils. In only 8 of 76 daughter colonies was a mixed population of hemopoietic cells observed. No differences were observed in the types of colony formed by cells from the various types of donor colonies. Another notable feature of the recloned cultures of mixed colony cells was the relative absence of clusters from the daughter cultures. What few colonies did develop tended to be present in culture dishes devoid of smaller proliferating aggregates of cells. Where clusters did develop, the numbers per culture dish were relatively low. These observations are of interest in view of the relatively high macrophage content of mixed colonies. When colony formation in cultures of fetal liver cells had been stimulated by mouse lung conditioned medium, a mixture of macrophage and/or neutrophil colonies developed. Recloning of these neutrophil colonies after seven days of incubation produced few daughter cells or clusters (table 6 ) . In sharp contrast, an exceptionally high proportion of the macrophage colonies in such cultures produced small macrophage daughter colonies (table 6, fig. 4) and large numbers of macrophage clusters. This latter experiment appears equivalent to the macrophage colony growth reported by Lin and Stewart ('74).

408

D. METCALF, G. R. JOHNSON AND T. E. MANDEL TABLE 5

Morphology of colonies grown from redispersed 7-daycolony cells Number and type of daughter colonies ' cells cultured Original stimulus

Fetal liver Bone marrow

Neutro- Neutrophil- Macrophi1 macrophage phage

Spleen conditioned medium Lung conditioned medium Spleen conditioned medium Lung conditioned medium

Erythroid

Megakaryocyte Mixed

Total

44

6

12

3

3

8

76

1

0

125

0

0

0

126

8

4

31

0

0

0

43

4

0

19

0

0

0

23

' All secondary cultures were stimulated by spleen conditioned medium and daughter colonies were typed after seven days of growth in the secondary cultures. TABLE 6

Colony- and cluster-foormingcellcontent of 7-day colonies grown from CBA fetal liver or bone marrow cells Colony-forming cell assays cells cultured

~~~

Stimulus

Type of colony

Lung conditioned medium

Neutrophil Neutrophilmacrophage Macrophage Neutrophil or mixed Macrophage Neutrophil or mixed Macrophage

Number positive/ number tested

Cluster-forming cell assays

Mean number per % Positive

colony

*

Number positive/ number tested

% positive

Mean number per colony

~

Fetal liver Bone marrow

Spleen conditioned medium Lung conditioned medium

1/44 3/17

2% 18%

2 221

7/44 11/17

16% 65%

929 25232

23/38

61%

10210

37/38

97%

138+.129

11/82 2/18

13% 11%

628 320

50182 17/18

61% 94%

1442208 59249

6/84 1/14

7% 7%

622 2

38/84 8/14

45% 57%

84284 42237

I Primary cultures were 2-20,000 12-day CBA fetal liver cells stimulated by 0.1 ml of lung conditioned medium or 75,000 CBA adult marrow cells stimulated either by 0.2 ml of spleen conditioned medium or 0.1 ml of lung conditioned medium. A Mean total colony content of colony-forming and cluster-forming cells perpositrue colony as calculated from colony and cluster numbers in secondary cultures. All secondary cultures stimulated by spleen conditioned medium.

In control experiments, individual 7-day neutrophil and/or macrophage colonies stimulated to develop in bone marrow cultures by spleen or lung conditioned medium were recloned in cultures containing spleen conditioned medium. Pure neutrophil colonies on recloning usually produced neither colonies nor clusters. A few of the neutrophil-macrophage colonies developing in cultures stimulated by spleen conditioned medium contained low numbers of colony-forming cells, some of which were able to generate neutrophil colonies of up to 300 cells (fig. 4, table 5). However the majority of daughter colonies were small and composed entirely of macrophages. Similar results were obtained with neutrophil and/or macrophage colonies stimulated by mouse lung conditioned medium. Few

of the macrophage colonies stimulated either by spleen or lung conditioned medium contained colony-forming cells and these always generated only small macrophage colonies. As before most macrophage colonies generated numerous small macrophage clusters (table 6). In repeated tests, 7-day mixed colonies were redispersed and assayed for their content of B-lymphocyte colony-forming cells in secondary cultures containing 2-mercaptoethanol and sheep red cells (Metcalf et al., '75a). No daughter colonies were observed. Addition of 2-mercaptoethanol to the cultures during the growth of the primary mixed colonies did not lead to the development of B-lymphoidcolonyforming cells. The recloning experiments failed to docu-

409

MULTIPOTENTIAL COLONY-FORMING CELLS TABLE 7

Spleen colony formation in irradiated CBA mice injected with pooled mixed colony cells

Mice injected with

Pooled green colony cells Pooled red colony cells Pooled white colony cells Intercolony agar Culture medium

Total cells injected '

Total number of spleen colonies

Erythroid

Undifferentiated

Neutrophi1

Mixed

Megakaryocyte

26

1,676,000

53

45

5

0

2

1

24

1,832,000

27

22

3

1

0

1

17 26 22

350,000

16 22

16 20 13

0 1

0

0

0

1 0

0

0

0

0

Number of mice

-

13

Number of colonies

0

' Total nucleated cell8 injected to whole groups of mice not

the number injected into each mouse. Spleens examined by serial section seven days after injection

ment the existence in mixed colonies of cells able to form the original type of large mixed colony but did reveal the existence in these colonies of neutrophil andlor macrophage colony-forming cells and a curious deficiency of cluster-forming cells. The other finding worthy of reiteration was that macrophage colonies generated by fetal liver cells more frequently contained colony-forming cells than did apparently similar colonies generated by adult marrow cells, emphasising the unusual absence of macrophage cluster-forming cells in mixed colonies grown from fetal liver cells.

Assays on mixed colonies for spleen colony-formingcells In 7-day cultures of 20,000 12-day CBA or CBA T6T6 fetal liver cells, green, red and white colonies considered likely to be mixed colonies were pooled, dispersed cell suspensions prepared and injected into irradiated CBA or CBA T6T6 mice. Control mice were injected (a) with a redispersed suspension of agar medium taken from the intercolony areas of the same donor cultures and equalling the volume of agar medium removed with the colonies or (b) with culture medium used in the preparation of the mixed colony cultures. Injection of control mice with pooled inter-colony agar was performed to check the possibility that CFU-S in the original population cultured might have survived culture in agar either in the inter-colony area or within the volume of agar occupied by the mixed colonies. Seven days after irradiation and injection, recipient mice were killed and their spleens serially sectioned and analysed for spleen colony formation using conventional criteria (Metcalf and Moore, '71). Pooled data from

seven such experiments are shown in table 7. It can be seen from this table that only mice injected with pooled green colony cells exhibited a clear rise in spleen colony numbers. The size of the colonies was similar to that of colonies generated by control fetal liver cells. As shown in the table, most colonies were erythroid and few neutrophil, mixed or megakaryocyte colonies were observed. Mice injected with pooled red or white colony cells exhibited only marginally higher numbers of spleen colonies than did control mice and mice injected with inter-colony agar similarly failed to exhibit increased numbers of spleen colonies. As different mice received differing numbers of injected cells, the spleen colony numbers need to be related to the numbers of cells injected after subtraction of background colony numbers as calculated from colony counts in control mice (35 colonies in 48 mice or 0.7 per mouse). This calculation has been performed in table 8 and the data have also been expressed as spleen colony-forming cells per mixed donor colony, using the figures for average colony size shown in table 2. Since the low number of spleen colony-forming cells observed in mixed colonies has so far prevented a formal seeding factor (f) determination on these cells, an arbitrary value of 0.2 was used in this calculation. For example, with 26 mice injected with green colony cells, 53 colonies were observed. Subtraction of 26 x 0.7 background coloniesleaves a net colony number of 34.8 for 1,676,000cells injected. With an f factor of 0.2 this represents 10.4 spleen colony-forming cells per lo5green colony cells. With an average size of 7,300 cells for 7-day green colonies,

410

D. METCALF, G . R. JOHNSON AND T. E. MANDEL

Thy-1 sera (1. Goldschneider, personal communication) failed to reveal positive cells. No evidence has been obtained therefore for t h e presence of lymphoid cells in these mixed Spleen colonycolonies. Type of mixed forming cells per Spleen colonycolony cells lo5 mixed colony forming cells per The exact frequency of cells forming mixed cells ' mixed colony hemopoietic colonies in cultures of fetal and Green colonies 10.4 0.76 adult tissues is difficult to assess unless Red colonies 2.8 0.09 sequential colonies are examined morphologiWhite colonies 5.9 0.11 cally. From such studies, approximately 10% ' Colony counts subtracted for background colonies (0.7 per mouse) of the colonies in cultures of all fetal tissues and an f factor of 0.2 used. appeared to be mixed, no one tissue exhibiting Based on mean colony size for 'I-day colonies shown in table 2. an outstandingly high frequency of such colonies. The necessity to stain and perform differenthis represents 0.76 spleen colony-forming tial cell counts on individual colonies to cercells per colony. tify their mixed nature somewhat restricts exKaryotypic analysis of spleen colonies in mice periments designed to determine the properinjected with mixed colony cells ties and frequencies of mixed colony-forming Analysis of spleen colonies was restricted to cells in various tissues and experimental irradiated mice injected with pooled 7-day situations. It aIso restricts the use of such green and/or red mixed colony cells. Colonies mixed colony cells for further biological tests. were grown from either CBA or CBA T6T6 As shown in the present study, mixed colonies fetal liver cells and injected into recipients in the culture dish could be roughly catewith the opposite karyotype. Individual col- gorized into three groups - red, green and onies were examined at intervals from 7 to 11 white-based on their size, color and general shape. "his method is suboptimal since some days after injection. Successful karyotypic analysis was per- purely erythroid colonies were mistakenly formed on nine colonies in mice injected with classified as mixed red colonies and some mixed colony cells and of these, five were neutrophil-macrophage colonies mistakenly shown to be exclusively of donor karyotype classified as mixed white colonies. However if and four exclusively of host karyotype. Two of only obvious colonies of each type were chosen, two spleen colonies in mice injected with agar all proved to be mixed in composition and the cells from such colonies were able to be subwere shown to be of host karyotype. jected to various bioassays. DISCUSSION Attempts to reclone 7-day mixed colony The present analysis of fetal liver cultures cells in cultures again stimulated by spleen stimulated by pokeweed mitogen-stimulated conditioned medium failed to reveal the presspleen conditioned medium has confirmed ence of mixed colony-forming cells equivalent that such cultures develop neutrophil-macro- to the cells initiating the original mixed colphage and erythroid colonies (Johnson and onies. However low numbers of colony-formMetcalf, '77, '78), and that some of the ing cells were detected, the colonies usually erythroid colonies developing contain, in addi- being composed only of neutrophils andlor tion, up to four other hemopoietic populations macrophages and these colonies were smaller - neutrophils, macrophages, eosinophils and than the original donor colonies. The data megakaryocytes. Identification of most of seemed best interpreted as indicating little these cells has been confirmed in the present capacity of mixed colony-forming cells for study by electron microscopic analysis of indi- genuine self-replication within developing vidual colonies. As is true of other hemopoietic colonies at least as assessed by the limited cricolonies grown in semisolid cultures (Parmley terion of analysis of 7-day colony populations. e t al., '76) differentiation of hemopoietic cells The cells forming mixed colonies were clearly in mixed colonies often appeared to fall short capable of generating some progenitor cells of complete maturation to mature end cells. B- assayed in agar cultures as neutrophil-macrolymphocyte colony-forming cells were not phage, erythroid or megakaryocytic colonydetected in mixed colonies and studies in this forming cells. The relative absence of macrophage colonylaboratory using fluorescein-conjugated antiTABLE 8

Calculated spleen colony-formingcell levels in mixed colonies

A

MULTIPOTENTIAL COI.ONY-FORMING CELLS

x

and cluster-forming cells from 7-day mixed colonies is noteworthy in view of the fact that 2-50%of mixed colony cells were macrophages (Johnson and Metcalf, '77) and as shown in the present study, macrophage colonies derived from fetal liver have an unusually high capacity to generate macrophage colonies and clusters on recloning in agar. Dicke e t al. ('71) reported the presence of low numbers of spleen colony-forming cells (CFU-S) in agar colonies grown from marrow cells from mice pretreated with nitrogen mustard and vinblastine. The present assays on 7-day mixed colonies also seem to have documented t h e presence of low numbers of CFU-S a t least in green mixed colonies, and again the frequency of such cells was low. Because seeding efficiency studies have yet to be performed, no firm estimate of the frequency of colony-forming cells is possible but assuming a conservative f factor of 0.2, an average of 0.76 spleen colony-forming cells per green mixed colony was obtained. In view of the known heterogeneity of spleen colony-forming cells in terms of their capacity for self-replication (Siminovitch et al., '63; Metcalf and Moore, '711, it would be of interest to know if similar heterogeneity existed in mixed colony cells grown in vitro since the above data represent mean figures from pools of colony cells. Most spleen colonies generated by mixed colony-forming cells were erythroid. Despite the early time of sampling, comparison of the data with published composition figures (see review, Metcalf and Moore, '71) for spleen colonies derived from fetal liver cells suggests that a higher proportion of neutrophil and mixed colonies would have been anticipated. This question needs further examination but raises the possibility that the in vitro culture conditions may have favored the generation of spleen colony-forming cells with a capacity for differentiation restricted to the erythroid pathway. It would also be of interest to determine the CFU-S content of these spleen colonies derived from injected in vitro colony cells since the cells forming the spleen colonies might have a quite restricted capacity for self-replication. Because onIy one set of culture conditions was used and only colonies of one age analysed, the observed frequencies of spleen ' colony-forming cells in mixed colonies are probably minimal figures. In this context, the content of CFU-S in spleen colonies is rela-

411

tively low in early colonies. Thus in 8-day colonies a frequency of only 1-20 CFU-S per lo5 colony cells was observed (Metcalf and Moore, '71) a frequency comparable with that observed in 7-day mixed colonies grown in agar. It seems of importance to vary the culture conditions and time of sampling to determine whether higher frequencies of spleen colonyforming cells can be documented in such colonies under different conditions. Studies are also required on the long-term repopulating potential of such mixed colony populations in irradiated recipients. The present studies can be regarded as initial evidence that the mixed colonies in agar cultures stimulated by spleen conditioned media are formed by multipotential hemopoietic cells some of which have a capacity for generating cells assayahle as spleen colonyforming cells - the conventional test for hemopoietic stem cells. On this basis, at least some of the cells forming mixed colonies in agar cultures can tentatively be categorised as hemopoietic stem cells (CFU-S). From the present data and that published previously (Johnson and Metcalf, '78) it can be calculated that approximately 70/105 12day fetal liver cells form mixed colonies in agar. This frequency agrees well enough with estimates of CFU-S frequency in fetal liver populations (5-15/105 12-day CBA fetal liver cells in this laboratory; 5-13/105cells (Silini et iuplan, '68; Vogel e t al., '70; Niewisch et a1.7 al., '68;'7 ; Lowenberg, '751, when an f factor correction is applied to the latter figures. However there is a major discrepancy between the incidence of the two types of cell in adult marrow. The frequency of cells forming mixed colonies in agar is only 2-3/105marrow cells, much lower than the frequency of CFU-S (1525/105 CBA marrow cells in this laboratory and 20-40 for the above authors) when an f factor correction is applied to the latter figures. It remains t o be determined therefore whether the cells forming mixed colonies in agar are a subset of hemopoietic stem cells or represent a fetal type of hemopoietic stem cell that persists in adult life but in relatively low numbers.

1

ACKNOWLEDGMENTS

The authors are indebted to Doctor P. Lala for assistance with the karyotype analyses and to Doctor A. W. Burgess for supplying the mouse lung conditioned medium. They are

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D. METCALF, G. R. JOHNSON AND T. E. MANDEL

indebted to Mrs. S. Webb and Misses M. A. Lonergan and K. Haynes for skillful technical assistance throughout this work. LITERATURE CITED Borsook, H., K. Ratner and B. Tattrie 1969 Studies on erythropoiesis. 11. A method of segregating immature from mature adult rabbit erythroblasts. Blood, 34: 32-41. Burgess, A. W., J. Camakaris and D. Metcalf 1977 Purification and properties of colony-stimulating factor from mouse lung-conditioned medium. J. Biol. Chem., 252: 1998-2003. Dicke, K. A,, M. G. C. Platenburg and D. W. Van Bekkum 1971 Colony formation in agar: in vitro assay for hemopoietic stem cells. Cell. Tiss. Kinet., 4: 463-477. Duplan, J. F. 1968 Etude comparative des cellules souches hematopoietiques de la moelle osseuse et du foie foetal. Nouvelle Revue Francaise d’Hematologie, 8: 445-456. Gregory, C. J., E. A. McCulloch and J. E. Till 1973 Erythropoietic progenitors capable of colony formation in culture. State of differentiation. J. Cell. Physiol., 81: 411-420. Hayat, M. A. 1970 Principles and Techniques of Electron Microscopy. Vol. I. Van Nostrand Reinhold Co., New York, p. 264. Heath, D. S., A. A. Axelrad, D. L. McLeod and M. M. Shreeve progen1976 Separation of t h e erythropoietin-responsive itors BFU-E and CFU-E in mouse bone marrow by unit gravity sedimentation. Blood, 47: 777-792. Johnson, G. R., and D. Metcalf 1977 Pure and mixed erythroid colony formation in vitro stimulated by spleen conditioned medium with no detectable erythropoietin. Proc. Natl. Acad. Sci. (U.S.A.), 74: 3879-3882. 1978 Nature of cells forming erythroid colonies in agar after stimulation by spleen conditioned medium. J. Cell. Physiol., 94: 243-252. Karnovsky, M. J. 1965 A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. Cell Biol., 27: 137A-138A. Lin, H. S., and C. C. Stewart 1974 Peritoneal exudate cells. I. Growth requirement of cells capable of forming colonies in soft agar. J. Cell. Physiol., 83: 369-378. Lowenberg, B. 1975 Fetal liver cell transplantation. Radiobiological Research Institute, TNO Rijswijk. Metcalf, D. 1977 Hemopietic Colonies. Springer-Verlag, Heidelberg, New York.

Metcalf, D., and G. R. Johnson 1978 Production by spleen and lymph node cells of conditioned medium with erythroid and other hemopoietic colony stimulating activity. J. Cell Physiol., 96: 31-42. Metcalf, D., H. R. MacDonald, N. Odartchenko and B. Sordat 1975 Growth of mouse megakaryocyte colonies in vitro. Proc. Natl. Acad. Sci. (U.S.A.), 72: 1744-1748. Metcalf, D., and M. A. S. Moore 1971 Haemopoietic Cells. North-Holland, Amsterdam. Metcalf, D., G. J. V. Nossal, N. L. Warner, J. F. A. P. Miller, T. E. Mandel, J. E. Layton and G. A. Gutman 1975a Growth of B lymphocyte colonies in vitro. J. Exp. Med., 142: 1534-1549. Metcalf, D., J. Parker, H. M. Chester and P. W. Kincade 1974 Formation of eosinophilic-like colonies by mouse bone marrow cells in vitro. J. Cell. Physiol., 84: 275-290. Moore, M. A. S.,and N. Williams 1973 Functional, morphologic and kinetic analysis of the granulocyte-macrophage progenitor cells. In: Hemopoiesis in Culture. W. A. Robinson, ed. DHEW Publication No. (NIH) 74-205 Washington, pp. 17-27. Moore, M. A. S., N. Williams and D. Metcalf 1972 Purification and characterization of the in vitro colony forming cell in monkey hemopoietic tissue. J. Cell. Physiol., 79: 283-292. Niewisch, H., I. Hajdik, I. Sultanian, H. Vogel and G. Matioli 1970 Hernopietic stem cell distribution in tissues of fetal and newborn mice. J. Cell. Physiol., 76: 107-116. Parmley, R. T., M. Ogawa, S. G. Spicer and N. J. Wright 1976 Ultrastructure and cytochemistry of bone marrow granulocytes in culture. Exp. Hematol., 4: 75-89. Reynolds, E. S. 1963 The use of lead citrate a t high pH as a n electron opaque stain in electron microscopy. J. Cell Biol., 17: 208-212. Silini, G., S. Pons and L. V. Pozzi 1968 Quantitative histology of spleen colonies in irradiated mice. Brit. J. Haeniatol., 14: 489-500. Siminovitch, L., E. A. McCulloch and J. E. Till 1963 The distribution of colony forming cells among spleen colonies. J. Cell. Comp. Physiol., 62: 327-336. Spurr, A. R. 1969 A low viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res., 26: 31-43. Vogel, H., I. Hajdik, H. Niewisch, I. Sultanian and G. Matioli 1970 Growth kinetics of hemopoietic fetal stem cells. J. Cell. Physiol., 76: 117-126.

PLATES

PLATE 1 EXPLANATION OF FIGURES

l a A light micrograph of a 1-p section of a 7-day mixed colony grown in agar from 12day CBA fetal liver cells showing the irregular size of colony cells and the cell-free area surrounding t h e colony. Toluidine blue stain. X 150.

h A thin section of the colony illustrated in figure la. In this electron micrograph the heterogeneity of colony cells is evident, the major groups being erythroid cells, macrophages and cell debris. x 2,000.

414

MULTIPOTENTIAL COLONY~FORMINGCELLS D. Metcalf, G. R. Johnson and T. E. Mandel

PLATE 1

415

PLATE 2 EXPLANATION OF FIGURES

In figures 2 and 3 are shown eight fields of a single 7-day mixed colony grown from 12-day CBA fetal liver. Horizontal bars in all figures indicate a length of one micron. 2a

Erythroid cells (E) in varying stages of differentiation, portion of a macrophage (M) with ingested cell debris and a polymorphonuclear leucocyte. X 3,680.

b

Erythroid cells showing various stages of differentiation ranging from a n early erythroblast (El) to more differentiated erythroblasts (E2 and E3) and reticulocyte (E4).x 8,000.

c

A well-differentiated neutrophil (N), a n early erythroblast monocyte-like cell. Fragments of cell debris are also present.

d

416

(E)and portion of a X

4,880.

A relatively mature granulocyte, probably of t h e neutrophilic series. The nucleus is multilobed but the cytoplasm is still relatively pale and contains fewer granules than a fully differentiated neutrophil. X 8,225.

MULTIPOTENTIAL COLONY-FORMING CELLS D Metcalf, G. R. Johnson and T. E. Mandel

PLATE 2

417

PLATE 3 EXPLANATION OF FIGURES

3a Two blast-like cells (B) of unknown lineage and a well-differentiated neutrophil is also present. This electron micrograph illustrates the presence within the same colony of cells of widely differing degrees of maturity. X 6,480.

b A granulocyte-like cell showing numerous large electron dense particles. Similar cells were readily identified in the light microscope in 1-psections by their content of numerous highly stained granules. The granules lack the characteristic bar characteristic of murine eosinophil granules, and the cell may be in the basophil lineage. x 9,400. c , d These two electron micrographs illustrate very large cells that appear to be megakaryocytes. Note their convoluted nucleus which in thin sections has the appearance of being multiple. c X 4,140;d X 3,680.

418

MULTIPOTENTIAL COLONY-FORMING CELLS D. Metcalf, G . R. Johnson and T. E. Mandel

PLATE 3

419

MULTIPOTENTIAL COLONY-FORMING CELLS D. Metcalf. G. R. Johnson and T. E. Mandel

FETAL LIVER SCM

PLATE 4

FETAL LIVER MLCM

BONE MARROW

BONE MARROW

SCM

MLCM

0

0

0 0

t?

0

0

0 0

m

4

-o a k - - -

EXPLANATION O F FIGURE

4

420

The size of individual daughter colonies recloned from 7-day colonles grown either from 12-day CBA fetal liver cells or adult CBA marrow cells stimulated either by spleen conditioned medium (SCM) or mouse lung conditioned medium (MLCM). Closed circles indicate macrophage daughter colonies, open circles, daughter colonies of other types.

& 0