Br. J. Cancer (1974) 30, 421

QUANTITATION OF HAEMOPOIETIC CELLS FROM NORMAL ANDLEUKAEMIC RFM MICE USING AN IN VIVO COLONY ASSAY

M. Y. GORDON

Froml the Department of Radiobiology, Medical College of St Bartholomew's Hospital, London

Received 12 June 1974. Accepted 26 June 1974

Summary.-The conventional diffusion chamber (CDC) as described by Benestad(1970) had been modified to assay the colony forming capacity of RFM bone marrowand spleen cells in agar diffusion chambers (ADCs). The colonies are morpho-logically identical to those formed by the CFUc in agar culture in vitro and have anincidence of approximately 1 in 103 normal nucleated bone marrow cells, and 1 in104 nucleated spleen cells. Comparison of the growth of normal bone marrow cellsin CDCs and in ADCs suggests that cell proliferation in diffusion chambers mayresult from the same precursor cell as detected by colony formation in agar culturein vitro. This proposal is supported by the suicide of approximately 46% of the ADCcolony precursor cells following incubation with 3H-labelled thymidine.

Colony formation by haemopoietic cells taken from leukaemic mice appears to bedue to the proliferation of a remaining normal cell population alone, while theleukaemic cells in the inoculum form a background of uniformly distributed blastcells. In the case of leukaemic cell culture, there are differences in the results fromCDCs and ADCs, and data from colonies in leukaemic ADC cultures are similar tothose from normal ADC colonies. These comparisons imply that the ADC techniquemay be used to monitor the functional capacity of normal bone marrow, by its abilityto form colonies, during the development of leukaemia. A humoral effect of a leukae -mic environment on the growth of normal bone marrow cells in ADCs has also beendetected.

HAEMOPOIETIC precursor cells are func-tionally detectable by a variety ofin vivo and in vitro methods, including thespleen colony (Till and McCulloch, 1961),agar colony (Pluznik and Sachs, 1]965;Bradley and Metcalf, 1966) and theintraperitoneal diffusion chamber (Bene-stad, 1970) techniques. The recent mor-phological identification of a possiblehaemopoietic stem cell in rodents and inprimates (Van Bekkum et al., 1971; Dickeet al., 1973b; Rubinstein and Trobaugh,1973), together with cell separation andcell kinetic studies (Metcalf and Moore,1971), has allowed speculation concerningthe identity of the cell types assayed bydifferent systems.

Until recently, the assay of haemo-poietic stem cells, depended upon in vivotechniques which may be divided intothose methods which involve cell graftingand those which do not. However, the

end-points used for these assays do notnecessarily measure the same aspect ofstem cell function (Lajtha and Schofield,1969). With the development of tissueculture techniques for haemopoietic pre-cursor cells, claims have been made forthe detection of pluripotential stem cellsboth in agar in vitro (Dicke, Platenbergand van Bekkum, 1971) and in intraperi-toneal diffusion chambers (Boyum andBorgstrom, 1970; Brievik, Benestad andBoyum, 1971). Other essentially similarculture systems appear to measure theincidence of a more committed precursorcell leading to the production of granulo-cytes and macrophages (Metcalf and Moore1971; Gordon and Coggle, 1974).

This paper describes a method com-bining the advantages of the conventionaldiffusion chamber (CDC) with those of theagar culture technique, which has beenused to assay bone marrow and spleen cells

M. Y. GORDON

from both normal and leukaemic RFAImice. CDCs and ADCs, containingaliquots of the same cell suspension, maybe inserted into host mice, thus allowingquantitation of colony forming capacityand total and differential cell produLctionunder the same physiological conditions.Furthermore, the complications intro-duced by the wide variety of colony stimu-lating factors (CSF) for agar colony growthin vitro, and their unikniown physiologicalsignificance (Metcalf, 1974) are avoided inthis in vivo culture system.

The use of both the agar (Pike ancdRobinson, 1970; Iscove et al., 1971)and the diffusion chamber methods (Car-sten, Boyum and Boecker, 1972; Boyumet al., 1972a) for human bone marrowculture indicates the potential usefulnessof combining the advantages of these tech-niques for haemokinetic studies in man.

MATERIALS AND METHODS

Experimental animials and cells for culture.Bone marroN cells flushed from the femurs

of 3-month old mnale RFM mice, or single cellsuspensions of their splenic tissue, wi-ere usedfor all diffusion chamber experiments. Theleukaemic donor mice had received 5 x 105leukaeinic spleen cells, from a cell line ofmyeloid leukaemia maintained by weeklypassage, which results in their death 8-9 daysafter i.v. injection.

SAS/4, RFM, CBA/T6T6 and C3H maleand female mice have been used as hosts indiffusion chamber experiments. Each seriesof experiments included a control assay of thecolony forming capacity of normal RFM bonemarrowT or spleen cells.

Diffusion chamber culture techniques.-CDCs are made by attaching a Alillipore filter(pore size 0-22 Eum) to each side of an acrylicring by the solvent action of acetone. Asmall hole in the ring allows injection of ahaemopoietic cell suspension into the cham-ber. Completed chambers are tested forleaks, sealed in Perspex containers andsterilized by exposure to 2-5 Mrad 15 MeV7x-rays: the chambers remain sterile until theseal is broken, and each chamber is filled tocapacity (350(ul) under sterile conditions.

The holes in the ring are sealed w%vith paraffinwax and one chamber is inserted into t,heperitoneal cavity of each host. Cells arerecovered in suspension from CDCs usingFicoll-pronase solution as described byBenestad (1970).

ADCs are chambers which have beenmodified by replacing the Millipore filter onone side of the acrylic ring w%vith a glasscoverslip. The medium used for culturewas Eagle's MEM supplemented wNith 20%horse serum, L-glutamine, L-asparagine andantibiotics (penicillin and streptomycin).

Molten agar, held in a wAater bath at 45°C,is added to the medium w%hich contains t,herequired number of cells for assay, to a finalagar concentration of 0-30. 350 pul of thismixture is injected into each chamber andallowed to set at room temperature beforesealing and insertion into host mice.

Examitination ofADC cultures. The ADCsare removed from the peritoneum of the host,wviped clean wvith a tissue and the singleMillipore filter gently peeled awNay from theacrylic ring. The agar gel is left supportedby the ring and coverslip. The transparencyof the coverslip facilitates the scoring ofcolony numbers using a dissecting microscopeat a magnification of x 25. Aggregates ofmore than 50 cells are scored as colonies,following the most frequently acceptedcriterion for scoring CFUc in agar culture invitro. Aggregates of fewer than 50 cells areconsidered as clusters.For histological examination, individual

colonies are picked out of the agar andsquashed between 2 glass slides w%ith a dropof horse serum. The slides are then gentlyseparated by sliding them apart, and theresulting smears air dried and stained wAithMay-Griinwald and Giemsa.

Thymidine suicide technique.-The methodused to investigate the level of 3H-labelledthymidine suicide of ADC precursor cellsw%as similar to t,hat described by Metcalf(1972). A sample from a bone marrow cellsuspension was incubated for 20 min at37°C with 3H-labelled thymidine (specificactivity 19 Ci mmol-1) at a concentration of250 ,uCi ml- 1 in Eagle's MEM. A secondsample was used as a control and was incu-bated under identical conditions in mediumcontaining unlabelled thymidine. Followingincubation, both the radioactive and thecontrol cell suspensions were wTashed 3 timesbefore in vivo incubation in ADCs.

422

QUANTITATION OF HAEMOPOIETIC CELLS

RESULTS

NAormal cell cultureSome of the nucleated bone inarrow or

spleen cells incubated in intraperitonealADCs proliferate to form colonies of granu-locytes and macrophages. The gross mor-phologies of these colonies are identicalto those seen in CSF stimulated agarcultures in vitro (Metcalf and Moore, 1971)

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and loose, mixed and compact types arepresent.

At the time of chamber insertion,single cells are seen in suspension in thesemi-solid agar medium. Thereafter, cer-tain precursor cells proliferate to formclusters and colonies, the largest of whichare visible to the naked eye 9 days afterimplantation, and contain at least 2000cells. This incubation period was usedfor routine scoring of colony numbers,although cultures have been maintainedfor 13 days with little sign of colonydegeneration. The range of colony sizefound in ADCs is as extensive as thatfound in agar cultures in vitro (Metcalfand Moore, 1971), indicating a similar

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FIG. la. The relatioinship between the number of intucleated bone marrow cells per chamber andthe number of ADC colonies scored 9 days after implantation. (SAS/4 male mice, :3 months old,wvere use(l as (liffusion chambei hosts in this experiment.)

Fi(e. lb. The relationship between the number of nucleated spleen cells per chamber and thenumber of ADC colonies scored 9 (lays after implantation. (SAS/4 male mice, : months old,w,ere used as (liffuision chamber hosts in this experiment.)

I

423

I I I I

M. Y. GORDON

TABLE I.-The Effect of SAS/4 Host Age and Sex on RFM Bone Marrow ColonyGrowth in ADCs

Age (months)369

Female hostsr

Coloiiies per 105cells No. of chambers

80-577±72192 83 ± 7 8291 73 ± 5 57

76

1 1

heterogeneity of precursor cells in bothsystems.

Promyelocytes and myelocytes arefound. in both compact and loose aggre-gates removed from 3-day old cultures.From the 5th to 13th days of incubation,loose colonies are the predominant morpho-logical type, although mixed and compactcolonies persist throughout tile cultuireperiod. Matture granulocytes are fouindin 6-day-old loose colonies, together withmacrophages and the earlier stages ofgranulocytic differentiation. Compactcolonies, together with macrophages andcolonies, which comprise approximately5%0 of the total colony number, containonly promyelocytes and myelocytes.

The (lata presented in Fig. la and bshow the linear relationship between thenumber of nucleated RFAI bone marrowor spleen cells per chamber and the numberof colonies scored. Using SAS/4 mice ashosts for the ADCs, RFM bone marrowcolony precursors have an incidence ofapproximately 1 in 103 nucleated cells,while the incidence of the spleen is 1 in 104nucleated cells.

Table I shows that neither the age niorthe sex of adult SAS/4 hosts has ainysignificant effect on the yield of coloniesfrom normal RFM bone marrow. How-ever, the strain of the host does influencethe efficiency of colony formation, and theuse of C3H mice as ADC hosts increasedthe number of precursor cells detected by5400 relative to SAS/4 hosts, while thereis no advantage in using syngeneic RFMhosts. The yield of colonies is alsoincreased when the CBA/T 6T 6 strain ofmice is used as the host (Table II).

Male hosts

Colonies per 105cells No. of chambers

86 83±9-39Not (lone93 18±7 64

6

1 1

TABLE II. The Effect of Host Strain onRFM Bone Marrow Colony Growth inADCCs

Strain (3-mnonithol(1 male mice)SAS/4RFAICBA/T 6T6CMH

Colonies per5 x 104 cells70 13 ± 7 - 7270*29± 8-01

105 17+ 8-30117 :38 -11 12

No. ofchambers

8768

Incubation with 3H-labelled thymidinecauses the suicide of the cells in S phase ofthe cell cycle at the time of administration.The surviving fraction provides an indica-tion of the nuimber of cells in cell cycle.Table III shows that approximately 46%of the ADC colony precursor cells aredestroyed by incubation with 3H-labelledthymidine, leaving a surviving fractionof 540% and indicating that most of theADC colony precursor cells are in activecell cycle.

The comparison of normal RFAI bonemarrow growth in ADCs and CDCs(Table I), initially containing 1 0 5nucleated cells, shows that the percen-tages of granulocytes and macrophagesrecovered in suspension (CDCs) or fromcolonies picked out of the agar (ADCs)are similar, especially when the data fromthe 3 colony types are pooled. Thisresult indicates that growth in CDCs maybe due to proliferation of the colony pre-cursor cells detected in ADCs.

Leukaemic cell cultutreThe development of colonies from

leukaemic bone marrow and spleen cellswas investigated using cells from terminalRFAI mice. Colonies which were normal

424

QUANTITATION OF HAEMOPOIETIC CELLS

TABLE III.-Reduction ofRFM Bone Marrow Colony Forming Capacity by ThymidineSuicide

No. of colonies per chamber±s.e.*

No. of cells Incubation without Incubation withper chamber 3H-thymidine 3H-thymidine Surviving fraction (0o)Ix 105 140 ± 6-36 74 67±9 43 53-34±11-377 5x104 96-4±10-48 47-60±6-07 49-38±12-115-0x104 60 3± 3-52 35 00±3 35 58-01±3 68

* C3H male mice, 3 months old, were used as diffusion chamber hosts for this experiment

TABLE IV.-Cornparison of the Growth of Normal RFM Bone Marrow in ADlCsand CDCs, 9 Days after Implantation

ADCsColonies/chamber 92 83 ± 7 82oo granulocytic cells 32 loose/0 macrophages 68 colonies00 granulocytic cells 51 mixed.0 macrophages 49 colonies0 granulocytic cells 100 compact0 macrophages colonies00 granulocytic cells 61 mean for00 macrophages 39f all colony

types

in their morphology, development andcytology appeared against a backgroundof leukaemic blast cells.

The changes in the colony formingcapacity of bone marrow and spleen cellsduring the development of leukaemia areshown in Fig. 2. The most markeddecrease is in the number of bone marrowcolonies per chamber, where the terminalyield is approximately 3%0 of the controlyield. The colony yield from aliquots ofterminally leukaemic spleen cells is 20%of the control yield. If the data fromleukaemic spleen ADC cultures are cor-rected for the increase in splenic cellularityduring the development of leukaemia(Gordon, 1974) the relative decrease incolony formation per spleen is 40%0(Fig. 2).

The data given in Table V may becompared with those given for normalcells in Table IV. In the case of leukaemicCDCs and ADCs, there are strikingdifferences in the cell types recovered.The predominance of granulocytic (leukae-mic blast) cells in aliquots of the totalCDC contents may be due to undetectablylow numbers of macrophages when cellsare scored from smears of cell suspensions.

29

CDCsTotal nucleate(d cellsx 105 7-53±0-650 granulocytic cells 460 macrophages 450 unidentified 9

The results for the ADCs are taken fromcolonies removed from the agar and notfrom the dense background of single cells.The relative numbers of granulocyticcells and macrophages are similar innormal and leukaemic ADCs, indicatingthat the colonies scored in leukaemic ADCcultures are derived from normal cells andthat this technique measures a parameterof the capacity of normal granulocyteand macrophage production during thedevelopment of leukaemia.

The effect of a leukaemic host environ-ment on normal bone marrow colonygrowth in ADCs was determined byincubating RFM bone marrow cells incontrol syngeneic hosts and in RFM miceimmediately after they had received ani.v. injection of 5 x 105 leukaemic spleencells. The lifespan of the leukaemic miceallowed an incubation period of 8 days, andthe results given in Table VI show a 280%stimulation of normal colony formationin leukaemic hosts.

DISCUSSIONMuch progress has been made in the

development of methods used for moni-toring the functional capacity of normal

425

M. Y. GORDON

--- 0 Spleen (colonies/106 cells)- 0O Spleen data corrected for increase

in splenic cellularity

- - av 8 9Time after transplantation (days)

FiG. 2.-Changes in the colony forming capacity of bone marrow and spleen cells during the develop-ment of leukaemia in RFM mice.

TABLE V.-Comparison of the Growth of Terminally Leulkaemic RFM Bone MarrowCells in ADCs and CDCs, 9 Days after Chamber Implantation

ADCsColonies/chambera3 * 47i O - 99% granulocytic cel)e 47) loose% macrophages 53 f colonies% granulocytic cells 441 mixed% macrophages 56f colonies% granulocytic cells 86 5 compact% macrophages 13*5f colonies% granulocytic cells 59) mean for% macrophagcs 41 f all colony

types

CDCsTotal nucleated cells x 10-5 71 5±0 6% granulocytic cells 100% macrophages

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M. Y. GORDON

TABLE VI.-The Effect of the Developmentof Leukaemia in Host Mice on RFMBone Marrow Colony Growth in ADCs

Colonies per No. of75 x 104 cells chambers

Normal RFM hosts 68-13+5-97 8Leukaemic RFM hosts 92-25±5-37 8

haemopoietic precursor cells during thedevelopment and treatment of diseases,such as leukaemia, in man. Until 1965,the spleen colony method was the onlytechnique available for the quantitationof haemopoietic stem cells (Till andMcCulloch, 1961) but its applicationremains restricted to rodents. The agarcolony (CFUc) assay (Pluznik and Sachs,1965; Bradley and AMetcalf, 1966) has beenused clinically to assay normal and leuk-aemic human bone marrow (Pike andRobinson, 1970; Iscove et al., 1971).However, the advantages of this in vitrosystem are accompanied by the difficultiesin the standardization of the techniqueand the complex terminology associatedwith different sources of colony stimulatingfactor (Metcalf, 1973). It is generallyaccepted that the agar colony precursorcell is a committed cell which is able toproduce both granulocytes and macro-phages in vitro (Metcalf and Moore, 1971).

The application of the intraperitonealdiffusion chamber technique to the growthof murine haemopoietic cells (Benestad,1970) and its later use in the culture ofhuman bone marrow cells (Carsten et al.,1972; Boyum et al., 1972b) avoided thecomplications of CSF, but allowed totaland differential cell production to bemeasured rather than colony formation.

It is clear that simultaneous measure-ments of total and differential cell produc-tion and of the colony forming capacityof haemopoietic precursor cells would beadvantageous to both experimental andclinical haematology. This paper hasdescribed a method in which CDCs andADCs, containing identical cell samplesincubated under the same conditions,may be used to measure these 3 parametersof bone marrow function.

Although the use of intraperitoneal

diffusion chambers (CDCs) does not requirethe addition of CSF, it has been shownthat cells in diffusion chambers are exposedto a humoral agent which can act as asource of CSF in agar in vitro duringintraperitoneal incubation (Gordon, Coggleand Lindop, unpublished data). Thelevel of this endogenous stimulating factormay vary under certain conditions, as inleukaemia (Table V) and between strains(Table II).

There are reports in the literature ofincreased CSF levels in leukaemic patientswhich have been reviewed by Metcalf(1971, 1973) and pretreatment of hostanimals has been shown to improve resultsfrom CDCs (Boyum et al., 1972a). Dicke,Riou and Platenberg (1973) pretreat thehost animals by irradiation before theyare used in their Millipore chamber agarsystem.

The growth of granulocytic and macro-phage colonies in ADCs suggests that theymay originate from the same precursorcell as the in vitro CFUc. This idea issupported by the data from the 3H-labelled thymidine experiments (TableIII). 3H-labelled thymidine has beenwidely used as an agent for killing cellsin S phase to give an indication of thefraction of cells in cell cycle, and has alsobeen applied to the spleen colony (CFUsor stem cell) and agar colony (CFUc)precursor cells. Information on the suicid-ing of CFUs and CFUc has been reviewedby Metcalf and Moore (1971). Approxi-mately 10% of CFUs are killed by expo-sure to 3H-labelled thymidine, while up to50% of CFUc are killed by this treatment.The suicide of some 46% of ADC colonyprecursor cells following incubation with3H-labelled thymidine suggests that theyhave a close relationship to the cellsdetected by colony formation (CFUc) invitro.

The results from cultures of leukaemiccells in ADCs show that there is a rapiddecline in bone marrow haemopoiesis,assayed by this particular technique,whereas the level of normal colony pro-duction falls more gradually in the spleen.

427

428 M. Y. GORDON

The differences in the rates of decline inthese 2 haemopoietic organs may bedue partly to the fact that the spleen isable to increase in size, so that the normalprecursor cells are diluted by leukaemicblast cells while the volume of the marrowcavity is constant.

Results from CDCs and ADCs arecompatible in normal bone marrow cellcultures (Table IV), while in leukaemiccell cultures the cell content of the coloniesdiffers from the cells recovered in suspen-sion from CDCs (Table VI). This findingmay be of diagnostic significance if humanbone marrow were to be cultured inCDCs and ADCs.

The ADC system provides a methodfor quantitating the functional capacityof normal haemopoietic precursor cells,by their ability to form colonies in leukae-mic RFM mice. Further experimentsare in progress to assess the significanceand timing of changes in the colonystimulating substance in RFM mice withmyeloid leukaemia.

The work was supported by grantsfrom the U.S.P.H.S. (Grant no. 00082)and the Cancer Research Campaign. Theauthor wishes to acknowledge the helpfuladvice of Professor Patricia J. Lindop.

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