Boeyum, A.

Carsten, A.L.

Laane, B.

Pettersen, E.O.

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BNL 17987THE DIFFUSION CHAMBER TECHNIC AS PROGENITOR (STEM) CELL ASSAYArne B(6yum , Erik 0 Petters»n , Blrger Laane , and2Arland L. Carsten-NOTICE-This report was prepared as an account of worksponsored by the United States Government. Neitherthe United States nor the United States Atomic EnergyCommission, nor any of their employees, nor any oftheir contractors, subcontractors, or their employees,makes any warranty, express or implied, or assumes anylegal liability or responsibility for the accuracy, com-pleteness or usefulness of any information, apparatus,product or process disclosed, or represents that its usewould not infringe privately owned rights.1. Norwegian Defence Research EstablishmentDivision for ToxicologyP.O. Box 25 - N-2007 KjellerNorway.2. Medical Research CenterBrookhaven National LaboratoryUpton, New York 11973MASTER*This work was supported in part by the U.S. Atomic Energy Commission.DISTRIBUTION OF THIS DOCUW1EN? IS UNLIM1TEJ2.ABSTRACTThe hemopoietic response in mice to X-irradiation and treatmentwith hydroxyurea or cyclophosphamide was studied by comparingthe diffusion chamber (DC) technic and the spleen colony technic.The hydroxyurea experiments showed that DCPCs (diffusion chamberprogenitor cells) had a higher prolIterative rate than spleencolony forming units (CFUs).The radiation response indicated that 7 day DC culturingmeasures two different types of progenitor cells, one relativelyradioresistant with D of 135 rads and one with radioser.sitivitycomparable to that of CFUs (50 rads).Two hours after cyclophoaphamide treatment, erythroid CF'J9were almost eradicated indicating that cyclophosphamide selectivelyblocked the ability of multlpotent stem cells to erythroid differentia-tion. The number of granuloid CFUs and DCPCs were less affected.It is concluded th«t DC culturing probably measures a mixtureof multipotent and unipotent stem cells.3.INTRODUCTIONThe environment of the diffusion chambers (DC) implantedintraperitoneally provides good growth conditions for stemcells, granulocytes and macrophages. After an initial loss ofspleen colony forming units (CFUs) amounting to approximately50 per cent, Che CFUs will multiply in the DC with a populationdoubling time of 20 hours. Their number on day 3 is equal to2or greater than the input value. The spleen colonies generatedby CFUs harvested from 1-day or 4-day DC cultures showed a type2distribution similar to that observed with normal marrow cells .In irradiated animals, the DC-CFUs proliferate faster than in normalhosts .The granulocytes and macrophages are growing rapidly from day2-3 of cultuving and after 7 days there may be a 10-fold increasein cellularity, when normal steady-state marrow cells have beeninoculated . Erythropoiesis has been observed when autologous4cells are cultured in rabbits . In mouse cell cultures there isno erythropoiesis unless the host animals are injected with erythro-2poietln or subjected to intermittert hypoxia . Even then the ery-thropoieflis is too scant to be useful for most practical purposes.The present paper describes attempts to characterize theprogenitor cells measured by the DC technlc. Hemopoiesis in themouse was studied by comparing the DC-technic and the spleencolony assay (SCA).MATERIALS AMD METHODS5 6 2Details of the DC technic ' and the SCA have been describedpreviously.An estimate of the number of stem cells (diffusion chamberprogenitor cells, DCPC) that are the ancestors of differen-tiated cells harvested after a given culture period may beobtained in two different ways:An absolute estimate of DCPC may be obtained by limitingdilution analysis ' . In short, the principles of this technicare as follows: Chambers containing small numbers of bonemarrow cells are implanted. If myeloblasts and promyelocytesare found in a chamber smear after a culture period of 7 days,it is assumed that the chamber contained one or more "effective"DCPC initially. Chambers which contain no granulocytes or onlymature granulocytes and/or macrophages are scored as empty.The probability, P(0) of having no DCPC in a chamber is givenby the equation applying to a Poisaon distribution.li P(01 - e "X where P((» - Number of empty chambers1) *(U) ere *< O ) Total number of chambersand X • the average number of DCPC per chamber which gives2) X - -In P(0)Thus, the average number of DCPC par DC can be estimated bydetermining the fraction of empty chambers. By this technicwe have found that in normal mouse marrow there is 1 DCPCper 1500 - 2000 bone marrow cells. This certainly is an under-5.estimate, but it is a reproducible procedure. Further, itis not necessary to count the cells, and the interpretationof the smears is quite rapidly performed. The disadvantageis that a large number of chambers is required to get areliable estimate.A relative estimate of the DCPC may be obtained from themean number of cells harvested per chamber. This estimatemay be expressed as,NCytopoietic capacicy (per femur) - jq—— fenrwhere N is the number of cells harvested, N, the numberof cells inoculated, and N, the number of cells per femur.r emIf the number of cells per femur in the experimental groupdeviates from the control, a relevant comparison can only bemade by calculating the cytopoietic capacity per femur, whichexpresses the number of cells that would have been harvestedif all cells in one femur had been cultured.RESULTS AND DISCUSSIONThe relationship between the number of cells inoculatedand the number of cells harvested is demonstrated in Fig. 1.In 7-day cultures grown in normal mice, the cytopoietic capacitydecreased significantly with increasing inocula , in particular4 5in the range from 2x10 to 1x10 cells per chamber. A similarbut less pronounced effect was seen in 4-day cultures. Thusoprevious findings indicating no feedback inhibition in 4-daycultures were not confirmed. The findings were almost similar6.when the cells were cultured in irradiated hosts (500 rads) . Theproblem of this cell-dose effect can largely be avoided by usinginocula In the range 1-2x10 ; it should be kept in mind that thegrowth is sub-maximal with these greater inocula.Table 1 shows the proliferative rate of progenitor cells,as determined with hydroxyurea injections. DCPCs had a higherprolIterative rate than CFUs, so obviously the two assaysystems do not assay identical populations. The CFUs in normal9 10marrow are predominantly multipotent stem cells (MSC) ' ,and it seems likely that the DC technic to a large extentmeasures unipotent (committed) stem cells (USC)Another comparative study is demonstrated in Fig. 2, whichshows the radiosensitivity of progenitor cells. Appropriatecorrections were made for the cell dose effect (Fig. 1) fromexperiments in which DC containing varying numbers of normal12bone marrow cells were cultured in irradiated hosts (500 rads)The CFUs were highly radiosensitive, and the survival curvewas exponential with a D of 50 rads. The radiation responseof DCPCs as measured with 7-day cultures was not very differentfrom that of CFUs for small doses, but above 60 rads Che curvewas exponential with a D of 135 rads. With 4-day cultures,the surviving fraction for small doses «a« •omevh&t largerthan for 7-day cultures. For doses between 80 and 200 radsthe curve exhibited a shoulder and for even larger doses thecurve was exponential with a D of 132 rads.These experiments Indicate a significant different radiationsensitivity for CFUs and DCPCs, and they show that differentprogenitor populations are assayed vith 4 and 7-day cultures.The survival curve for 7-day cultures may be a composite of7.two exponential dose response curves with different D ,corresponding to a very sensitive and a lesa sensitive celltype. The survival curve of the most resistant cell hasa D equal to the D of our experimental curve for highdoses, i.e. 135 rads. It follows from geometrical considera-tions that the extrapolation number for this part of the curve,which is 0.59, expresses the fraction of resistant cells. Thus,in 7-day cultures, we may be measuring a mixture of 40 percentvery sensitive and 60 percent less sensitive progenitor cells.Using these values it is possible to show that a combination oftwo exponential curves, one with a D of 135 rads and the otherwith a D of 50 rads will give a curve that is almost identialto the experimental one for 7-day DC culturing. ThiE mayindicate that the most radiosensitive cell giving rise to differ-entiating cells in DC are CFUa.The survival curve obtained with 4-day cultures refiectean even more complex radiation response than 7-day cultures.The broad shoulder .aay indicate the presence of an additionalprecursor cell type, which to some extent is able to accumu-late sublethal radiation damage.The use of cytotoxic agents may also be helpful in attemptsto characterize progenitor cells by comparing the effect indifferent assay systems. We have examined the effect of oneor two injections of cyclophosphamid* on CFUs and DCPCa (Fig. 3).8.Two hours after the injection, the number of erythroid CFUswas reduced to 2.5 percent of the control. Thereafter, erythroidCFUs increased exponentially with a doubling time of 20 hours.A second injection 4 days later had a similar effect, showingthat it was independent of their proliferative rate. The findingsindicate that cyclophosphamide preferentially impairs the abilityof MSC to differentiate towards the erythron. Granuloid CFUs andDCPCs were less affected. These two types of progenitor cellsshowed a similar pattern of depletion end regeneration, butthe curves were not identical, again suggesting that theDCPC and CFUs are not identical.In conclusion, the diffusion chamber technic apparentlyassays a mixture of MSC and USC. We are planning to extendthe irradiation experiments in order to examine whether theCFU fraction of the DCPC population will Increase when theculture period is prolonged.9.REFERENCES1. Bdyum, A,, C*rsten, A.L., Lae rum, O.D. and Cronkite, E.P.:Kinetics of cell proliferation of murine bone marrow cellscultured in diffusion chambers: Effect of hypoxia, bleeding,erythropoietin injection, polycythemia, and irradiation ofthe host. Blood 40, 174, 1972. '2. Breivik, H., Ben eat ad, H.B., and Bdyura, A.: Diffusionchamber and spleen colony aasay of murine hematopoietic stemcella. J. Cell Phyaiol. J_, 65, 1971.3. Bdyum, A. and Breivik, H.: Kinetics of murine haemopotettccell proliferation in diffusion chambers. Cell Ties. Kinet.S, 101, 1973.4. Grigoriu, G., Antonescu, M. and lercan, E.: Evidence fora circulating stem cell: Newly formed erythroblasCs found inautologous leukocyte-filled diffusion chambers inserted intobled rabbits. Blood 37, 187, 1971.5. Benestad, H.B.: Formation of granulocytes and macro-phages in diffusion chamber cultures of mouse blood leukocytes.Scand. J. Haemat. T_, 279, 1970.6. Bdyum, A., and BorgstrAn, R : The concentration ofgranulocytic stem cells in raouie bone marrow, determined withdiffusion chamoer technique. Scand. J. Haemat. T_t 294, 1970.10.7, Brelvik, H.: Haematopoietic stem cell content of murinebone marrow, spleen end blood. Limiting dilution analysis ofdiffusion chamber cultures. J. Cell Physiol. 1_, 73, 1971.8, Breivik, H., and Benestad, H.8.: Regulation of granu-locyte and macrophage formation in diffusion chamber culturesof mouse haematopoietic cell9. Sxp. Cell. Res. 22. 340» 1972.9, Wu, A.M., Till, J.E., Siminovitch", L. and McCulloch, E.A.:A cytological study of the capacity for differentiation ofnormal hemopoletic colony-forming cells. Ccll.Phyaioi. 69,177, 1967.10. Curry, J.L., Trentin, J.J., aad Wolf, N.: Hemopoieticcolony studies II. Erythropoiesis. J. Ex^. Med. 125, 703,1967.11. Benestad, H.B. and Breivik, H.: Properties of haema-topoietic progenitor cells. Acta Physiol. Scand. j$3, 339,1971.12. Pettersen, E.O., B<5yum, A., I.aane, B.F.M.: X-ray inactivationof murine bone narrow cells as measured by the spleen colonyassay and the diffusion chamber technique. (To be published1*.13. B^yum, A., Carsten, A.L- and Lae rum, O.D.: Haem&jopoiesis inmice treated with one or two injections of eyelophosphamide,as measured by the spleen colony and the diffusion chambertechniques. (To be published).Table 1Per cent of progenitor cells (per femur) surviving hydroxyurea treatmentCFU Absolute number Relative number82 * 11 53 * 12 38 ! 8(46) (138) (34)Male (C,H x DBA)F, hybrid mice were injected with hydroxyurea (900 mg/kg)or saline. Bone marrow was sampled two hours later to be tested with thespleen colony and the diffusion chamber techniques. Diffusion chamberprogenitor cells (= DCPC) were determined in absolute number by limitingdilution analysis and in relative number from culture cellularity. Thevalues are given as mean t SE in per cent of saline-injected controls.The number of observations (spleens or chambers) are shown in parenthesisand the controls comprised a similar number.LEGENDS TO FIGURESFigure 1. The figure shows the cytopoietic capacity of various size inocularelative to the cytopoietic capacity of control chambersinoculated with 10 bone marrow cells. Each experiment comprised7 chambers per cell dose and the number of experiments is indicatedat each point. Means and their SE are shown.Figure 2. The radiosensitivity of CFUs and DCPCs 'diffusionchamber progenitor cells) of (CUHxDBA) Fj hybrid mice. The hostanimals were irradiated before and after chamber implantation Insuch a way that all mice received a total dose of 500 rads,whereas the implanted cells were irradiated with varying doses.Bach point represents 4°-5 experiments each comprising 7 chambers.In the spleen colony experiments the animals were similarlyirradiated before and after the injection of cells and receiveda total dose of approximately 900 rads (12-13 spleens per point).Figure 3. Hale-Stoner-Brookhaven mice were injected with eyelophosphamide(200 nag/kg) and the bone marrow cells were harvested from twohours to 4 days after the injection and tested with spleen colonyassay and by DC culturing (limiting dilution method). Groupsof mice received a second injection of eyelophosphamide 4 daysafter the first injection. The spleens were examined hi.sto-logically and each point represents 3-4 spleens.uuUJOo.ouUJ>1UJ1.816|U1210080.604iU1.2100.80.67 DAY DC-CULTURESriDAY DC-CULTURES203210* ^io1 io* 210s / /U~lo6NUMBER OF CELLS INOCULATEDFigure 2n=1 75DOSE, RAD• 4 DAY DC-CULTURES* 7 DAY DC ̂ CULTURES•CFUFigure 3toCFUs AND DCPCs PER FEMUR2 ^YCLOPHOSPHAMIDE.b lOCPCsCOLONY TYPEI l l6 7DAYS

Diffusion chamber technic as progenitor (stem) cell assay

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