Proc. Natl. Acad. Sci. USAVol. 90, pp. 4354-4358, May 1993Cell Biology

Hemopoietic stem-cell compartment of the SCID mouse:Double-exponential survival curve after y irradiation

(radiation sensitivity/scid mutation/severe combined immunodeficiency)

SATOSHI TANIGUCHI*t, YOKO HIRABAYASHI*, TOHRU INOUE**, MASAYOSHI KANISAWA*, HIDEKI SASAKI§,KENSHI KOMATSU1, AND KAZUHIRO J. MORItDepartments of *Pathology and §Pediatrics, Yokohama City University School of Medicine, Yokohama-236, Japan; tDepartment of Biology, Faculty ofScience, Niigata University, Niigata-950-21, Japan; and lRadiation Biophysics, Atomic Disease Institute, Nagasaki University, Nagasaki-852, Japan

Communicated by Eugene P. Cronkite, February 10, 1993

ABSTRACT It has been reported that SCID (severe com-bined immunodeficiency, scid/scid) mice are more radiosen-sitive than normal mice. In the present studies, graded doses ofradiation were given to bone marrow cells from SCID mice,and double-exponential survival curves were observed forday-9 and day-12 colony-forming units in the spleen (CFU-S).Single-exponential curves were found for SCID CFU in in vitroassays for granulocyte/macrophage-CFUs and erythroidburst-forming units, as reported elsewhere. Since the size ofthis more resistant fraction seems to decrease with stem-cellmaturation, the finding implies that this fraction is a primitivesubpopulation of the stem-cell compartment. The mean lethaldose (Do), however, of this less sensitive SCID CFU-S is muchless than the Do of regular CFU-S in normal littermates. Spleencolonies produced by SCID bone marrow were relatively smalland abortive. The size of these colonies decreased nearlyexponentially with increasing doses of radiation. These colonieswere believed to be produced by this less sensitive fraction ofthe stem cells, which carried residual injuries. The coloniesproduced by the sensitive fraction have disappeared, beingkilled by a relatively low dose of radiation. This observationmight account for the high lymphomagenesis arising fromprimitive hemopoietic stem cells in SCID mice, because thesmallness of the colonies suggests that there is unrepaired ormisrepaired damage. Furthermore, this less sensitive fractionmight be a source of the "leaky" change of T and B cells,possibly due to the induction of an equivocal repair systemwhich appears in the later stages of life in the SCID mice.

Homozygous scid/scid mice (SCID mice) have a severecombined immunodeficiency arising from a lack of mature Tand B lymphocytes (1-4). A defect in the mechanism forrepairing DNA double-strand breaks (DSBs) is responsiblefor the impairment, involving not only a developmentalrearrangement in the lymphocytes of the T-cell receptorgenes and the B-cell immunogloublin genes but also a de-creased recovery from general radiation damage (5, 6). Fi-broblasts of SCID mice grown in vitro are highly sensitive toionizing radiation (4, 5). In addition, intestinal crypt cells andepithelial skin cells are highly sensitive compared with suchcells in congenic BALB/c mice and in counterpart controlmice, C.B-17-+/+ (4). Radiosensitivity of the hemopoieticsystem also has been studied (4, 5); in figure 1 of ref. 4, themean lethal dose (Do) of the hemopoietic stem cells (colony-forming units in the spleen, CFU-S) appears to be about 25.4cGy (4). When the survival of CFU-S is calculated from thisvalue, there is a big discrepancy between the dose whereendogenous spleen colonies disappear, >500 cGy (5), and thedose where <1 CFU-S per individual remains, about 320

cGy; based on the total number ofCFU-S in the SCID mouse,50,406, and the number of CFU-S in femoral marrow, 3100(7), the proportion of femoral marrow CFU-S to the wholebody CFU-S compartment is 6.15% (8). This discrepancyimplies that the radiosensitivity ofCFU-S in the SCID mousedoes not follow a single exponential but may contain anadditional fraction that is less sensitive to radiation. Further-more, heterogeneity of the stem-cell family during its matu-ration and development is known to be the rule (9, 10). Moreknowledge of the biology of SCID mice is needed. In thisreport, we discuss the radiobiology of the hemopoietic stemcells (HSCs).There is a higher incidence of hemopoietic malignancies in

mice with the scid mutation (2). Whether this is due to adefect in the repair pathway for DSB in HSCs or to thecombined lymphoid deficiencies is not established.To study these questions, we have measured the radiosen-

sitivity of the committed stem-cell compartment, the granu-locyte/macrophage-colony-forming units (CFU-GM) and theerythroid burst-forming units (BFU-E), and the day-9 andday-12 CFU-S. We found the CFU-GM and BFU-E fromSCID bone marrow to be much more radiosensitive thanthese entities from the bone marrow of C.B-17-+/+ mice.The 9- and 12-day CFU-S were also much more radiosensi-tive; however, the survival curve is not a single exponentialbut, rather, a double exponential with a steeper slope for thesensitive component associated with a flatter slope for theless-sensitive component. This less-sensitive fraction mayplay an important role in the immunohemopoietic system ofmice with the scid mutation, especially in respect to their hightumorigenicity and their "leaky" change of lymphocytes(11).

MATERIALS AND METHODSMice. Seven-week-old male C.B-17-scid/scid mice and

their counterpart controls, C.B-17-+/+ mice, purchasedfrom CLEA Japan (Tokyo), were used for sources of bonemarrow cells (hereafter C.B-17-scid/scid mice are designatedsimply as SCID, and C.B-17-+/+ as +/+). As recipient micefor the spleen colony assay to evaluate CFU-S, we used maleBALB/c mice of the same age, also from CLEA Japan.Throughout each experiment, the mice were housed in lam-inar-flow cage racks in an environmentally controlled clean-room with a 12-hr light/dark cycle at the Laboratory AnimalCenter, Yokohama City University School of Medicine.Medium. a modified Eagle's medium (a-MEM; Flow Lab-

oratories) supplemented with penicillin (100 units/ml) and

Abbreviations: CFU, colony-forming unit(s); CFU-S, CFU in thespleen; CFU-GM, granulocyte/macrophage-CFU; BFU-E, eryth-roid burst-forming unit; DSB, double-strand break; HSC, hemopoi-etic stem cell; Do, mean lethal dose.tTo whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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streptomycin sulfate (100 pg/ml) (both from Meiji Pharma-ceutical, Tokyo) were used in all experiments, unless other-wise stated.

Cell Preparation. Bone marrow cells were harvested fromthe femur and tibia of both hindlegs of about 50 SCID miceand 3 +/+ mice by flushing the bone with an appropriatevolume of medium through a 27-gauge needle. Since thenumber of spleen colonies was expected to fall exponentiallywith an increasing dose of radiation, especially in the case ofSCID CFU-S, the number ofbone marrow cells injected wasincreased with the increasing dose of radiation. Table 1shows our protocol for establishing the number of cells to beinjected.

Radiation. The mice were irradiated in an experimental137CS y irradiator (Gamma-Cell 40, CSR, Toronto). Micewere placed in an irradiation chamber whose inner surfacewas coated with an aluminum/copper film, each layer 0.5mmthick, and were irradiated at 132 cGy/min. For cell suspen-sions, the tubes were radially arranged in an ice-cold plasticbox placed in the center of the chamber and exposed to from0 to 300 cGy; the tubes were quickly and gently agitated every45 sec to keep oxygen deficiency to a minimum duringirradiation. To assay the number of spleen colonies, therecipient mice were exposed to a lethal dose of radiation, 912cGy for day 9 CFU-S (CFU-S9) and 895 cGy for day 12CFU-S (CFU-S12) in an irradiation chamber similar to thatdescribed above.

Assays of CFU-GM and BFU-E. Irradiated or unirradiatedcells were incubated in 35-mm Petri dishes (Nunc) at 37°C inhumidified air with 5% CO2. Iscove's modified Dulbecco'smedium (GIBCO) was supplemented with 0.9%o methylcel-lulose (Dow), 30o fetal bovine serum (HyClone), 1% bovineserum albumin (Sigma), and 10 AM 2-mercaptoethanol (To-kyo Chemical, Tokyo). As a source of colony-stimulatingfactors, we added 5% WEHI-3 conditioned medium (12) andrecombinant human erythropoietin (2 units/ml, 140,000units/mg of protein: Kirin-Amgen, Thousand Oaks, CA) forBFU-E (13) and 5% lung conditioned medium (14) and 5%L-cell conditioned medium (15) for CFU-GM. Colonies thatstained positively with 3,3'-diaminobenzidine 12 days afterplating were counted as BFU-E. Granulocyte/macrophagecolonies were counted on day 7 under an inverted micro-scope. Representative results obtained from two separateexperiments are plotted in Fig. 1 for five different exposures.Assay of CFU-S. The assay has been described (16). Le-

thally irradiated BALB/c mice were injected through the tailvein with irradiated or nonirradiated cell suspensions. Therecipient mice were killed by cervical dislocation 9 days afterirradiation and the injection of marrow cells to assay forCFU-S, or on day 12 for the more primitive CFU-S (CFU-S12) that produce late-appearing colonies. The spleens wereexcised and fixed in Bouin's solution to visualize the colo-nies, which then were counted under a dissecting micro-

Table 1. Number of bone marrow cells (BMCs) injected permouse in the assays of CFU-S

BMCs injected, no. x 10-5 per mouse

Dose given to SCID +/+donor BMCs, cGy CFU-Sg CFU-S12 CFU-Ss CFU-S12

0 0.62 0.20 0.50 0.3025 ND 0.25 ND ND50 7.40 1.33 ND 0.30100 8.89 8.50 1.00 0.36150 17.80 ND ND 0.60200 32.00 ND 2.80 1.00250 64.00 375.00 ND ND300 ND ND 6.25 ND

ND, not done.

scope. The data given in the text are all from triplicateexperiments for day 9 and from duplicate experiments for day12.Histomorphometric Examination of Spleen Colonies. After

the spleen colonies were counted, all of the spleens, as wellas the femoral bones, were embedded routinely in paraffin,sectioned at 5 ,um, and stained with hematoxylin and eosin.This procedure allowed us to make accurate morphometriccomparisons of spleen colonies from SCID and +/+ mice.The size of the spleen colonies was measured under themicroscope, and the numbers of cells in colonies of large andsmall diameter were calculated with an equation found else-where (17, 18).

Statistial Analysis. All the data were computerized andprocessed with STATVIEW II (Abacus Concepts, Berkeley,CA). The mean and standard error of the mean, the 95%confidence limits, and the statistical significance ofdifferenceand/or similarity were mechanically calibrated and drawn ineach figure.

RESULTSRadiosensitivity of CFU-GM and BFU-E. The data are

shown in Fig. 1. Bone marrow suspensions were irradiated at50, 100, 200, 250, and 300 cGy. Survival curves for CFU-GMand BFU-E are shown with the 95% confidence limits. Thereis a statistically significant difference between the curves forSCID and +/+ bone marrow, with a much steeper exponen-tial for the SCID data. The slopes of each regression' line arelisted in Table 2, and the significance of differences istabulated in Table 3. The Do was 48.0 cGy for SCID CFU-GMand 39.3 cGy for the BFU-E. These values can be comparedwith the Do values of 215.9 and 143.9 cGy for +/+ bonemarrow. Thus, for cells irradiated in vitro, the radiosensitiv-ity of the +/+ BFU-E is less than that for CFU-GM but thedifference is not statistically significant. Studies by othershave shown that BFU-E are more radiosensitive thanCFU-GM (19-21).

Radiosensitivity ofDay-9 CFU-S. The cell suspensions usedfor assaying day-9 CFU-S (CFU-S) were simultaneouslyprepared and irradiated. Fig. 2 shows the survival curves forCFU-S of SCID and +/+ mice after various doses ofradiation. Surprisingly, the survival curve for SCID CFU-S5

Radiation dose, cGy0 100 200 300 400

01

.001

FIG. 1. Survival curves of CFU-GM and BFU-E after gradeddoses of radiation given in vitro. Filled symbols are for data fromSCID bone marrow, whereas open symbols are for the +/+ control.Triangles with heavy dashed lines show BFU-E, and squares withsolid lines show CFU-GM. The 95% confidence limits are shown bydotted lines for BFU-E and dashed lines for CFU-GM. Data fromduplicate experiments are plotted together.

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Table 2. Slopes of regression lines for each radiationsurvival curveType of Slope of regression line No. ofCFU Source of BMCs (mean ± SE x 10-2) data

CFU-GM +/+ -0.2 ± 0.03 5SCID -0.9 ± 0.1 6

BFU-E +/+ -0.3 ± 0.1 5SCID -1.1 ± 0.2 5

CFU-S +/+ -0.4 ± 0.029 11SCID (25-300*) -1.1 ± 0.1 13SCID (25-100*) -1.8 ± 0.1 8SCID (100-300*) -0.9 ± 0.1 8

CFU-S12 +/+ -0.4 ± 0.1 6SCID (25-250*) -1.1 ± 0.2 5SCID (25-100*) -2.0 ± 0.2 3

BMCs, bone marrow cells. All data points were calculated bycomputer. Results of CFU-S12 SCID (100-250 cGy) were not testedbecause of insufficient data points between these doses.*Data points between these doses (cGy) were applied.

consists oftwo components; an initial steeper slope, followedby a flatter one. The control +/+, by contrast, shows asingle, flatter exponential curve, with a slight shoulder. Whenthe Do values were calculated for the steeper curve forradiation doses ranging between 0 and 100 cGy, the result wassimilar to that reported elsewhere (4)-i.e., about 24.0 cGy,which is much smaller than the 107.9 cGy obtained from the+/+ control. Although this value for +/+ was a little high incomparison with reported values of other normal strains (22),similar, reproducible results were found with BALB/c mice.Another notable feature of the SCID survival curve was theflatter slope at radiation doses between about 100 and 300cGy, with a larger Do, 48.0 cGy, than the value derived fromthe steeper part of the curve; furthermore, this larger valuealso reflects a greater sensitivity than the +/+ control.Triplicate sets of data for CFU-Sg give essentially the sameresults. The difference between the slopes ofboth regressionlines in the SCID CFU-S9 is statistically significant (Table 3).The extrapolated number of the flatter curve, crossing aradiation dose point at 0 cGy, showed that the cells formingthis fraction were about 10.4% of the total CFU-Sg of theSCID bone marrow.The basis for this relatively radioresistant fraction in the

SCID CFU-S9 compartment is not known, nor is it knownwhy there is not a radioresistant fraction in either CFU-GMor BFU-E. However, even if in vitro and SCID CFU-Sg hadthe same content (about 10%) of less resistant cells with the

Table 3. Statistical significance of slopes of regression linesbetween pairs of survival curvesCFU-

associated Pair of curves t value* d.f. to(0.001) DifferencetCFU-GMBFU-ECFU-5sCFU-5sCFU-5sCFU-SgCFU-S9CFU-S9CFU-S12CFU-S12CFU-S12

+/+, SCID+/+, SCID+/+, w-SCID+/+, s-SCID+/+, r-SCIDw-SCID, s-SCIDw-SCID, r-SCIDs-SCID, r-SCID+/+, w-SCID+/+, s-SCIDw-SCID, s-SCID

16.138.00

10.3238.4513.7315.58-4.45

-18.007.12

13.646.16

6.776.82

14.718.108.10

16.8916.8916.006.453.026.26

4.7815.4084.7035.0415.0413.9653.9654.0155.959

12.9245.959

YesYesYesYesYesYesYesYesYesYesYes

d.f., degrees offreedom; w-SCID, all data points ofSCID CFU-S;r-SCID, data points of radioresistant SCID CFU-S; s-SCID, datapoints of radiosensitive SCID CFU-S.*Data obtained were results of t test from values in Table 2.tStatistical difference of slopes for regression lines between each pairof curves after graded doses of radiation.

c

Cl

Radiation dose, cGy0 100 200 300 400

FiG. 2. Survival curves for day-9 CFU-S after graded doses ofradiation. Filled symbols are for data from SCID bone marrow,whereas open symbols are for the +/+ control. Data from threeindependent experiments are shown. Dashed lines show the 95%confidence limits.

middle range of resistance as seen in SCID CFU-S9, thiswould not be detectable within the span of exposures used,due to the restricted number of cells available in our culturesystem.

Radiosensitivity of Day-12 CFU-S. CFU-Sg are consideredto be more primitive than CFU-GM and BFU-E (10). Further,the CFU-S for day 12 is considered to be even more primitivethan CFU-S7.9, because the late-appearing colonies producedby CFU-S12 have the potential to form greater numbers ofsecondary colonies in the spleen than the early-appearingcolonies; also, they are much more radioprotective whenreseeded by injection into secondary, lethally irradiatedrecipients.

Fig. 3 shows, again, a double-exponential curve for CFU-S12 from the SCID bone marrow. The curve for CFU-S12 from+/+ is flatter and is a single exponential with a tiny shoulder.

Radiation dose, cGy0 100 200 300 400

1

.001 .

FIG. 3. Survival curves for day-12 CFU-S after graded doses ofradiation. Filled symbols, SCID bone marrow; open symbols, +/+control. Two representative experiments are combined. Dashed linesshow the 95% confidence limits. Asterisk indicates that the datainclude those from moribund mice before scheduled killing.

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Although the data points for the flatter part were limited, asshown in the bottom row of Table 3, the slope for the steeperpart [s (sensitive)-SCID) was significantly different from aslope [w (whole)-SCID] in which the data at 250 cGy wereincluded in the whole (a < 0.001). In other words, a possiblemaximum slope of 95% confidence for the steeper portiongives a crossing point with a dose of 250 cGy at about 5.62 x10-4, which is much lower than the variance ofthe actual datapresented at a dose of 250 cGy, 1.17 ± 0.96 x 10-3. Thesurviving fractions ofcolonies were slightly higher than thoseat CFU-S9, and the percent survival of SCID CFU-S12 washigher than that of SCID CFU-Sg at any radiation dose; inother words, the availability ofthis SCID CFU-S12 was about12.9% of the total CFU-S, obtained by the incidence of theradioresistant fraction, which can be estimated by an extrap-olation toward the ordinate axis.

Abortive and Smaller Spleen Colonies In SCID CFU-S. Theflatter survival curve for SCID CFU-S, representing therelatively radioresistant fraction, suggests the possible exis-tence of incomplete cell and tissue damage after irradiation.Because residual damages in CFU-S produce abortive andsmaller colonies (17), to test this hypothesis we measured thesize of the day-9 spleen colonies (Fig. 4). Spleen coloniesfrom the 200 cGy-irradiated SCID CFU-Ss were muchsmaller: 1.49 + 1.06 mm3 compared with 2.42 + 0.73 mm3 in+/+ controls. There were prominent abortive colonies inthese sections, and an example of such a colony is shown inFig. 5. Although the survival curve showed a biphasiclogarithmic curve, the size distribution was not biphasic.

DISCUSSIONA mutant mouse with a severe combined immunodeficiencywas discovered in 1983 by Bosma et al. (1). Mice homozygousfor this mutation lack the ability to develop functional T andB cells; however, the development of both lymphocyticlineages proceeds normally until the stage requiring generearrangement (3, 7, 23, 24); therefore, it is thought that therequisite mechanisms have been deleted in mice homozygousfor the scid mutation. Biedermann et al. (4) incidentally foundthat these mice had an extraordinary hypersensitivity toionizing radiation, and they considered that both the immu-nodeficiency and hyperradiosensitivity were due to a defectof the DSB repair system. A strange phenomenon that hasbeen recorded, since Bosma et al. (1) found the SCID mice,is the "leaky" phenomenon (11), which might cause a partialif not a complete normalization, due to the induction ofequivocal recombination and ofthe DNA repair system. Thisidea implies the possible existence of heterogeneous subpop-ulations in the HSC family in this mutation. The impetus for

E

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0

0 100 200 300 400Radiation dose, cGy

FIG. 4. Size of spleen colonies from SCID and control +/+ bonemarrow cells versus graded dose of radiation. Surface colonies weremeasured by a sliding microcaliper under a dissecting microscope.Filled symbols, SCID colonies; open symbols, +/+ control. Verticalbars show the standard error of the mean.

FIG. 5. Histological sections showing spleen colonies from 200cGy-irradiated SCID marrow cells (A), and from nonirradiatedcontrol +/+ marrow cells (B). Insets show low-power views ofeachcolony; cells are scanty and dispersed in A, whereas they are morepacked and denser in B. The main part of each panel shows ahigh-power view of part of the field shown in the Inset. (xlS andx70.)

us to start this study was to provide accurate radiobiologicaldata on the SCID stem cells.As shown in Fig. 1, the radiosensitivities of CFU-GM and

BFU-E from SCID mice are 3-4 times as high as those of+/+ mice. These data are compatible with the report ofFulop and Phillips (5), although there was a small differencein actual values. However, it was surprising that the survivalcurves for both CFU-Sg and CFU-S12 in the present studywere double (Figs. 2 and 3) rather than a single exponential.A hypothesis which we rejected statistically is that there wasa regression line of SCID based on the whole range (25-300cGy) which was similar to either the slope ofthe steeper parts(25-100 cGy) or the slope of the flatter part (100-300 cGy)(Table 3). Thus, the less sensitive fraction in the SCID miceis evident.What role does this less radiosensitive fraction play? It is

generally known that potential lethal damage after irradiationoccurs in the less sensitive fraction (25). In fact, the averagesize of SCID spleen colonies formed after relatively highdoses of radiation was smaller than that of the controls. Fig.4 shows this remarkable decrease in size of SCID colonieswhen the cell suspensions had been given >150 cGy. Thesurvival of the sensitive fraction for CFU-S9 at a dose of 200cGy is 0.02%, extrapolated from the curve in Fig. 2, whereasthe survival ofthe less sensitive CFU-S9 is 0.17%. Therefore,the lower average size of colonies apparently representscolonies from the less sensitive CFU-S; the residual damageafter irradiation, as a result of incomplete DSB repair, couldbe reflected in such small and abortive colonies.

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The heterogeneity ofHSCs is often explained on the basisof the time that they appear after transplantation (26, 27) orby their differential sensitivity to some cytotoxic drugs (28,29) or to ionizing radiation and ultraviolet light (19-21, 30,31). The radiosensitivity of HSCs seems to depend on theirorigin (fetal liver, adult bone marrow, or spleen) or the levelof differentiation (31). The radiosensitivity of CFU-S in fetalliver was reported to be higher than those in adult bonemarrow and the spleen. This observation led to the idea thatHSCs may not be a homogeneous population. The radiosen-sitivities of different HSC compartments have been directlymeasured by in vitro and in vivo colony-forming techniques(19-21). These studies suggested a possible correlation be-tween radiosensitivity and differentiation status.As we expected, a larger fraction of the less radiosensitive

cells was observed in CFU-S12 than CFU-S9. However, it isstill uncertain why the less sensitive fraction was not ob-served in the CFU in vitro, CFU-GM and BFU-E, of SCIDmice. A possible reason is that the less sensitive fractionobserved in CFU-S decreased during differentiation towardthe CFU in vitro, and the fraction may not be present in theCFU in vitro. Indeed, a majority of blood cells might bederived from the sensitive fraction, and that would explainwhy most lymphocytic elements are lacking in SCID mice,and why any descendant cells derived from a less sensitivefraction cannot appear in peripheral blood. In other words, ifsuch blood cells, derived from the less sensitive fraction,were present in peripheral blood, there also should be lym-phocytes which had successfully undergone gene rearrange-ment during their maturation. Such a subpopulation might nothave been available at a detectable level in the present studyin CFU in vitro, although it should again be asked why theless sensitive fraction in CFU in vitro is less frequent thanthat in CFU-S.The lower sensitivities of this relatively radioresistant

subpopulation of CFU-S suggest that the DSB repair systemof these cells is not completely defective. This characteristicprobably participates in the production of neoplastic clonesin SCID immunohemopoietic systems, and the absence offunctional lymphocytes may allow tumor development. Inaddition, the leaky change of SCID mice (11) might beexplained by the existence of less defective stem cells,probably with nearly normal developmental ability. This is sobecause some CFU-S are known to develop into a lymphoidlineage (32), and the existence of lymphoid-restricted stemcells is questionable (33). The reason that myeloid cellsremain normal (34) is probably not that the stem cells remainnormal but that myeloid cells do not require any repair orrecombination system during their maturation and differen-tiation.

We are very grateful to Mr. Hideaki Mitsui for excellent technicalsupport in pathology and statistical data processing. We thankMessrs. Masaichi Ikeda and Takehisa Suzuki for processing thepathology data and Ms. Yuko Takada for secretarial help. We thankDr. Avril Woodhead, Biology Department, and Dr. Eugene P.Cronkite, Medical Department, Brookhaven National Laboratory,Upton, NY, for critical reading ofthe manuscript. This research wassupported in part by two Grants-in-Aid for Scientific Research fromthe Japanese Ministry of Education, Science, and Culture (02807042,04247210).

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