1967 29: 102-113

J. E. TILL, L. SIMINOVITCH and E. A. McCULLOCH

vW/WColony-Forming Cells Transplanted in Mice of Genotype The Effect of Plethora on Growth and Differentiation of Normal Hemopoietic

http://bloodjournal.hematologylibrary.org/misc/rights.dtl#repub_requestsInformation about reproducing this article in parts or in its entirety may be found online at:

http://bloodjournal.hematologylibrary.org/misc/rights.dtl#reprintsInformation about ordering reprints may be found online at:

http://bloodjournal.hematologylibrary.org/subscriptions/index.dtlInformation about subscriptions and ASH membership may be found online at:

. Hematology; all rights reservedCopyright 2011 by The American Society of 20036.the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by

The Effect of Plethora on Growth and Differentiation ofNormal Hemopoietic Colony-Forming Cells Transplanted

in Mice of Genotype W/W”

By J. E. TILL, L. SI�IINovrrCim AND E. A. MCCULLOCH

T � FUNCTIONAL ASSAYS are, at present, commonly used to investi-

gate stem cells. First, Gurney and his collaborators, making use of findings

which indicate that the hormone erythropoietin acts on relatively undiffer-

entiated stem cells,1� devised an assay for such stem cells based on respon-

siveness to erythropoietin.4’� Second, we have suggested that the cells that

give rise to spleen colonies in heavily irradiated#{176} or genetically anemic re-

cipient mice7 may be considered as stem cells,8 and that the spleen colony

method provides a quantitative assay for such cells. Evidence has been

summarized elsewhere” that the stem cells which form spleen colonies are not

identical with the cells which respond to erythropoietin. For example, it has

been shown that exposure of mice to hypoxia, a procedure which might be

expected to increase erythropoietin levels, had little effect on the colony-

forming efficiency of cells from the marrow or spleen of these animals at a

time when erythropoiesis was greatly increased.10

Transfusion-induced plethora suppresses erythropoiesis, presumably by de-

creasing the production of erythropoietin.3’� A number of workers�13 have

studied the formation of spleen colonies in irradiated plethoric mice, and.

in most cases, plethora has been found to cause a reduction in the number of

spleen colonies and an increase in the proportion of colonies which are

primarily granulocytic in composition. Such findings might be interpreted as

indicating that a proportion of the colony-forming cells are indeed sensitive

to erythropoietin, and so fail to proliferate in the plethoric animals. However,

an alternative interpretation is also possible. Suppression of erythropoiesis

might prevent differentiation of enough ervthroblasts to form macroscopically

visible colonies, and the predominantly granulocytic colonies which were

observed might result from stimulation of the growth of multipotent’4 colony-

From the Departments of Medical Biophysics and Medicine, University of Toronto,

and The Ontario Cancer Institute, Toronto, Ontario, Canada.

This work was supported by the Medical Research Council (Grant 1420), The National

Cancer Institute of Canada, The Defence Research Board (Grant 9350-14, G and C),

and the Jane Coffin Childs Memorial Fund for Medical Research.

First submitted March 21, 1966; accepted for publication July 8, 1966.

J. E. TILL, PH.D.: Department of Medical Biophysics, University of Toronto, and The

Ontario Cancer lnstitute, Toronto, Ontario, Canada. L. SI?s!INOVITCII, PH.D.: Department of

Medical Biophysics, University of Toronto, and The Ontario Cancer Institute, Toronto,

Ontario, Canada. E. A. MCCULLOCH, M.D.: Departments of Medical Biophysics and

Medicine, University of Toronto, and The Ontario Cancer Institute, Toronto, Ontario,

Canada.

[02

BLOOD, \TOL. 29, No. 1 (JANUARY), 1967

EFFECT OF PLEThORA ON COLONY-FORMING CELLS 103

forming cells by radiation-induced granulocytopenia in the irradiated plethoric

hosts.

The difficulty in interpretation of the results obtained when irradiated

plethoric mice are used as hosts for the formation of splenie colonies might

be avoided by using genetically anemic mice of genotype W/W’ as recipients.

Such animals have a severe macrocytic anemia but their peripheral leukocyte

and thrombocyte counts are nearly normal.15 The hemopoietic colony-forming

cells of these genetically anemic mice are defective and unable to give rise

to normal numbers of spleen colonies. However, the tissues of mice of genotype

W/W� are competent and unirradiated mice of this genotype support colony

formation when cells derived from coisogenic normal mice are injected into

them.7 In the present paper, we report the results of a series of experiments

in which the process of colony formation and the growth of colony-forming

cells was studied following the injection of normal coisogenic bone marrow

cells into plethoric mice of genotype W/W�.

MATERIALS AND METHODS

Mice. Genetically anemic mice and their normal coisogenic littermates were either

obtained from the Jackson Laboratory. Bar harbor, Maine, or were bred in the animal

colony of the Ontario Cancer Institute. The breeding stock in the O.C.I. colony was

obtained from the Jackson Laboratory and therefore mice from either source were used,

as available. Mice of genotype W/Wv were obtained by crossing mice from the inbred

strain WB maintained heterozygous at the W locus (WB-W/+) and C57BL/6 maintained

heterozygous at the W locus by repeated backcrossing (B6-WV+). This cross yields normal

(WB x B6)F1 hybrid mice ( WBB6F1 ) which served as donors of heinopoietic cells, and

genetically anemic WBB6F1�W/Wv mice which served as recipients of these cells. In

addition, mice of genotype Wx/Wv were obtained b�’ crossing animals of genotype

C3H�Wx/+ with animals of genotype C57BL/6-W�/+. This cross yields normal

C3B6F1-+/+ mice as donors and genetically anemic mice of genotype WVWV to

serve as recipients. Similar results were obtained with WIW” and W1/W�’ recipients and

these will not be designated separately. Following injection with red blood cells anti

marrow cells the animals were housed 2 to 3 per cage and allowed food (Rockland

Mouse Diet) and water freely.Transfusion Procedure. Red blood cells for transfusion were obtained either from normal

coisogenic mice or from heterozvgous littermates (W/+, Wx/+ or Wv/+). Red cell donors

were lightly anesthetized and bled from the carotid artery. The red cells were washed

three times in the cold with phosphate buffered saline. Anemic mice to be transfused

received 1 nil. of washed packed red cells intraperitoneally on each of 3 or 4 successive

days. One ml. of packed ret! cells usually contained in excess of 1.4 X 10�� red cells.

Genetically anemic mice did not tolerate the procedure well, and in some experiments

as many as 50 per cent of the transfused animals died during the course of the experiment.

Some of the survivors were found to have failed to retain elevated red blood cell counts

(see “Results”). However, less extensive transfusion procedures, while permitting a greater

survival, did not yield adequate suppression of erythropoiesis.

Colony Formation. The spleen colony technic using recipients of genotype W/Wv has

been described previously.7 In brief, hemopoietic cells were obtained from the inarrows of

coisogenic normal littermates, or from the spleens of transfused or control animals which

had received transplants of marrow cells from normal coisogenic donors (see below).

An appropriate number of these cells was injected intravenously into the recipient animals,which had been given a sublethal dose of irradiation (150 rads) prior to injection

of the cells. After 10 days, the spleens were removed, fixed in Bouin’s solution, and the

104 TILL, SIMINOVITCH AND MC CULLOCH

macroscopic spleen colonies were counted. Results were expressed as colons-forming units

(CFU) per 10� nucleated cells injected.

Assay for the Growth of Colony-Forming Units. The technic for obtaining growth

curves for CFU in irradiated recipients1� was adapted for measuring growth curves of

normal hemopoietic cells transplanted into unirradiated mice of genotype W/Wv or

Wx/Wv. The colon�’-forming efficiency of a marrow cell suspension was first tested Iw

injecting 8 X 10� nucleated cells into lightly irradiated genetically anemic mice. Then.

2 x iO� nucleated marrow cells from the same cell suspension were injected into

unirradiated recipients of genotype W/Wv or Wx/Wv. These unirradiated recipients

consisted of animals which had been transfused prior to injection of the marrow cells,

along with their nontransfused controls. At various times thereafter, individual mice

from these groups were killed anti cell suspensions prepared from spleen anti marrow.

The spleen cell suspension was tested for colony-forming efficiency by injecting appropriate

numl)ers of cells into groups of lightly irradiated anemic mice of the appropriate W

genotype. Tests for iron-incorporating capacity anti for peroxidase-positive cells were also

clone on the spleen and marrow cell suspensions. At the time of the sacrifice a peripheral

red cell count and reticulocyte blood count were niade on each recipient animal.

Incorporation of Radioactive Iron. Ervthropoiesis in spleen and marrow cell suspensions

was tested Iw measuring the capacity of such cells �o incorporate radioactive Fe�” into

heme in vitro, as described elsewhere.’7 In brief. the technic used was as follosvs:

1.2 x 10� nucleated niarrow cells were incubated at 35 C. for 45 or 90 minutes in a

medium consisting of CMRL 1066 anti mouse serum labelled with Fe59 (obtained as

the chloride from Abbott Laboratories. specific activity 10-25 me/mg.). The amountof Fe59 incorporated into heme per unit time was found to be linearly related to the

number of cells present in the incliI)ation mixture. Results were expressed as percentages

of the activity added to the incubation tube which was incorporated into heme by

I .2 x 10� marrow cells in 45 minutes.

Peroxidase Staining Technic. The number of granulocvtes present in the spleen cell

suspensions was determined using the peroxicIase-staining technic of Rvtomaa’ � as

nioclifieci l)Y Fowler.19 using cells deposited on Millipore filters.

Irradiation Procedures. Radiation was delivered to recipient mice of genOt\�)e W/\%v,

using a Cs1�7 irradiator designed 1w Cunningham. Bruce, an(l \Vebb2#{176} at a close rate of

approximately 1 15 rads per minute.

Red Cell Counts and Reticuloc,�ie Counts. Ret! cell counts were made using a Coulter

counter (Coulter Electronics, Hialeah, Fla.), and reticulocytes were counted directly on

smears stained with brilliant cresvl violet.

RESULTS

Effect of Plethora on Spleen Colony Formation in Mice of Genotype W/Wr

Preliminary experiments were designed to test whether or not the suppres-

sion of erythropoiesis in unirradiated mice of genotype W/W’ would affect

the number of colonies observed in the spleens of such animals following

the transplantation of marrow cells from coisogenic normal mice. Groups

of five animals were rendered plethoric by the injection of 1 ml. of packed

washed red cells on four successive days. Three days after the last intra-

peritoneal injection of red cells these animals and control anemic mice were

injected with 8 x 10� normal nucleated marrow cells. After 10 or 14 days,

the recipient animals were killed. Peripheral red cell counts and reticulocyte

counts were done on each animal, and marrow from each group was pooled

and its capacity to incorporate radioactive iron into heme was tested. The

spleen of each animal was fixed in Bouin’s solution and examined for spleen

colonies. The results are presented in Table 1. From the table it is apparent

EFFECT OF PLEThORA ON COLONY-FORMING CELLS 105

Table 1.-Effect of Transfusion.lnduced Suppression of Erythropoiesis

on the Formation of Spleen Colonies

Transfused Not Transfused

Item Exp. 1 Exp. 2 Exp. 1 Exp. 2

No. of animals tested 4 8 5 9

Mean red cells/ird. ( X 109) 9.23 9.67 5.89 5.60

Mean reticulocytes ( %) 0.2 - 3.6 -

Fe59 uptake in marrow cells (%) 0.03 0.02 0.67 0.48

Cells/2 femora ( X 10 7 ) 5.2 3.0 3.3 3.2

Cells/spleen ( >< 10-s) - 3.6 - 2.9

Granulocytes/spleen (%) - 3.8 - 3.6

CFU/10� marrow cells, d�iy 10 - 0.0 24.6 7.2

day 14 0.0 0.3 - -

that the transfused animals had elevated red cell counts and markedly reduced

numbers of circulating reticulocytes, compared with mice which had not

been transfused. In addition, the pool of marrow cells obtained from the

transfused animals showed a very low capacity to incorporate radioiron into

heme, in comparison with the nontransfused controls. These findings indicate

that the transfusion procedure was successful in raising the red cell count of

mice of genotype W/Wv and in suppressing erythropoiesis. The numbers

of cells obtained from spleen and marrow, and the percentages of granulocytes

present in the spleen, were not significantly changed by the suppression of

erythropoiesis in these experiments. In contrast, it is apparent from the table

that very few spleen colonies were seen in the transfused mice, and that

those which were seen only became visible on the fourteenth day after in-

jection of the marrow cells. Thus, suppression of erythropoiesis in mice of

genotype W/W� inhibits the development of detectable macroscopic spleen

colonies by marrow cells from normal coisogenic donors.

Effect of Plethora on the Growth of Hemopoietic CelLc in Mice of Genotype

WIW’

Macroscopic colonies might fail to develop in plethoric mice of genotype

Wi W’ following marrow cell transplantation, either because the transplanted

colony-forming cells failed to grow in the recipient animals or because

suppression of erythropoiesis prevented formation of erythroblasts in numbers

sufficient to give rise to macroscopic colonies. To distinguish between these

two alternatives, the growth of colony-forming cells in plethoric and anemic

mice of genotype WI W� was measured. In these experiments groups of mice

received 1 ml. of packed washed red cells on three successive days. Three

days later these animals and anemic controls received 2 x 106 nucleated

marrow cells derived from coisogenic normal mice. At intervals from day 7 to

day 10 after injection of the marrow cells, individual mice were killed,

samples of peripheral blood were taken for red cell and reticulocyte counts,

and cell suspensions were made from spleen and bone marrow. The bone

marrow and spleen cell suspensions were tested for their ability to incorporate

106 TILL, SIMINOV1TCH AND MC CULLOCH

��l5- I � i � I � I I�--�i� � i � -

� ,0 - - , ...� � �- .. � � �-. V Reticulocytes

.� �

�%

‘�5 �

�‘I

0 I � I � I � � -$�� �

0.8 - -

.� . . � . A 0 Iron incorporation

�0.6 - � LA -

q,�0.4 - A � -

�o: � � � 0 � � I�;-

RBC per ml. x109

Fig. 1.-Effect of erythrocyte transfusion on erythropoiesis in individual unirra-diated mice of genotype W ‘Wv. Upper: Reticulocvte count versus red cell count inperipheral blood 10 to 13 days after transfusion. V = transfused; V = not trans-

fused. Lower: F&11 incorporation into heme versus peripheral red cell count 10 to 13

days after transfusion. A = transfused, spleen cells; A = not transfused, spleen

cells; #{149}= transfused, marrow cells; 0 = not transfused, marrow cells. Results of 4

separate experiments. Stippled areas indicate the zones occupied b� 85 per centof the points obtained for animals having red counts of less than 7.5 x 10� RBC

p#{128}’�’nil.

radioiron into heme; in addition, the percentages of peroxidase-positive cells in

the spleen cell suspensions were determined. The colony-forming efficiencies

of the cells in the spleen cell Suspensions were measured by injecting suitable

numbers of cells into groups of lightly irradiated mice of genotype W/Wv and

counting the numbers of spleen colonies seen after 10 days.

The effect of the transfusion procedure on the peripheral red cell and

reticulocyte counts, and on the capacity of spleen and marrow cells to

incorporate radioiron into heme, is shown in Figure 1. Results obtained from

transfused animals are shown as closed symbols, while results obtained from

control anemic animals are shown as open symbols. It is evident from this

figure that individual mice were not uniformly affected by transfusion. In

some animals which received transfusion of red cells, plethora was successfully

achieved, in that low peripheral reticulocyte counts and low levels of incor-

poration of radioiron into heme in spleen or marrow cells were observed 10

to 13 days after the last transfusion of red cells. In other transfused animals,

plethora was not achieved, in that suppression of erythropoiesis was not

maintained throughout the entire duration of the experiment. On the basis of

the results shown in Figure 1, erythropoiesis in individual animals was con-

/

Irradiatednormal

/

fi

2l0

Unirradiated

W/WV

yerspjeen

0 2 4 6 8 0

Days after transplantation

EFFECT OF PLETHORA ON COLONY-FORMING CELLS 107

#{188}.

Q)

t4�

L)

I I

$2 $4

Fig. 2.-Growth of CFU in the spleens of unirradiated recipient animals of geno-

types W � WV which had been given 2 X lO� marrow cells derived from normal lit-

termates. A = recipients successfully plethorized; A = recipients not successfullyplethorized; 0 � nontransfused controls. Data shown are the means of results ob-tamed for 3 to 8 individual mice per point. The horizontal dotted line indicates the

initial number of CFU per spleen, obtained b�’ multiplying the mean iiuinber of

CFU per 2 x 10� marrow cells b�’ 0.17, the fraction of injected marrow CFU which

lodge in the spleen.�

sidered to have been suppressed successfully only if their peripheral red counts

were 7.5 x 10� cells per ml. or greater, at the time of test.

The results of the assays of spleen cells for their colony-forming ability

are shown in Figure 2 as a function of the time when the animals were

killed. Time zero in this case was taken as the time the marrow cells were

injected, 3 days after the last transfusion of erythrocytes. The animals were

tested individually for successful suppression of erythropoiesis ( Fig. 1 ), and

on the basis of these tests they were separated into 3 groups. These groups

consisted of animals in which erythropoiesis was successfully suppressed

(closed triangles in Fig. 2), animals in which transfusion was unsuccessful

in suppressing erythropoiesis (open triangles), and animals which were not

transfused (open circles). The values shown in Figure 2 are means of the

results obtained for the individual animals. The solid curve shown in Figure 2

represents the results obtained previously’6 for the growth of normal marrow

CFU in the spleens of irradiated normal C3B6F1 recipient mice. Also shown

108 TILL, SIMINOVITCH AND MC CULLOCH

in the figure is the mean initial numl)er of CFIJ present in the spleen

( dashed horizontal line ) , calculated from the measured colony-forming effi-

ciency of the original marrow cell suspensions by assuming that 17 per cent

Of the CFU initially injected lodged in the spleens of the recipient animals.8

An experiment, carried out in the same way as those described previously,8

confirmed that this value is also applicable to unirradiated, genetically anemic

recipient mice, as well as to the irradiated normal recipients used in the

previous experiments.

It is evident from the data shown in Figure 2 that although the growth

of injected normal CFU in the spleens of unirradiated W/W” recipients

was delayed in comparison with the growth of CFU in normal, heavily

irradiated animals, growth occurred in the W/W� recipients whether or not

erythropoiesis had been suppressed. Indeed, the number of CFU per spleen

obtained from animals in which erythropoiesis had been successfully suppressed

did not differ significantly (p > 0.1 )21 from the number of CFU per spleen

obtained from control animals or from animals in which transfusion did not

result in suppression of erythropoiesis. Thus, the growth of normal CFU in

animals of genotype W/Wv appears to be controlled independently from the

control of erythropoiesis.

Effect of the Transfusion Procedure on Granulopoiesis

Hemopoietic colony-forming cells appear to be capable of giving rise to

granulocytic as well as erythrocytic descendants.6’14 It was of interest to

see whether or not the suppression of erythropoiesis by plethora would

affect granulopoiesis. In the same experiments which yielded the results

given in Figures 1 and 2, the histochemical test for peroxidase was used as

an assay for granulocytic cells in the spleens of individual transfused and

anemic mice of genotype WI W� which had been injected with marrow cells

from normal donors. The results of these tests are presented in Figure 3.

In the figure, granulopoiesis in individual animals, as measured by the total

number of peroxidase-positive cells in their spleens, is compared with erythro-

poiesis in the same animals, as measured by percentage iron incorporation into

heme in marrow cells. Data from animals in which suppression of erythropoiesis

was successfully achieved by transfusion of erythrocytes are shown along

with data from nontransfused controls and from animals in which suppression

of erythropoiesis was not successfully achieved. As shown in Figure 1,

animals in which erythropoiesis had been effectively suppressed by trans-

fusion all yielded cells which incorporated very little radioiron, in com-

parison with cells from control animals and animals in which transfusion

had been ineffective, which showed a high level of iron incorporation. How-

ever, there was no correlation between successful suppression of erythro-

poiesis and numbers of granulocytes per spleen. Although animals which had

been transfused had a significantly higher total granulocyte count per spleen

than control animals (p < 0.02), this increase in granulocytes was observed

whether or not the transfusion procedure had been effective in suppressing

0 0.2 0.4 0.6 0.8 1.0

Fe59uptake, percent

EFFECT OF PLETHORA ON COLONY-FORMING CELLS 109

(i�

Fig. 3.-Number of granulocytes per spleen as a function of Fe5#{176}incorporationinto the heme of marrow cells obtained from unirradiated recipient mice of geno-type W/W� which received 2 X 106 normal marrow cells 7 to 10 days prior to test.

A recipients successfully plethorized; A recipients not successfully plethorized;0 = nontransfused controls. The stippled area indicates the zone occupied by 85per cent of the points.

erythropoiesis. The granulocyte counts in the two groups of animals whichreceived transfusions did not differ significantly (p > 0.5).21

The design used in the experiments reported above does not permit oneto distinguish between host erythropoiesis and granulopoiesis, and that derivedfrom the transplanted cells. However, cells with colony-forming capacity foundin the recipient animals may be considered to he of graft origin since en-dogenous cells of mice of genotype WI WV are ineffective in the processof colony formation.7 One would expect, therefore, that if granulopoiesis oh-served in the recipient mice of genotype WI W� were largely derived from thetransplanted cells, then the number of peroxidase-positive cells observedshould be correlated with numbers of CFU; however, if granulopoiesis werelargely derived from the host, no such correlation should be observed. Sincein the experiments described above, determinations were made of both thenumber of peroxidase-positive cells per spleen in individual animals (Fig. 3)and the number of CFU per spleen in these same animals (averaged toobtain the results of Fig. 2), it was possible to test for such a correlation. Theresults are given in Figure 4. Data are shown for animals in which erythropoie-sis was successfully suppressed, along with data for nontransfused controls and

$02

10’

Granulocytes per spleen

110 TILL, SIMINOVITCH AND MC CULLOCH

L)

Fig. 4.-Number of CFU p�r spleen versus number of granulocytes per spleen

in individual unirradiated recipient animals of genotype W/W� which received2 x 10� normal marrow cells 7-10 days prior to test. A recipients successfully

plethorizeci; A recipients unsuccessfully plethorized; 0 = nontransfused con-

trols. The stippled area indicates the zone occupied by 85 per cent of the points.

for animals in which suppression of ervthropoiesis was unsuccessful. It is

evident from the figure that a correlation exists between the granulocyte count

per spleen and the number of CFU per spleen in individual recipients. A test

of association between the two measurenients21 showed a highly significant

association (p < 0.001 ) l)etween granulocytes and CFU, although neither

granulopoiesis nor colony-forming ability was correlated with erythropoiesis.

DIsaissIoN

Results 1)resented in this paper show that suppression of erythropoiesis by

transfusion will prevent the appearance of macroscopic colonies in the spleens

of mice of genotype W/W� following the transplantation of marrow cells

from normal coisogenic donors. However, this suppression of colony formation

appears to result from a failure to develop the large numbers of erythropoietic

cells required for the recognition of macroscopic colonies and not from any

direct effect on the growth of colony-forming cells.

It seems �vell established that erythropoiesis is suppressed in plethoric

animals because production of the hormone erythropoietin is inhibited under

these conditions.3’5 It has been suggested previously9”0 that although the

progeny of hemopoietic colony-forming cells may respond to the action of

erythropoietin, the colony forming cells themselves are not sensitive to

EFFECT OF PLETHORA ON COLONY-FORMING CELLS 111

the hormone. The observations presented in this paper provide further support

for this view, since the absence of a stimulus for erythropoiesis did not inhibit

the proliferation of colony-forming cells (Fig. 2). Moreover, the observation

that suppression of erythropoiesis prevented the development of macroscopic

colonies (Table 1 ) is consistent with the view9’10 that cells responsive to

erythropoietin (“erythropoietin-sensitive cells”) are included among the pro-

geny of colony-forming cells.

It should he pointed out that no correlation was observed in our results

between the suppression of erythropoiesis and granulopoietic activity ( Fig. 3).

This lack of correlation was not due to a complete suppression of granulopoiesis

in unirradiated \V/%V’ mice, since an increase in granulocytes was observed

in the spleens of WI W� animals given transplants of genetically normal marrow

( Fig. 4). It is possible that the growth of transplanted colony-forming cells

in W/W’ hosts ( Fig. 2) was related to a continuing stimulus for granulopoiesis

even in the absence of a stimulus for erythropoiesis. This finding is consistent

with the view’9 that the control of granulopoiesis is independent of the con-

trol of erythropoiesis.

Previous results’#{176} had shown that a stimulation of erythropoiesis by expo-

sure of animals to hypoxia did not produce any significant increase in the num-

hers of colony-forming cells in spleen or marrow. The observations presented

in this paper show that the proliferation of colony-forming cells is not inhibited

in the absence of a stimulus for erythropoiesis. Taken together, these findings

provide strong support for the view9’10 that colony-forming cells differ from

er�thropoietin-sensitive cells.

SUMMARY

Suppression of erythropoiesis by transfusion of animals of genotype WIW�

��‘as found to prevent the development of macroscopic spleen colonies following

injection of normal coisogenic marrow cells. This inhibition of colony-formation

was not due to a failure of colony-forming cells to proliferate in the absence

of erythropoietic stimulation, since the growth rate of normal colony-forming

cells in plethoric animals did not differ significantly from that seen in anemic

hosts. It is likely that, in plethoric hosts, insufficient differentiated erythroblasts

were produced to permit the development of macroscopically visible spleen

colonies. Evidence was obtained that granulocytic differentiation proceeded

during the growth of the transplanted colony-forming cells, and that this mode

of differentiation was not affected by the suppression of erythropoiesis. These

results indicate that both granulocytic differentiation and the process of self-

renewal by which colony-forming cells increase in numbers are controlled

independently of the control of erythropoiesis. These experiments provide

additional support for the view that colony-forming cells differ from cry-

thropoietin-sensitive cells.

SUMMARIO IN INTERLINGUA

Le suppression, per transfusiones, del erythropoiese in animales del genotypo

IVIWV se ha provate capace a prevenir le disveloppamento de macroscopic

colonias splenic post le injection de normal cellulas medullari coisogene. Iste

112 TILL, SIMINOVIT�H AND MC CULLOCH

inhibition del formation de colonias non esseva un effecto del non-proliferation

de cellulas a formation de colonias in le absentia de stimulation erythropoietic,

viste que le intensitate crescential de normal cellulas a formation de colonias in

animales plethoric non differe significativemente ab illo incontrate in hospites

anemic. Ii es probabile que, in hospites plethoric, insufficiente erythroblastos

differentiate esseva producite pro render possibile le disveloppamento de mac-

roscopicamente visibile colonias splenic. Esseva obtenite evidentia in supporto

del conclusion que Ic differentiation granulocytic procedeva durante Ic cres-

centia del transplantate cellulas a formation de colonias e que iste modo de

differentiation non esseva afficite per Ic suppression del erythropoiese. Iste

resultatos indica que tanto Ic differentiation granulocytic como etiam le pro-

cesso del auto-renovamento per le qual le cellulas a formation de colonias aug-

menta br numeros es regulate de maniera independente del regulation del

erythropoiese. Le experimentos hic reportate supporta additionalmente le con-

ception que cellulas a formation de colonias differe ab cellulas sensihile pro

erythropoietina.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the excellent technical assistance of Miss R. Wyncoll,

Mrs. M. Bulmer, Miss J. Sheldon, Mrs. C. Spencer, and Messrs. R. Course, A. Galberg,

R. Kuba, F. Mallory, and P. Winter.

REFERENCES

1. Alpen, E. L., and Cranmore, D.: Ob-

servations on the regulation of cry-

thropoiesis and on cellular dynamics

by Fe59 autoradiography. In: TheKinetics of Cellular Proliferation (F.

Stohlman, Jr., Ed.). New York, Grune

& Stratton, 1959, p. 290.

2. Erslev, A. J.: The affect of anemic anoxia

on the cellular development of nu-

cleated red cells. Blood 14:386, 1959.3. Jacobson, L. 0., Goldwasser, E., and

Gurney, C. W.: Transfusion-induced

polycythaemia as a model far studying

factors influencing erythropoiesis. In:

Ciba Foundation Symposium on

Haemopoiesis (C. E. W. Wolsten-holme and M. O’Connor, Eds.). Lon-

don, Churchill, 1960, p. 423.

4. Gurney, C. W., Lajtha, L. C., and Oli-ver, R.: A method for investigation of

stem cell kinetics. Brit. J. Haemat.

8:461, 1962.

5. Gurney, C. W., Degowin, R., Hofstra, D.,

and Byron, J.: Applications of erythro-

poietin to biological investigation. In:

Erythropoiesis (L. 0. Jacobson and M.

Doyle, Eds.). New York, Grune &Stratton, 1962, p. 151.

6. Till, J. E., and McCulloch, E. A.: A

direct measurement of the radiation

sensitivity of normal mouse bone mar-

row cells. Rad. Res. 14:213, 1961.7. McCulloch, E. A., Siminovitch, L., and

Till, J. E.: Spleen-colony formation in

anemic mice of genotype WW�. Sci-

ence 144:844, 1964.

8. Siminovitch, L., McCulloch, E. A., and

Till, J. E.: The distribution of colony-

forming cells among spleen colonies.J. Cell Comp. Physiol. 62:327, 1963.

9. McCulloch, E. A., Till, J. E., and Simi-

novitch, L.: The role of independentand dependent stem cells in the con-

trol of hemopoietic and immunologic

responses. ln: Methodological Ap-

proaches to the Study of Leukemias

(V. Defendi, Ed.). Philadelphia, The

Wistar Institute Press, 1965, p. 61.10. Bruce, W. R., and McCulloch, E. A.:

The effect of erythropoietic stimulation

on the hemopoietic colony-forming

cells of mice. Blooci 23:216, 1964.11. Liron, M., and Feldman, M.: The specif-

ic suppression of the differentiation

of erythroid clones in polycythemic

animals. Transplantation 3:509, 1965.

EFFECT OF PLETHORA ON COLONY-FORMING CELLS 113

12. Schooley, J. C., Cantor, L. N., and

Havens, V. W.: Relationship between

growth of colony-forming cells anderythropoietin-sensitive cells in mice

irradiated with 200 R of Co�� gamma

rays. Exp. Hemat. 9:55, 1965.

13. Curry, J., Trentin, J., and Wolf, N.: Con-trol of spleen colony histology by

erythropoietin, cobalt, and hypertrans-

fusion (abstract). Exp. Hemat. 7:80,

1964.

14. Becker, A. J., McCulloch, E. A., and Till,J. E.: Cytological demonstration of the

clonal nature of spleen colonies derivedfrom transplanted mouse marrow cells.

Nature (London) 197:452, 1963.

15. Russell, E. S.: Problems and potentiali-

ties in the study of gene action in themouse. In: Methodology in Mammal-

ian Genetics (W. J. Burdette, Ed.).

San Francisco, Holden-Day, 1963, p.

217.16. McCulloch, E. A., and Till, J. E.: Pro-

liferation of hemopoietic colony-

forming cells transplanted into irradi-

ated mice. Radiat. Res. 22:383. 1964.

17. Thompson, M. W., McCulloch, E. A.,

Siminovitch, L., and Till, J. E.: The

cellular basis for the defect in haemo-

poiesis in flexed-tailed mice. I. The

nature and persistence of the defect.Brit. J. Haemat. 12:152, 1966.

18. Rvt#{246}maa, T.: Identification and counting

of granubocytes by peroxidase reaction.

Blood 19:439, 1962.19. Fowler, J. H., Till, J. E., McCulloch,

E. A., and Siminovitch, L.: The cell-

ular basis for the defect in haemo-

poiesis in flexed-tailed mice. II. The

specificity’ of the defect for erythro-poiesis. Brit. J. Haemat. (in press).

20. Cunningham, J. R.. Bruce, W. R., andWebb, H. P.: A convenient l37Cs

unit for irradiating cell suspensions

and small laboratory animals. Physics

in Med. and Biol. 10:381, 1965.21. Wilcoxon, F., and Wilcox, R. A.: Some

Rapid Approximate Statistical Proce-

dures. Pearl River, N. Y., LederleLaboratories, 1964.