Erythroid Stem Cells in Friend-Virus Infected Mice UTA OPITZ,HANS-JOACHIM SEIDEL AND IVAN BERTONCELLO Department of Clinieal Physiology, University of Ulm, 7900 Ulm/Germany

I

ABSTRACT The erythropoietic stem cell compartment was studied in Friend-virus (polycythemic strain, FV-P) infected DBA/2 and NMRI mice with the CFUE and BFUE technique. Early after infection there was a depression in CFUE number in bone marrow and spleen, followed by an increase of the CFUE concentration, earlier and more pronounced in the spleen than in the marrow. Three days after FV-P infection an erythropoietin (Ep) independent CFUE population started to grow and replaced the normal Ep-dependent population within 8 to 12 days. The shift to Ep independency was not gradual. CFUE colonies of FV-P infected bone marrow cells were two to three times larger than control colonies after three days in vitro incubation. BFUE colonies increased in number during the first days of infection, but were totally lost after more than ten days. After velocity sedimentation of bone marrow cells of FV-P infected animals, however, the BFUE containing fractions showed normal BFUE colony growth and normal Ep sensitivity. In unfractionated bone marrow cell cultures BFUE colony growth could be observed later than ten days post infection when the cultures were refed with medium. It was therefore concluded that the loss of BFUE colony growth after FV-P infection was an in vitro artefact due to inadequate culture conditions. Studies of the erythropoietic stem cell com- partment size and with respect to the EPpa'rtment have been greatly facilitated by the dependency of these early precursor cells. The development of two in vitro colony assays. The results of this part of the study indicate that CFU-E assay (colony forming units - erythro- BFU-E of FV-P infected mice remain EPpoietic) (Stephenson e t al., '71; Iscove e t al., dependent, but need special conditions for '74) identifies a cell one or two division steps growth in the in vitro cultures. before the beginning of hemoglobin synthesis MATERIALS AND METHODS (Cormack, '76) and the BFU-E assay (burst forming units - erythropoietic) (Axelrad et 1. Mice al., '74) refers to a more immature erythropoiFemale NMRI mice (Suddeutsche Veretin sensitive cell, closely related to the suchstierfarm, Tuttlingen) or DBAI2 mice pluripotent stem cell (CFU-S) (Gregory, '76). (Zentrale Tierversuchsanstalt, Hannover) of We used the CFU-E and BFU-E assays as de- 20-23 gm weight were used. Ten animals per scribed by Iscove et al. ('74, '75) to study the cage were kept in artificial light 12 hours changes in the erythropoietic stem cell com- daily. They were fed commercial pellets and partment after infection of mice with the water ad libitum. polycythemic Friend virus (FV-P). Results on the CFU-Egrowth in mice after FV-P infec- 2. Virus tion by Horoszewicz e t al. ('75) and by Liao The N-B tropic polycythemia inducing and Axelrad ('75) had shown that an Erythro- Friend virus (FV-P) was kindly donated by poietin (EP) independency developed in bone Professor Doctor W. Schafer, Tiibingen. This marrow and spleen cell cultures. FV-P has been serially passaged in DBA/2 In the present paper we further characterize the growth pattern of these autonomous Received Aug. 2, '77. Accepted Dec. 22, '77. Supported by the Deutsche Forscbungsgemeinschaft (Sondercolonies and the development of their EPforschungsbereich 112, Zellsystemphysiologie). independency. In addition, the BFU-E comI. Bertoncello, Fellow of the Alexander von Humboldt partment was studied for changes of the com- Foundation. I

J. CELL. PHYSIOL. (1978)96: 95-104.

95

96

U. OPITZ, H.-J. SEIDEL AND I. BERTONCELLO

mice. Mice were infected i.p. with 0.2 ml of a 10%cell free homogenate of leukemic spleens in saline.

tions of 30 ml were collected, concentrated by centrifugation and then cultured for BFU-E as described.

3. Cell suspensions Four mice per point were killed by cervical dislocation, the spleen weight was determined and a single cell suspension of the spleens, and one femur (9 mm length) of each mouse was prepared in a-medium (Flow Laboratories) containing 2% fetal calf serum (Seromed).

7. Irradiation of cell suspension

4. Colony forming units/erythropoietic (CFU-E) The method described by Iscove et al. ('74, '75) was used. 0.8% methylcellulose, 30%fetal calf serum, erythropoietin (EP) Step I11 (Connaught Laboratories), 0.2-0.4 U/ml, depending on the optimal activity of the batch, a-Thioglycerol a t a n end concentration of lo-' M and bone marrow or spleen cells in a-medium were mixed. Four parallel petri dishes (Greiner) containing 1ml with 3 X lo5celldm1 were set up, and incubated for 48 hours at 37"C, in a humidified atmosphere containing 5% CO,. Erythroid colonies containing more than eight small cells were scored without staining at a magnification of 80. To check the scoring accuracy erythroid colonies were transferred to slides and stained for hemoglobin by the method of Borsook et al. ('69). 5. Burst forming units/erythropoietic (BFU-E) Cells were cultured for ten days at a concentration of 3-4 x lo5 celldm1 in the same medium as used for the CFU-E, except that the concentration of E P was increased to 1-3 U/ ml. The E P was extensively dialysed against a-medium before use. Colonies of more than 200 cells with CFU-E like aggregates were scored a t a magnification of 40. To check the cell types within the colonies, they were transferred to slides and stained by the Pappenheim method. 6. Unit gravity sedimentation The method described by Miller and Philipps ('69) was used. One and two percent solutions of BSA (Behring) were prepared in Hanks solution (Seromed) and sterilized by filtration through a 0.45 p Millipore filter. The chamber (12 cm diameter) was loaded with 20 ml of a bone marrow cell suspension containing 1-2 x 10' cells and sedimentation was carried out a t 4"C, for four hours. Frac-

Cells were kept in a n icewater bath and irradiated with 1,200 rads (280 kV, 15 mA, 1 mm-Cu filter, focal distance 40 cm, and a dose rate of 100 R/min). RESULTS

Characterization of Friend Leukemia NMRI and DBA/2 mice were infected with FV-P and the development of the Friend disease was studied. The data concerning the increase of reticulocyte numbers and hematocrits and spleen weight showed that the virus and the strains of mice used gave results compatible with those of previous authors (Mirand, '67a,b; Mirand e t al., '68; Tambourin e t al., '73). Analysis of the CFU-E compartment The CFU-E technique as described by Iscove and Sieber ('74) was used to study NMRI mice after FV-P infection. Early after infection the numbers of EP-dependent CFU-E in the bone marrow and spleen were depressed. Twenty days after infection however the CFU-E concentration of the bone marrow had doubled and t h a t of the spleen increased to a 10-fold control value (figs. la,b). Without E P only background numbers of erythroid colonies (110/105cells) developed in normal control cultures. FV-P infection of the mice however induced t h e growth of E P independent erythroid colonies (fig. 1).The first E P independent erythroid colonies appeared in bone marrow and spleen cell cultures three to five days after infection and then progressively replaced the E P dependent colonies. To see whether the shift to E P independency occurred gradually, a n E P dose response curve was established seven days after FV-P infection (table 1).At t h a t stage after infection there were 28 and 334 EP independent CFU-E colonies per lo5 bone marrow and spleen cells respectively. After addition of E P additional colonies were stimulated indicating the presence of a n E P dependent CFU-E population. The dose response curve for the growth of these additional colonies was not different from that of control mice. In both assays the maximal colony numbers were obtained using the E P doses which were optimal for control cultures.

97

ERYTHROID STEM CELL IN FRIEND-VIRUS INFECTED MICE

(BUNE MRRRUW)

EFFECT UF FV-P INFECTION

D R Y 5 R T T E R FV-P 0

FV-P 0 EP

INFECTION + FV-P t EP

EFFECT OF FV-P INFECTIUN

(5PLEEN)

-&-b

- -0

9

I0

D R Y 5 R F T E R FV-P

112

;I

I6

I8

20

22

INFECTIUN

A FV-P 0 EP A FV-P t EP Fig. 1 Effect of FV-P infection on CFU-E concentration in bone marrow (fig. la) and spleen (fig. lb) of NMRI mice: The mean & 1 SEM of all controls is shown. Infected mice: CFU-E in cell cultures without addition of EP 0 ,A and with EP +, A respectively.

98

U. OPITZ. H.-J. SEIDEL AND I. BERTONCELLO

BONE MARROW SPLEEN 10.

q 75

Y3LL LL

u

0 5.

(y

X

51 25

10

30

70

50

xi04 CELLS Fig. 2 Effect of varying cell concentration on number of CFU-E. Cultures were set up seven days after FV-P infection and stimulated with the optimal EP dose. The mean f 1 SEM of four dishes is shown. TABLE 1

Erythropoietin dose response in normal and FV-P infected DBAI2 mice CFUEllO' cells

EP Dose

Control mice

FV-P mice

(Ulml)

0

0.02 0.04 0.1 0.2 0.4 0.8

BM

Spleen

2 84 124 245 370 558 570

0 33 42 87 124 156 168

BM

28; 60 55 126 212 248 297

Spleen

334" 112 228 336 510 554 572

Values = X of three dishes. Values minus number of EP independent CFUE 128). for the bone marrow and (3341" for the spleen.

In control cultures there is a linear relationship between the number of CFU-E colonies and cells plated (Opitz e t al., '77). Seven days (fig. 2) and also later after FV-P infection this response seemed to be linear over a wide range for both bone marrow and spleen cell cultures. As seen in figure 1, the first EP independent CFU-E appear soon after infection. To test

Fig. 3 Erythroid colonies in (fig.3a) normal bone marrow cell cultures after two days of incubation; (fig. 3b) FV-P infected bone marrow cell cultures after three days of incubation. Magnification 200.

whether a longer in vitro incubation would increase the number of EP-independent colonies, cultures of FV-P infected bone marrow and spleen cells were set up two to five days after infection and incubated up to 11 days. As seen in table 2 the number of EP independent colonies did not increase after a longer in vitro incubation period. The increase of the EP independent colonies was only seen after prolonged infection in vivo. Normal CFU-E colonies reached their maximal numbers after 48 hours of in vitro incubation (table 3). To determine whether EPindependent CFU-E showed a similar growth characteristic, bone marrow and spleen cell cultures were set up and counted 1 , 2 , 3 , 4and 7 days later. As in the controls, infected cells gave maximal colony numbers after 48 hours of incubation. One and two days after infection colony size did not differ from controls. Spleen colonies were always smaller than those of the marrow. In contrast t o the normal cultures however, two-thirds of the colonies of infected bone marrow cells continued to grow until day 3 and reached about three times the normal colony size (fig. 3). Only 5 1 0 %of the colonies in infected spleens present a t day 3 of in vitro incubation reached a size comparable

99

ERYTHROID STEM CELL IN FRIEND-VIRUS INFECTED MICE TABLE 2

Development of EP independent CFUE after FV-P infection (DBAI2 mice) In vitro incubation in days Days after FV-P infection

2 3 4 5

CFU-El2 x

lo5 cells

Bone marrow cells Spleen cells Bone marrow cells Spleen cells Bone marrow cells Spleen cells Bone marrow cells Spleen cells

3

5

7

2 1 17 26 20 26 15 34

2 2 19 23

8

2 0 9 18 6

12 14 35

13 20

0

9

11

0 0

0 1 5 9

0

0

0 0

0

0

-

0 0

20

Values are the mean of three dishes. TABLE 3

Influence of in vitro incubation on number of CFUE NMRI mice 19 days after FV-P infection normal NMRI mice Number of CFUEW Days of in vitro incubation

Bone marrow cells + Ep FV-P infected Bone marrow cells 0 Ep FV-P infected Spleen cells + EP FV-P infected Spleen cells 0 EP FV-P infected Bone marrow cells + Ep control Spleen cells + EP

X

lo5 cells

2

3

4

7

9042 52

2,4362 156

1,512%204

1,4822 510

450% 140

1,0562204

2,1542 38

1,5602191

1.398% 54

6482 144

624% 60

2,9882 84

2,8582 78

1,6382198

8882 136

714% 66

2,6792124

2,4842180

1,4522 36

498% 134

1,406% 78

7982 50

4732 76

341% 66

4402 54

3432 44

1022 28

51% 26

1

Values are the mean of four dishes

%

S.D.

to these marrow colonies. Later there was no further enlargement of the colony size. As in normal cultures colonies then disintegrated. The presence or absence of E P in the plates was of no influence as all colonies were EP independent 19 days after FV-P infection.

Analysis of the BFU-E compartment With normal cell cultures containing three units of E P BFU-E numbers were in the range of 10-20/105 bone marrow cells and 8-20/106 spleen cells. Without addition of E P no colony growth occurred. The colony counts depended on the lot of E P and on the dose. The batch of the fetal calf serum was also critical. Some batches which supported good CFU-E growth were unsuitable for BFU-E cultures and vice versa. Normally there was one BFU-E/105 bone marrow cells with 0.5 U EP/ml, five to

eight BFU-E’s with 1 U EP/ml and 8 to 15 with two units. A linear dose response for BFU-E numbers was found over a range from 8 x lo4to 5 x lo5bone marrow cells per plate. Most of the bone marrow BFU-E colonies were larger than those of the spleen and contained CFU-E-like aggregates in the periphery and some large cells. Erythroblasts of the different maturation stages were found. Spleen BFU-E colonies often resembled those colonies described by Gregory (’76)as “3-4 day BFU-E colonies,” or very big CFU-E colonies (fig. 4). After FV-P infection there was a slight increase in BFU-E colony numbers at days 1and 3 but later they decreased in bone marrow and in spleen cell cultures (see table 4 for bone marrow cells). When the dishes were fed with 50 p l double strength a-medium at days 2 and

100

U. OPITZ, H.-J. SEIDEL AND I. BERTONCELLO

Fig. 4 BFU-E colonies from (a) normal bone marrow cells, magnification 100; (b) normal spleen cells, magnification 200.

5 after onset of the cultures and incubated with a fetal calf serum, which gave poor CFUE growth but enhanced BFU-E growth in normal cultures, the BFU-E colony numbers were in the range of the controls even late after infection, indicating that the normal culture conditions were insufficient for the growth of BFU-E from FV-P infected mice.

In a further analysis of this in vitro loss of BFU-E normal bone marrow cells were cocultured with cells from FV-P infected mice. This reduced the colony numbers of the normal cells to 52% after addition of infected marrow cells and to 29% after addition of spleen cells (table 5). Irradiation of cells before addition removed the inhibitory poten-

101

ERYTHROID STEM CELL IN FRIEND-VIRUS INFECTED MICE TABLE 4

Decline of BFU-E colony numbers from bone marrow cells of FV-P infected NMRI mice under normal culture conditions. Normal growth is obtained after refeeding ofthe cultures at days 2 and 5 of incubation (BFU-E per lo5 cells) ~~

FV-P infected mice Feeding of cell cultures Control mice

2

X

No 50 fil double strength medium

Days after infection

1

3

8

15

26

20* 4

20%5

252 3

162 3

5%2

322

272 4

33%3

35%4

34%6

2825

23%5

~~

Values are th e mean

f

1SEM of four dishes

TABLE 5

BFU-E numbers in normal. FV-P infected and mixed cell cultures NMRI mice Bone marrow cells

Bone marrow cells

Spleen cells

26 days after FV-P inf.

26 days after FV-P inf.

irradiated

irradiated

BFU-Eldish (no feeding of cultures)

+i.6 x 105

38* 4 0 0 20%4 28f 6 11%3 392 7

3 x 105 3 x 105 3 x 105 3 3 3 3

x 105 x 105 x 105 x 105

+i.6 x 105 f i . 6 x 105

Values are the mean

f

+ 1.6 x 105

1SEM of four dishes

TABLE 6

Unit gravity sedimentation of normal and FV-P infected bone marrow cells Modal sedimentation velocities (mm/hr)

Normal bone marrow cells (unfract.) Normal bone marrow cells (fract.)

Infected bone marl‘OW cells 26 days after FV-P (fract.)

BFUElfraction

Nucleated cells/ fraction

BFUE/

lo5 cells

Nucleated cells loaded on gradient

21 7.21 6.56 5.91 5.27 4.63 4.00 3.38 5.79 5.15 4.52 3.89 3.27

300 676 912 923 1401 1315 69 438 693 1024 810 31

1.20 x 1.47 3.38 10.26 9.34 5.98 5.68 1.75 X 4.33 5.69 4.50 3.06

lo6

25 46 27 9 15 22

lo6

25 16 18 18

1.33 X

lo*

8.6

10’

1 X

1

Values are th e mean of three dishes

tial. That the BFU-E loss was mediated by living cells was further seen in a cell separation experiment using the velocity sedimentation. There was normal BFU-E growth in the fractions where BFU-E colony growth from normal bone marrow cells was observed, the modal sedimentation velocity was not different from controls (table 6). There was

always an absolute Ep requirement and no difference in the Ep sensitivity nor-in the size of the colonies. DISCUSSION

Erythropoietic cell growth becomes independent from erythropoietin after FV-P infection (Horoszewicz e t al., ’75; Liao and Axelrad,

102

U. OPITZ, H.-J. SEIDEL AND I. BERTONCELLO

'75). I t is also not regulated by the normal negative feedback from plethorism (McGarry and Mirand, '73). This Ep independency starts one to four days after virus infection and is completely developed after 7 to 14 days. Our results confirm these findings and demonstrate that the development of the Ep independency is only dependent on the time after infection in vivo. Prolonged in vitro cultivation of the cells after virus infection in vivo did not lead to a n increase of Ep independent CFU-E. This excludes a conversion of Ep dependent CFU-E to Ep independency during the in vitro cultivation in methylcellulose. In principle, however, such a conversion seems to be possible with a special strain of Friend virus and under special culture conditions (Clarke e t al., '75). CFU-E colonies from bone marrow cells of controls attain their maximal size after two days of in vitro incubation. Bone marrow CFU-E colonies from infected mice however reach their maximal size after three days and two to three times larger than control colonies. This could indicate a better proliferative potential of these CFU-E, or the colony forming cell could be one to two division steps more immature than normal CFU-E. Cell cycle times seem to be similar in both instances since the colonies have the same size a t day 2 in our experiments. A cell cycle time of ten hours as described by Cormack ('76) would lead to the formation of colonies of about 128 cells after three days which indeed was in the range of these large CFU-E colonies. These large colonies predominately developed in the bone marrow, and only very few were present in the spleen. Whether this reflects a migration of more differentiated precursors from the marrow to the spleen has to be further analysed. The proliferative capacity of the FV-P infected erythropoietic cells, however, is also very limited as seen from the disintegration of the colonies. Our findings with the dose response curve of the Ep responsive CFU-E a t day 7 after infection support the view that the conversion to Ep independency is not gradual. No cells with intermediate response were found. Antiserum against Ep does not reduce the number of the independent colonies (Liao and Axelrad, '75). There is, however, a period of 7 to 14 days until all CFU-E are Ep independent. At this time after infection the virus titer may be high enough to infect all developing erythropoietic precursor cells which are targets for the virus.

On the other hand, this period could be the time which is necessary for the erythropoietic descendents of infected pluripotent stem cells to replace normal erythropoietic cells. This second hypothesis seems unlikely, as many reports suggest t h a t the target cell for FV-P is in the Ep responsive compartment (Tambourin and Wendling, '71; Frederickson et al., '75; Nasrallah and McGarry, '76). Studies on chemotherapy from our laboratory clearly demonstrate t h a t normal precursor cells of CFU-E's must be present in FV-P infected mice even when they only have Ep independent CFU-E. After treatment with hydroxyurea normal Ep dependent CFU-E appear in the regenerative phase (Seidel and Opitz, '78). A comparative analysis of the stem cell compartments revealed a situation where CFU-S were only slightly depressed (Wendling e t al., '72, '73, '74; Tambourin and Wendling, '71, '75), the CFU-E number increased, and BFU-E not detectable. As the BFU-E is assumed to be closely related to CFU-S (Gregory, '76) and a precursor cell for CFU-E, we asked whether this loss of BFU-E could be a n in vitro artefact due to special culture conditions. In fact it could be shown that the BFUE were present in about normal concentrations, that they were dependent on Ep and that their lack of growth under normal BFU-E culture conditions might have something to do with nutritional or metabolic requirements. One might also think of a negative feedback mechanism exerted by the high number of CFU-E's and erythroblasts in the plate, since the refeeding experiment was only done with a batch of fetal calf serum which gave poor growth for CFU-E. This has to be further analysed, but since the feedback regulation of plethorism does not exist in vivo after FV-P infection, it is unlikely that such a mechanism exists under in vitro culture conditions. Cellular interactions, however, do influence the BFU-E growth as demonstrated by Heath et al. ('76) by mixing fractions after velocity sedimentation. In conclusion, on the basis of our data we believe that the alterations in the hematopoietic stem cell compartments after FV-P infection occur mainly in the erythropoietic compartment a s demonstrated by the EP independency of the CFU-E population. The BFU-E population however remained E P dependent in growth and seemed to be not directly affected. The EP independency of the CFU-E growth is also the main characteristic of this disease

ERYTHROID STEM CELL IN FRIEND-VIRUS INFECTED MICE

103

etic cells of mice infected with Friend virus. Int. J. Cancer, 15: 467-482. McGarry, M. P., and E. A. Mirand 1973 Altered responsiveness to erythropoietin in mice following infection with polycythemia inducing Friend virus. Exp. Hemat., 1: 174-182. Miller, R. G., and R. A. Philipps 1969 Separation of cells by ACKNOWLEDGMENTS velocity sedimentation. J. Cell. Physiol., 73: 191-202. Mirand, E. A. 1967a Erythropoietin-like effect of a We gratefully acknowledge the excellent polycythemic virus. Proc. SOC. Exp. Biol. Med., 125: technical assistance of Mrs. E. Barthel and 562-565. 1967b Virus-induced erythropoiesis in hyperMrs. K. Steinhoff. transfused-polycythemic mice. Science, 156: 832-833. Mirand, E. A., R. A. Steeves, L. Avila and J. T. Grace, Jr. LITERATURE CITED 1968 Spleen focus formation by polycythemic strains of Axelrad, A. A,, D. L. McLeod, M. M. Shreeve and D. S. Heath Friend leukemia virus. P. S. E. B. M., 127: 900-904. 1974 Properties of cells t h a t produce erythrocytic colMirand, E. A., R. A. Steeves, R. D. Lange and J. T. Grace onies in vitro. In: Proc. Second International Workshop 1968 Polycythemia in mice: erythropoiesis without on Hematopoiesis in Culture. W. A. Robinson, ed. U. S. erythropoietin. Proc. SOC.Exp. Biol., Med., 128: 844-849. Government Printing Office, Washington, pp. 226-234. Nasrallah, A. G., and M. P. McGarry 1976 In vivo distincBorsook, H., K. Ratner and B. Tattric 1969 Studies on tion between a target cell for Friend virus (FV-P) and erythropoiesis. 11. A method of segregating immature murine hematopoietic stem cells. J. Nat. Cancer Inst., 57: from mature adult rabbit erythroblasts. Blood, 34: 32-41. 443-445. Clarke, B. J., A. A. Axelrad, M. M. Shreeve and D. L. McLeod Opitz, U., H. J. Seidel and I. Rich 1977 Erythroid stem cells 1975 Erythroid colony induction without erythropoietin in Rauscher virus infected mice. Blut, 35: 35-44. by Friend leukemia virus in vitro. Proc. Nat. Acad. Sci. Seidel, H. J., and U. Opitz (1978,submitted) Hemopoietic U.S.A., 72: 3556-3560. stem cells in Friend virus infected mice under chemoCormack, D. 1976 Time-lapse characterization of eryththerapy. 111. Effects of Hydroxyurea. rocytic colony-forming cells in plasma cultures. Exp. Stephenson, J. R., A. A. Axelrad, D. L. McLeod and M. M. Hemat., 4: 319-327. Shreeve 1971 Induction of colonies of hemoglobin-synFrederickson, T., P. Tambourin, F. Wendling, C. Jasmin and thesizing cells by erythropoietin in vitro. Proc. Nat. Acad. F. Smajda 1975 Target cell of the polycythemia inducing Sci. (U.S.A.), 68: 1542-1546. Friend virus: Studies with Myleran. J. Nat. Cancer Inst., Tambourin, P., 0.Gallien-Lartigue, F. Wendling and D. 55: 443-446. Huaulme 1973 Erythrocyte production in mice infected Gregory, C. S. 1976 Erythropoietin sensitivity as a difby the polycythaemia-inducing Friend virus or by the ferentiation marker in the hemopoietic system: Studies anemia-inducing Friend virus. Brit. J. Haemat., 24: of three erythropoietic colony responses in culture. J. 511-520. Cell. Physiol., 89: 289-302. Tambourin, P. E., and F. Wendling 1971 Malignant transHeath, D. S.,A. A. Axelrad, D. L. McLeod and M. M. Shreeve formation and erythroid differentiation by polycythae1976 Separation of the erythropoietin-responsiveprogenmia-inducing Friend virus. Nature New Biol., 234: itors BFU-E and CFU-E in mouse bone marrow by unit 230-233. gravity sedimentation. Blood, 47: 777-791. 1975 Target cell for oncogenic action of polycyHoroszewicz, 3. S., S. S. Leong and W. A. Carter 1975 thaemia-inducing Friend virus. Nature, 256: 320-322. Friend leukemia: rapid development of erythropoietinWendling, F., P. E. Tambourin and P. Jullien 1972 independent hematopoietic precursors. J. Nat. Cancer Haematopoietic CFU in mice infected by the polythemia Inst., 54: 265-267. inducing Friend virus. I. Number of CFU, and differentiaIscove, N. N., and F. Sieber 1975 Erythroid progenitors in tion pattern in the spleen colonies. Int. J. Cancer, 9: mouse bone marrow detected by macroscopic colony for554-566. mation in culture. Exp. Hemat., 3: 32-43. 1973 Hematopoietic CFU in mice infected by the Iscove, N. N., F. Sieber and K. H. Winterhalter 1974 polycythaemia inducing Friend virus. IV. Pattern of Erythroid colony formation in cultures of mouse and blood recovery in irradiated mice grafted with normal or human bone marrow: Analysis of the requirement for infected bone marrow cells. Biomedicine, 18: 521-529. erythropoietin by gel filtration and affinity chromatogra- Wendling, F., P. E. Tambourin, 0. Gallien-Lartigue and M. phy on agarose-concanavalin A. J. Cell. Physiol., 83: Charon 1974 Comparative differentiation and enumera309-320. tion of CFU-S from mice infected either by the anemia Liao, S. K.,and A. A. Axelrad 1975 Erythropoietin-indeor polycythemia inducing strains of Friend virus. Int. J. pendent erythroid colony formation in vitro by hemopoiCancer, 13: 454-462.

compared to the anemia and erythroblastosis which is induced by the Rauscher virus, where only EP dependent CFU-E are found in our laboratory with the same techniques (Opitz e t al., '77).

Erythroid stem cells in Friend-virus infected mice.

Erythroid Stem Cells in Friend-Virus Infected Mice UTA OPITZ,HANS-JOACHIM SEIDEL AND IVAN BERTONCELLO Department of Clinieal Physiology, University of...
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