Ontogeny and Evolution of Normal and Malignant Hemato-poietic Cells Chairmen: D. Metcalf and R. S. Metzgar
Comparative Leukemia Research 1975, Bibi. Haemat., No. 43 Ed. J. Clemmesen and D. S. Yohn, pp. 1-5 (Karger, Basel 1976)
Proliferation of Hemopoietic Stem Cells and Development of Potentially Leukemic Cells in vitro T. M. Dexter and L. G. Laitha1 Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester
We have reported that in some `co-cultures' of thymus and bone marrow cells there was proliferation and differentiation of hemopoietic stem cells for several months (Dexter and Lajtha, 1974). The role of the thymus cells in these cultures is difficult to understand and for this reason we have recently modified the technique and developed a system where proliferation of bone marrow stem cells occurs without the addition of thymus cells. These cultures have been used to study the characteristics of potentially leukemic hemopoietic cells in vitro. Materials and Methods To set up the monolayer system of `attaching' cells 107 bone marrow cells obtained from BDF, mice were inoculated into glass bottles, in 10 ml of Fischer's medium supplemented with 1 Supported by the Medical Research Council and the Cancer Research Campaign.
200 /0 horse serum (Flow Labs.) and cultured as described previously for thymus cells (Dexter and Lajtha, 1974). The cultures were fed at weekly intervals by removal of half of the growth medium and addition of an equal volume of fresh medium at weekly intervals, for 3 weeks. During this 3-week culture period, a population of attaching cells becomes established in the cultures. To initiate the culture `proper', at the end of the third week, half of the growth medium was again removed but now 5 ml of fresh growth medium containing a new inoculum of 107 syngeneic-nucleated bone marrow cells was added to the established bone marrow monolager cultures. At weekly intervals thereafter the cultures were fed as before and the nonattaching cells removed were assayed for morphology, stem cell concentration (CFUg) (Till and McCulloch, 1961) and behavior, granulocyte precursor cells (CFUC ) (Bradley and Metcalf, 1966; Pluznik and Sachs, 1966) and the ability to produce leukemia in various `conditioned' recipients. The collection and assay of the nonattaching population of cells has been described in detail elsewhere (Dexter et al., 1973).
Results The characteristics of a typical culture of normal bone marrow cells are shown in ta-
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Introduction
ble I. Cell proliferation is obviously occurring since after each feeding (which removes half of the nonattaching cells) the number of cells recovers by the following week. (For the duration of the culture the number of adhering cells showed little change and was maintained at a fairly constant level.) Similarly, there is proliferation of CFUg, at least for 12 weeks, since the number increases after successive depopulations (it has previously been established that CFUg — at least in short-term cultures — occur only in suspension and are not detectable in the adhering population of cells) (Dexter et al., 1973). The CFUg present in these cultures form both erythroid and granulocytic spleen colonies and can protect potentially lethal irradiated mice from hemopoietic death. CFU, are also being produced for several months. These cells form CSF-dependent colonies and clusters of granulocytes and macrophages in soft agar. For the first few weeks these cultures contain mainly granulocytes and macrophages. Subsequently, however, an accumulation of blast cells occurs — which is not associated with an increase in cell density in the cultures — and cultures containing more than 704/ο blast cells are regularly seen. With prolonged culturing the percentage of blast cells decreases. It is also worthy of comment that this accumulation of blast cells occurs concomitantly with proliferation of apparently normal stem cells. Preliminary experiments have shown that these cells express neither T nor B lymphocyte markers and their origin and nature awaits elucidation. After various times in vitro (9-34 weeks) the cells were tested for their ability to produce leukemias after injection into syngeneíc recipients. Different routes of inoculation were used and the cells were injected into either normal, immunosuppressed (600 rad X-rays) or T lymphocyte-deficient mice. So far, out of a total of 39 mice injected, no leukemias have developed. How-
2
ever, the number of animals inoculated with the cultured cells is low and the postinoculation observation times somewhat variable (14-50 weeks). Hence, no definite conclusions can yet be drawn regarding the frequency of `spontaneous' leukemic transformation of normal bone marrow cells in vitro. Culture of ‚NU-treated bone marrow. We have also studied the characteristics in culture of bone marrow cells derived from mice treated in vivo with methylnitrosourea (MNU). ΒDFI 9 mice were injected intravenously with a single dose of 50 mg/kg MNU. 3 weeks later some mice were killed, the femoral marrow cells removed and injected immediately in vivo to test the leukemic potential. Additionally 107-nucleated cells were inoculated into several culture bottles, and a monolayer allowed to form over a 3-week culture period. At this time a further group of the same mice were killed, the marrow cells again assayed in vivo for leukemogenicity or added to the established MNU bone marrow monolayer cultures (107-nucleated cells/culture). The growth conditions and method of feeding were as for normal bone marrow cells. The characteristics of a typical culture of 'NU-treated bone marrow are shown in table II. As with cultures of normal bone marrow cells, cell proliferation, production of CFUg and CFUC and accumulation of blast cells occurs. For the first 10 weeks in culture the CFUg are apparently normal, forming erythroid and granulocytic spleen nodules and can reconstitute the hemopoietic systems of potentially lethal irradiated mice. After 10 weeks in culture, however, spleen colonies containing mainly undifferentiated cells are found and the cells lose their protective ability. The CFUe produced throughout the culture period form colonies and clusters containing differentiated granulocytes. Macrophage colonies are only occasionally seen. Injection of these cultured 'NU-treated
Downloaded by: Université de Paris 193.51.85.197 - 2/5/2020 2:36:37 AM
DexterlLajtha
Culture of Hemopoietíc Cells in vitro
3
Table I., Culture of normal bone marrow cells; proliferative capacity, morphology and production of stem cells Weeks in culture
Total suspension cells/culture (x106)
Total CFU./ culture
Total CFUC/ culture
Morphology, % blasts granulocytes
1 2 4 5 7 9 12 13 17 20 34
0.9 0.8 0.6 0.7 0.9 0.9 0.7 1.1 0.6 0.6 12
160+10 140+18 180+40 160+20 110+8 240+18 190+20 ND 0 0 0
1,450+40 1,400+86 ND ND 1,600+90 ND 2,500+170 ND 0 50+4 0
3 2 62 ND 93 72 70 60 ND 0 5
74 59 31 ND 3 4 2 0 ND 0 0
macrophages
23 37 6 ND 3 24 28 40 ND 100 100
ND = No data.
Table ΙΙ. Culture of 'NU-treated bone marrow cells ; proliferation of cells, morphology and stem cell production Weeks in culture
Total suspension cells/culture (x 106)
Total CFUS/ culture
Total CFUe/ culture
Morphology blasts granulocytes
macrophages
1 3 6 7 10 12 15 17 20 30
2.8 1.6 0.9 1.1 0.9 1.0 1.1 1.5 1.0 1.0
600+ 100 350+20 70+10 280+50 240+ 70 480+40 300+20 300+30 0 0
4,300+200 ND ND ND ND ND 2,100+ 180 1,500+200 0 0
4 ND 24 22 74 ND 40 60 47 10
14 ND 16 30 17 ND 59 31 43 90
82 ND 60 48 9 ND 1 8 10 0
cells regularly produced leukemia (table III) — 15/22 animals developed leukemia between 11 and 18 weeks after inoculation of the cells (with longer observation times the final incidence may well be higher). The leukemias probably arise from the cells inoculated (rather than from transformation of host cells by some virus present in the injected cells or in the growth medium) since no leukemias were produced when 18- or 30-week cultured cells were ir-
radiated with 2,000 rad X-rays before injection, or when only the cell-free growth medium was injected. Of the 15 leukemias produced, 13 were characterized by massive and widespread lymph node enlargement along with splenic enlargement. The thymus was not involved. All these leukemias arose in 9 recipients. Five of these leukemias were tested for the presence of surface immunoglobulín (Ig) determinants (characteristic of bone marrow derived or
Downloaded by: Université de Paris 193.51.85.197 - 2/5/2020 2:36:37 AM
ND = No data.
Dexter/Lajtha
4
Table III. Leukemia-producing ability of 'NU-treated bone marrow cells cultured for various time intervals Weeks in culture
01 02 10 15 17 18 22 29
Cells injected (route)
Pretreatment of recipient mice (rad X-rays)
Observation time, weeks
Leukemias
107 (i.v.) 107 (i.v.) 107 (iv.) 107 (i.v.) 107 (i.v.) 5 χ 104 (intrasplenic) 2.5 x 105 (1.ν.) 5 χ 104 (intrasplenic) 6 x 10δ (í.v.) 6 x 105 (1.Υ.) 9 x 105 (i.v.) 9 x 105 (i.v.) 1.6 x 105 (i.v.) 1.6 x 105 (i.v.)
600 800 none 600 none 600
50 50 50 47 47 23
0/15 0/15 0/10 0/15 0/15 0/3
600
18
2/2
17, 18
600 none 600 600 none none 600
13 16 16 11 11 12 13
4/4 2/2 2/2 0/2 0/2 2/2 3/3
11,12,12,13 14, 16 15, 16
Latent periods
7
12, 12 12, 12, 13
B lymphocytes) by an indirect immunofluorescence technique (Moller, 1961). They were tested also for 8-antigen (characteristic of thymus-dependent, T lymphocytes) using a cytotoxícity assay based on release of 51Cr from cells treated with AKR anti O C3H serum (Wigzell, 1965; Reif and Allen, 1964) and guinea pig serum as a source of complement. All five leukemias were negative for O and Ig. They were also peroxidase-negative. Interestingly, the other two leukemias were produced by injection of the cultured cells (derived from mice) into d recipients. These leukemias were characterized by spleen and liver enlargement, and were O+ve (indicating T cells). Discussion We have previously reported that stem cell proliferation occurs in co-cultures of thy-
mus and bone marrow cells (Dexter and Lajtha, 1974) and have now shown that this proliferation also occurs in BM alone cultures (without the addition of thymus cells) provided that these cells are cultured on an adhering population of bone marrow-derived cells. However, the adhering cells are a heterogenous population and it is not known which particular population(s) of cells (nor the `factors' produced) is required for stem cell maintenance. What is apparent, however, is that whilst factor(s) are present facilitating stem cell maintenance and proliferation as well as production of CFUC, the cultures appear to be deficient in those factors allowing sustained granulocyte maturation, hence possibly the blast cell accumulation. This maturation block is readily overcome in agar cultures when CSF is present, suggesting that the decline in granulopoiesis in the liquid cultures may be associated with a deficiency in CSF. Indeed, preliminary experiments
Downloaded by: Université de Paris 193.51.85.197 - 2/5/2020 2:36:37 AM
01 and 02 injections of freshly isolated marrow obtained from mice respectively on day 21 and 42 post-MNU.
Culture of Hemopoietic Cells in vitro
an observation period of 330-450 days. The inability of these freshly isolated cells to produce leukemia when injected immediately in vivo remains an enigma and host factors are presumably having a major part to play. Only following a period of culture in vitro, do the bone marrow cells appear able to overcome the host resistance and produce leukemia when injected. The events occurring in the culture remain to be elucidated but the culture system allows a step-by-step analysis where hopefully some mechanisms of leukemogenesis can be investigated. References Bradley, T. R. and Metcalf, D.: flust. J. exp. Biol. med. Sci. 44: 287-300 (1966). Dexter, T. M.; Allen, T. D.; Lajtha, L. G.; Schofield, R., and Lord, B. 1.: J. Cell Physiol. 82: 461-474 (1973). Dexter, T. M.; Schofield, R.; Lajtha, L. G., and Moore, M.: Br. J. Cancer 30: 325-331 (1974). Dexter, T. M. and Lajtha, L. G.: Brit. J. Haemat. 28: 525-530 (1974). Moller, G.: J. exp. Med. 114: 415-432 (1961). Pluznik, D. H. and Sachs, L.: Expl Cell Res. 43: 553-563 (1966). Reif, A. E. and Allen, J. M. V.: J. exp. Med. 120: 413-433 (1964). Till, J. E. and McCulloch, E. A.: Radiat. Res. 14: 213-222 (1961). Wigzell, H.: Transplantation 3: 423-431 (1965).
Dr. T. M. Dexter, Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester Μ20 9ΒΧ (England)
Downloaded by: Université de Paris 193.51.85.197 - 2/5/2020 2:36:37 AM
have established that little of any CSF is produced in the bone marrow cultures (unlike in cultures of thymus and bone marrow) (Dexter and Lajtha, 1974). However, addition of CSF to the established monolayer cultures (where blast accumulation has occurred) does not reverse the blastic morphology. It should be pointed out, however, that at this time we have no indication that these blast cells belong either to the myeloid or lymphoid series and their exact nature still remains to be clarified. Culture of 'NU-treated bone marrow follows a similar pattern to that observed in cultures of untreated marrow cells (i.e., stem cell proliferation — although at a somewhat higher level and for longer periods than occurs generally with cultures of normal bone marrow cells — and blast cell accumulation) but injection in vivo of the cultured 'NU-treated cells, produces leukemia. The data presented here indicate, therefore, that the original 'NU-treated bone marrow cells contain a potentially leukemic lesion. However, previous studies (Dexter et al., 1974) have shown that whilst MNU is a powerful leukemogen (producing a high incidence of predominantly T lymphocyte leukemias after a single injection) the freshly isolated bone marrow appeared not to be a major source of potentially leukemic cells. Indeed, when the femoral bone marrow was removed from 'NU-treated mice 1, 21, 42 or 70 days post-MNU treatment (Dexter et al., 1974, and table III) and injected into normal or immunosuppressed mice, no leukemias developed in a total of 105 recipients during
5
Comparative Leukemia Research 1975, Bibi. Haemat., No. 43 Ed. J. Clemmesen and D. S. Yohn, pp. 1-5 (Karger, Basel 1976)
Proliferation of Hemopoietic Stem Cells and Development of Potentially Leukemic Cells in vitro T. M. Dexter and L. G. Laitha1 Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester
We have reported that in some `co-cultures' of thymus and bone marrow cells there was proliferation and differentiation of hemopoietic stem cells for several months (Dexter and Lajtha, 1974). The role of the thymus cells in these cultures is difficult to understand and for this reason we have recently modified the technique and developed a system where proliferation of bone marrow stem cells occurs without the addition of thymus cells. These cultures have been used to study the characteristics of potentially leukemic hemopoietic cells in vitro. Materials and Methods To set up the monolayer system of `attaching' cells 107 bone marrow cells obtained from BDF, mice were inoculated into glass bottles, in 10 ml of Fischer's medium supplemented with 1 Supported by the Medical Research Council and the Cancer Research Campaign.
200 /0 horse serum (Flow Labs.) and cultured as described previously for thymus cells (Dexter and Lajtha, 1974). The cultures were fed at weekly intervals by removal of half of the growth medium and addition of an equal volume of fresh medium at weekly intervals, for 3 weeks. During this 3-week culture period, a population of attaching cells becomes established in the cultures. To initiate the culture `proper', at the end of the third week, half of the growth medium was again removed but now 5 ml of fresh growth medium containing a new inoculum of 107 syngeneic-nucleated bone marrow cells was added to the established bone marrow monolager cultures. At weekly intervals thereafter the cultures were fed as before and the nonattaching cells removed were assayed for morphology, stem cell concentration (CFUg) (Till and McCulloch, 1961) and behavior, granulocyte precursor cells (CFUC ) (Bradley and Metcalf, 1966; Pluznik and Sachs, 1966) and the ability to produce leukemia in various `conditioned' recipients. The collection and assay of the nonattaching population of cells has been described in detail elsewhere (Dexter et al., 1973).
Results The characteristics of a typical culture of normal bone marrow cells are shown in ta-
Downloaded by: Université de Paris 193.51.85.197 - 2/5/2020 2:36:37 AM
Introduction
ble I. Cell proliferation is obviously occurring since after each feeding (which removes half of the nonattaching cells) the number of cells recovers by the following week. (For the duration of the culture the number of adhering cells showed little change and was maintained at a fairly constant level.) Similarly, there is proliferation of CFUg, at least for 12 weeks, since the number increases after successive depopulations (it has previously been established that CFUg — at least in short-term cultures — occur only in suspension and are not detectable in the adhering population of cells) (Dexter et al., 1973). The CFUg present in these cultures form both erythroid and granulocytic spleen colonies and can protect potentially lethal irradiated mice from hemopoietic death. CFU, are also being produced for several months. These cells form CSF-dependent colonies and clusters of granulocytes and macrophages in soft agar. For the first few weeks these cultures contain mainly granulocytes and macrophages. Subsequently, however, an accumulation of blast cells occurs — which is not associated with an increase in cell density in the cultures — and cultures containing more than 704/ο blast cells are regularly seen. With prolonged culturing the percentage of blast cells decreases. It is also worthy of comment that this accumulation of blast cells occurs concomitantly with proliferation of apparently normal stem cells. Preliminary experiments have shown that these cells express neither T nor B lymphocyte markers and their origin and nature awaits elucidation. After various times in vitro (9-34 weeks) the cells were tested for their ability to produce leukemias after injection into syngeneíc recipients. Different routes of inoculation were used and the cells were injected into either normal, immunosuppressed (600 rad X-rays) or T lymphocyte-deficient mice. So far, out of a total of 39 mice injected, no leukemias have developed. How-
2
ever, the number of animals inoculated with the cultured cells is low and the postinoculation observation times somewhat variable (14-50 weeks). Hence, no definite conclusions can yet be drawn regarding the frequency of `spontaneous' leukemic transformation of normal bone marrow cells in vitro. Culture of ‚NU-treated bone marrow. We have also studied the characteristics in culture of bone marrow cells derived from mice treated in vivo with methylnitrosourea (MNU). ΒDFI 9 mice were injected intravenously with a single dose of 50 mg/kg MNU. 3 weeks later some mice were killed, the femoral marrow cells removed and injected immediately in vivo to test the leukemic potential. Additionally 107-nucleated cells were inoculated into several culture bottles, and a monolayer allowed to form over a 3-week culture period. At this time a further group of the same mice were killed, the marrow cells again assayed in vivo for leukemogenicity or added to the established MNU bone marrow monolayer cultures (107-nucleated cells/culture). The growth conditions and method of feeding were as for normal bone marrow cells. The characteristics of a typical culture of 'NU-treated bone marrow are shown in table II. As with cultures of normal bone marrow cells, cell proliferation, production of CFUg and CFUC and accumulation of blast cells occurs. For the first 10 weeks in culture the CFUg are apparently normal, forming erythroid and granulocytic spleen nodules and can reconstitute the hemopoietic systems of potentially lethal irradiated mice. After 10 weeks in culture, however, spleen colonies containing mainly undifferentiated cells are found and the cells lose their protective ability. The CFUe produced throughout the culture period form colonies and clusters containing differentiated granulocytes. Macrophage colonies are only occasionally seen. Injection of these cultured 'NU-treated
Downloaded by: Université de Paris 193.51.85.197 - 2/5/2020 2:36:37 AM
DexterlLajtha
Culture of Hemopoietíc Cells in vitro
3
Table I., Culture of normal bone marrow cells; proliferative capacity, morphology and production of stem cells Weeks in culture
Total suspension cells/culture (x106)
Total CFU./ culture
Total CFUC/ culture
Morphology, % blasts granulocytes
1 2 4 5 7 9 12 13 17 20 34
0.9 0.8 0.6 0.7 0.9 0.9 0.7 1.1 0.6 0.6 12
160+10 140+18 180+40 160+20 110+8 240+18 190+20 ND 0 0 0
1,450+40 1,400+86 ND ND 1,600+90 ND 2,500+170 ND 0 50+4 0
3 2 62 ND 93 72 70 60 ND 0 5
74 59 31 ND 3 4 2 0 ND 0 0
macrophages
23 37 6 ND 3 24 28 40 ND 100 100
ND = No data.
Table ΙΙ. Culture of 'NU-treated bone marrow cells ; proliferation of cells, morphology and stem cell production Weeks in culture
Total suspension cells/culture (x 106)
Total CFUS/ culture
Total CFUe/ culture
Morphology blasts granulocytes
macrophages
1 3 6 7 10 12 15 17 20 30
2.8 1.6 0.9 1.1 0.9 1.0 1.1 1.5 1.0 1.0
600+ 100 350+20 70+10 280+50 240+ 70 480+40 300+20 300+30 0 0
4,300+200 ND ND ND ND ND 2,100+ 180 1,500+200 0 0
4 ND 24 22 74 ND 40 60 47 10
14 ND 16 30 17 ND 59 31 43 90
82 ND 60 48 9 ND 1 8 10 0
cells regularly produced leukemia (table III) — 15/22 animals developed leukemia between 11 and 18 weeks after inoculation of the cells (with longer observation times the final incidence may well be higher). The leukemias probably arise from the cells inoculated (rather than from transformation of host cells by some virus present in the injected cells or in the growth medium) since no leukemias were produced when 18- or 30-week cultured cells were ir-
radiated with 2,000 rad X-rays before injection, or when only the cell-free growth medium was injected. Of the 15 leukemias produced, 13 were characterized by massive and widespread lymph node enlargement along with splenic enlargement. The thymus was not involved. All these leukemias arose in 9 recipients. Five of these leukemias were tested for the presence of surface immunoglobulín (Ig) determinants (characteristic of bone marrow derived or
Downloaded by: Université de Paris 193.51.85.197 - 2/5/2020 2:36:37 AM
ND = No data.
Dexter/Lajtha
4
Table III. Leukemia-producing ability of 'NU-treated bone marrow cells cultured for various time intervals Weeks in culture
01 02 10 15 17 18 22 29
Cells injected (route)
Pretreatment of recipient mice (rad X-rays)
Observation time, weeks
Leukemias
107 (i.v.) 107 (i.v.) 107 (iv.) 107 (i.v.) 107 (i.v.) 5 χ 104 (intrasplenic) 2.5 x 105 (1.ν.) 5 χ 104 (intrasplenic) 6 x 10δ (í.v.) 6 x 105 (1.Υ.) 9 x 105 (i.v.) 9 x 105 (i.v.) 1.6 x 105 (i.v.) 1.6 x 105 (i.v.)
600 800 none 600 none 600
50 50 50 47 47 23
0/15 0/15 0/10 0/15 0/15 0/3
600
18
2/2
17, 18
600 none 600 600 none none 600
13 16 16 11 11 12 13
4/4 2/2 2/2 0/2 0/2 2/2 3/3
11,12,12,13 14, 16 15, 16
Latent periods
7
12, 12 12, 12, 13
B lymphocytes) by an indirect immunofluorescence technique (Moller, 1961). They were tested also for 8-antigen (characteristic of thymus-dependent, T lymphocytes) using a cytotoxícity assay based on release of 51Cr from cells treated with AKR anti O C3H serum (Wigzell, 1965; Reif and Allen, 1964) and guinea pig serum as a source of complement. All five leukemias were negative for O and Ig. They were also peroxidase-negative. Interestingly, the other two leukemias were produced by injection of the cultured cells (derived from mice) into d recipients. These leukemias were characterized by spleen and liver enlargement, and were O+ve (indicating T cells). Discussion We have previously reported that stem cell proliferation occurs in co-cultures of thy-
mus and bone marrow cells (Dexter and Lajtha, 1974) and have now shown that this proliferation also occurs in BM alone cultures (without the addition of thymus cells) provided that these cells are cultured on an adhering population of bone marrow-derived cells. However, the adhering cells are a heterogenous population and it is not known which particular population(s) of cells (nor the `factors' produced) is required for stem cell maintenance. What is apparent, however, is that whilst factor(s) are present facilitating stem cell maintenance and proliferation as well as production of CFUC, the cultures appear to be deficient in those factors allowing sustained granulocyte maturation, hence possibly the blast cell accumulation. This maturation block is readily overcome in agar cultures when CSF is present, suggesting that the decline in granulopoiesis in the liquid cultures may be associated with a deficiency in CSF. Indeed, preliminary experiments
Downloaded by: Université de Paris 193.51.85.197 - 2/5/2020 2:36:37 AM
01 and 02 injections of freshly isolated marrow obtained from mice respectively on day 21 and 42 post-MNU.
Culture of Hemopoietic Cells in vitro
an observation period of 330-450 days. The inability of these freshly isolated cells to produce leukemia when injected immediately in vivo remains an enigma and host factors are presumably having a major part to play. Only following a period of culture in vitro, do the bone marrow cells appear able to overcome the host resistance and produce leukemia when injected. The events occurring in the culture remain to be elucidated but the culture system allows a step-by-step analysis where hopefully some mechanisms of leukemogenesis can be investigated. References Bradley, T. R. and Metcalf, D.: flust. J. exp. Biol. med. Sci. 44: 287-300 (1966). Dexter, T. M.; Allen, T. D.; Lajtha, L. G.; Schofield, R., and Lord, B. 1.: J. Cell Physiol. 82: 461-474 (1973). Dexter, T. M.; Schofield, R.; Lajtha, L. G., and Moore, M.: Br. J. Cancer 30: 325-331 (1974). Dexter, T. M. and Lajtha, L. G.: Brit. J. Haemat. 28: 525-530 (1974). Moller, G.: J. exp. Med. 114: 415-432 (1961). Pluznik, D. H. and Sachs, L.: Expl Cell Res. 43: 553-563 (1966). Reif, A. E. and Allen, J. M. V.: J. exp. Med. 120: 413-433 (1964). Till, J. E. and McCulloch, E. A.: Radiat. Res. 14: 213-222 (1961). Wigzell, H.: Transplantation 3: 423-431 (1965).
Dr. T. M. Dexter, Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester Μ20 9ΒΧ (England)
Downloaded by: Université de Paris 193.51.85.197 - 2/5/2020 2:36:37 AM
have established that little of any CSF is produced in the bone marrow cultures (unlike in cultures of thymus and bone marrow) (Dexter and Lajtha, 1974). However, addition of CSF to the established monolayer cultures (where blast accumulation has occurred) does not reverse the blastic morphology. It should be pointed out, however, that at this time we have no indication that these blast cells belong either to the myeloid or lymphoid series and their exact nature still remains to be clarified. Culture of 'NU-treated bone marrow follows a similar pattern to that observed in cultures of untreated marrow cells (i.e., stem cell proliferation — although at a somewhat higher level and for longer periods than occurs generally with cultures of normal bone marrow cells — and blast cell accumulation) but injection in vivo of the cultured 'NU-treated cells, produces leukemia. The data presented here indicate, therefore, that the original 'NU-treated bone marrow cells contain a potentially leukemic lesion. However, previous studies (Dexter et al., 1974) have shown that whilst MNU is a powerful leukemogen (producing a high incidence of predominantly T lymphocyte leukemias after a single injection) the freshly isolated bone marrow appeared not to be a major source of potentially leukemic cells. Indeed, when the femoral bone marrow was removed from 'NU-treated mice 1, 21, 42 or 70 days post-MNU treatment (Dexter et al., 1974, and table III) and injected into normal or immunosuppressed mice, no leukemias developed in a total of 105 recipients during
5
