Proceeding8 of the National Academy of Sciences Vol. 67, No. 3, pp. 1542-1549, November 1970

In Vitro Induction of Differentiation in Hematopoietic Cells from Leukemic and Non-Leukemic Patients Michael Paran, Leo Sachs, Yigal Barak*, and Peretz Resnitzky* DEPARTMENT OF GENETICS, WEIZMANN INSTITUTE OF SCIENCE, REHOVOT, ISRAEL, AND KAPLAN HOSPITAL*, REHOVOT, ISRAEL Communicated by Albert B. Sabin, August 17, 1970 Abstract. Human spleen-conditioned medium can induce the formation in vitro of large granulocyte colonies from normal human cells. The granulocyte colonies contained cells in various stages of differentiation, from myeloblasts to mature neutrophile . Human spleen-conditioned medium also induced colony formation with rodent bone-marrow cells, whereas rodent spleen-conditioned medium induced colony formation with rodent bone marrow but not with human cells. This in vitro system has been used to determine the potentialities for cell differentiation in bone-marrow and peripheral blood cells from patients with a block in granulocyte differentiation in vivo. The cloning efficiency, colony size, and number of mature granulocytes in bone-marrow colonies from patients with congenital neutropenia, whose bone marrow contained only 1% mature granulo- cytes, were not less than in people whose bone marrow had the normal level of about 40% mature granulocytes. The cloning efficiency of peripheral blood cells from patients with acute myeloid was 350 times higher, with 10 times larger colonies, than the cloning efficiency of peripheral blood cells from normal people. The cytochemical properties and number of mature granulocytes in colonies from the leukemic patients were the same as in colonies from non- leukemic people. The results indicate that a block in cell differentiation in vivo, in these cases with neutropenia and acute , was overcome in vitro, in the presence of an inducer in the conditioned medium. In patients with chronic myeloid leukemia, colony formation was induced only in some of the cases. This indicates that there are blast cells with different potentialities for the develop- ment of colonies in different patients.

The development of. an in vitro system to study the control mechanism for growth and differentiation of normal hematopoietic cells has previously been reported'-. The results obtained with this system, using hematopoietic cells from rodents, have indicated that a substance released into the tissue culture medium by various types of cells can induce the formation in vitro of colonies of normal macrophages and granulocytes. It has been shown that the macrophageI and granulocyte4 colonies can be derived from single cells, and that the granulo- 1542 Downloaded by guest on September 28, 2021 VOL.67,1970 GRANULOCYTE DIFFERENTIATION 1543

cyte colonies contain cells in various stages of differentiation from myeloblasts to mature neutrophile granulocytes'. The present experiments were undertaken (1) to determine the conditions re- quired for the development of hematopoietic cell colonies from normal human bone marrow, and (2) to use this in vitro system to determine the potentialities for growth and differentiation of bone marrow and peripheral blood cells from leu- kemic and non-leukemic patients. Materials and Methods. Cloning of hematopoietic cells: The cells used for cloning were taken from the bone marrow of 2-month-old male Swiss mice and Wistar rats as described' or from human bone marrow and peripheral blood. Human bone marrow was obtained by aspiration from the iliac crest, sternum, or tibia. The cells were transferred to a glass tube containing a heparin solution [Evans Medical Ltd., Heparin (Mucous) pyrogen free, without preservative] at 5000 units/ml, and allowed to sediment for 1 hr. After centrifugation at 150 X g for 5 min, the buffy coat was removed, the cells were resuspended in serum-free EM (Eagle's medium with a 4-fold concentration of amino acids and vita- mins) and counted by diluting 1:20 in Turck diluting fluid (methylene blue in 3% acetic acid). Peripheral blood cells were handled in the same way. 2 X 105 nucleated cells were usually seeded for cloning per 50-mm plate. Mature granulocytes were not counted as they were presumably no longer able to multiply. Cell suspensions were made for cloning by dilution with serum-free EM. The cells were cloned in soft agar (0.33%) on a harder agar base (0.5%), as previously described', in EM (usually with 20% calf serum). Colonies were counted microscopically with an inverted microscope containing a X 5 objective and X 10 eye piece. Unless otherwise stated, colonies from human cells were counted 10 days after seeding. Each calculation of the number of cells per colony was based on a count of at least 30 pooled colonies. For the differential cell count, a sample from the cell suspension used for seeding was centrifuged, the pellet was re- suspended in 0.1 ml of EM; the cells were smeared on a slide and stained with May- Grunwald-Giemsa solution. Cell staining: Colonies were picked from the agar with a capillary tube. The cells were used for staining with May-Grunwald-Giemsa and for the cytochemical tests after smearing on slides and air drying. For orcein staining, colonies were picked by the same method and placed on slides without smearing. A few drops of aceto-orcein (0.4% orcein in 60% acetic acid) were added and the slides were covered with a Petri dish. The colonies were covered 1 hr later with a coverglass and examined microscopically. Conditioned medium containing the inducer: Human spleen-conditioned medium was prepared from mass cultures of human spleen cells after splenectomy from patients having thalassemia major, hereditary spherocytosis, or traumatic rupture of the spleen. Spleen cells were seeded at 108 nucleated cells per 100-mm Petri dish (Falcon Co.) in EM, usually with 10% calf serum. The medium harvested at 4 days after seeding ("0-4 day medium") was centrifuged and stored at -20'C. The cells were reseeded to new Petri dishes at 108 cells per 100-mm plate, and the medium ("4-6 day medium") was harvested 2 days later and stored at -20'C. Conditioned medium was always added to the lower agar layer in the assay for colony formation. Results. Development of granulocyte colonies from normal bone marrow cells: Results obtained with human spleen-conditioned medium have shown that this medium contains an inducer of colonies from normal human bone- marrow cells, with about the same cloning efficiency as that obtained with mouse and rat bone-marrow cells with spleen-conditioned medium from these rodents. No colonies were obtained without the inducer. Although horse serum has given high cloning efficiencies with rodent cells2 3 a high cloning efficiency with human hematopoietic cells was found with calf or human serum. Conditioned medium Downloaded by guest on September 28, 2021 1544 MEDICAL SCIENCES: PARAN ET AL. PROC. N. A. S.

from the four human spleens tested had similar activity, and 4-6 day spleen-con- ditioned medium was generally more active than 0-4 day medium. Human spleen-conditioned medium induced colony formation with rat and mouse bone- marrow cells, but no colonies were obtained with human bone-marrow cells by spleen-conditioned medium from these rodents. In contrast to the development of colonies of macrophage or granulocytes by the hematopoietic cells of rodents with inducer from either rodents or humans, the human bone-marrow cells pro- duced only granulocyte colonies which, however, survived longer in culture than the rodent granulocyte colonies. The human colonies (Fig. 1) contained cells in various stages of differentiation, some from myeloblasts to mature granulocytes, and reached their maximum size about 2 weeks after seeding. There was a straight-line relationship between the number of colonies and the number of cells seeded. In all the following experiments, a 25% concentration of human spleen- conditioned medium was used with calf serum, and colonies were counted only if they contained more than 50 cells. Cloning efficiency and colony size with bone-marrow cells from non-leukemic patients: Bone marrow was collected from 15 non-leukemic patients. The re- sults indicate (Table 1) that granulocyte colonies were obtained with human spleen-conditioned medium in all cases, with an average cloning efficiency of about one colony per 103 cells seeded, and an average colony size of about 800 cells 14 days after seeding. In two cases with congenital neutropenia, where the differential cell count with bone marrow showed only 1% mature granulocytes, the cloning efficiency and number of cells per colony was not less than in other patients with up to 40% mature granulocytes. 100 colonies examined from the neutropenic patients contained a similar number of mature granulocytes as colonies for normal bone marrow. Addition of 20% of one of these patients' serum did not inhibit colony formation or differentiation with cells from the patient's or normal marrow. Cloning efficiency and colony size with bone-marrow and peripheral blood cells from leukemic patients: The eight cases of leukemia examined, none of which (except no. 5) was undergoing treatment at the time the cells were collected, included two cases with acute neutrophilic myeloid leukemia (Table 2). Bone marrow was available from one of these patients: the cloning efficiency, about one colony per 30 cells seeded, was about 30 times higher than the average cloning efficiency from non-leukemic patients, and the colonies were also larger. The peripheral blood cells from both patients gave 1560 and 1989 colonies per 2 X 105 cells seeded with 1250 and 750 cells per colony, compared to 1-9 colonies with 50- 200 (average, 85) cells per colony with peripheral blood from normal people (Table 2). The study of cell smears stained with orcein and May-Grunwald- Giemsa, and cytochemical tests for peroxidase6, alkaline phosphatase7, the periodic acid-Schiff reaction', and esterase activity (using naphthol AS-D chloroacetate as substrate)9, showed no obvious differences between colonies from leukemic and non-leukemic patients. 100 colonies examined per patient contained a similar number of mature granulocytes as colonies from non-leukemic cases, and the cells were positive in these tests. A third case of was classified as eosinophilic. This patient had about a 40-fold lower cloning efficiency with bone-marrow and Downloaded by guest on September 28, 2021 VOL. 67, 1970 GRANULOCYTE DIFFERENTIATION 1545

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1). One of the cases that gave colonies, and the two without colonies, were in a state of blast crisis, so that there appear to be blast cells with different poten- tialities in different patients. Addition of 20% serum from leukemic patients nos. 1 and 5 did not inhibit colony formation or differentiation with the patient's or normal hematopoietic cells. The very low cloning efficiency, four granulocyte colonies per 2 X 105 cells seeded, with bone-marrow cells from a patient with acute indicates that most of the blasts from this patient did not form colonies. No colonies were found with any of the leukemic or non-leukemic patients in the absence of the spleen-conditioned medium. Discussion. The present results have shown that an inducer in human spleen- conditioned medium can cause large granulocyte colonies to develop in vitro from normal human hematopoietic cells. The formation of these colonies is a sensitive assay for immature cells able to multiply and differentiate, which may be of value in the early detection of hematological disorders. The observations that human spleen-conditioned medium induced the formation of granulocyte colonies from human and rodent cells, whereas rodent spleen-conditioned medium induced colony formation with rodent but not with human cells, indicates that the inducer has some species specificity, apparently in only one direction. This is also in- dicated by the observations that although mouse bone-marrow colonies have been obtained with some human sera and urine10, human normal bone marrow cloned with mouse kidney cells only gave colonies with 10-50 cells11. The present in vitro system has proved useful in determining the potentialities for growth and differentiation in bone-marrow and peripheral blood cells in pa- tients with a block in granulocyte differentiation in vivo. The cloning efficiency and number of mature granulocytes in bone-marrow colonies from patients with congenital neutropenia were no less than with the normal bone marrow. This indicates that the block in cell differentiation in these patients did not seem to be due to an inherent inability of the cells to differentiate to mature granulocytes. A similar conclusion can be drawn from the results with acute myeloid leukemia, where the peripheral blood cells showed a cloning efficiency 350-fold higher than that found with normal people, but where the degree of maturation in these colonies was the same as in the colonies from normal persons. A block in cell differentiation in vivo thus appeared to be overcome in vitro in the cells from these patients in the presence of the inducer. The results suggest that the difference between normal people and the patients with acute myeloid leukemia is an in- sufficient supply of inducer in the leukemic patients resulting in the accumula- tion of blast cells. There may be a different degree of insufficiency of inducer in some patients with chronic leukemia. It will be of interest to chemically characterize the inducer for colony formation from non-leukemic and leukemic patients, compared with the conditioned medium from rodent cells3"2. It will also be of interest to determine the conditions required to induce differentiation in such patients in vivo, and the therapeutic value of treatment with inducer. The results obtained with the chronic myeloid leukemias, where there was an induction of colony formation in only some of the cases, indicates that blast cells in different leukemias can show a difference in their ability to respond to the inducer. This difference in the potentialities of blast cells from leukemias is Downloaded by guest on September 28, 2021 VOL. 67, 1970 GRANULOCYTE DIFFERENTIATION 1549

supported by observations with mouse leukemia, which indicate that some cells cloned in vitro differentiated to mature granulocytes in one cell line' and only to immature granulocytes in another cell line14. Although the human spleen-conditioned medium induced the formation of colonies with neutrophile granulocytes, no colonies were obtained with eosino- philic granulocytes, or with cells from the monocytic leukemia. Further studies should be able to determine to what extent this was due to a lack of the appro- priate colony-forming cells in these patients, or to a requirement for a different type of inducer. 1 Pluznik, D. H., and L. Sachs, J. Cell. Comp. Physiol., 66, 319 (1965); Pluznik, D. H., and L. Sachs, Exp. Cell Res., 43, 553 (1966). 2Ichikawa, Y., D. H. Pluznik, and L. Sachs, Proc. Nat. Acad. Sci. USA, 56, 488 (1966); Paran, M., Y. Ichikawa, and L. Sachs, Proc. Nat. Acad. Sci. USA, 62, 81 (1969). ' Sachs, L., Proc. 8th Canad. Cancer Conference, (Canada: Pergamon Press, 1968), p. 146. 4Paran, M., and L. Sachs, J. Cell. Physiol., 73, 91 (1969). 5 Paran, M., and L. Sachs, J. Cell. Physiol., 72, 247 (1968). 6 Cartwright, G. E., Diagnostic Laboratory , 2nd. Ed. (New York: Grune and Stratton, 1958), p. 87. 7Kaplow, L. S., Amer. J. Clin. Pathol., 39, 439 (1963). 8 Hotchkiss, R. D., Arch. Biochem., 16, 131 (1948). 9 Moloney, W. C., K. McPherson, and L. Fliegelman, J. Histochem. Cytochemn., 8, 200 (1960). 10 Foster, R., D. Metcalf, W. A. Robinson, and T. R. Bradley, Brit. J. Haematol., 15, 147 (1968); Robinson, W. A., E. R. Stanely, and D. Metcalf, Blood, 33, 396 (1969). 11 Senn, J. S., E. A. McCulloch, and J. E. Till, Lancet, ii, 597 (1967). 12 Ichikawa, Y., M. Paran, and L. Sachs, J. Cell. Physiol., 73, 43 (1969). '3 Ichikawa, Y., J. Cell. Physiol., 74, 223 (1969). 14Metcalf, D., M. A. S. Moore, and N. L. Warner, J. Nat. Cancer Inst., 43, 983 (1969). Downloaded by guest on September 28, 2021