Leukemia Research Vol. 3. No. 3, p p . 109-116. Pergamon Press, Ltd. 1979. Printed in Great Britain


REPORT ON THE 1978 ANNUAL MEETING OF THE INTERNATIONAL SOCIETY OF EXPERIMENTAL HEMATOLOGY HAL E. BROXMEYER* Sloan Kettering Institute for Cancer Research, Laboratories of Developmental Hematopoiesis New York, New York 10021, U.S.A. THIS report summarizes some of the studies relating to hematopoietic stem cells, progenitor cells and granulopoiesis which were presented in Chicago, Illinois, U.S.A., during August 1978. Abstracts of this meeting can be found in a supplemental issue to Experimental Hematology (Vol. 6, Suppl. No. 3, 1978). The page number in the supplemental issue which corresponds to the study summarized will be placed in parentheses following the names of the investigators (e.g. Experimental Hematology page no., E . H . p . x). This report will be presented in seven separate but not mutually exclusive categories. I. I N V I V O SPLEEN C O L O N Y F O R M I N G CELL ( C F U - s ) - - P L U R I P O T E N T I A L

STEM CELL The #7 vivo assay for pluripotential stem cells as performed by Till and McCulloch [i] has not yet been adapted for human cells. However, Johnson and Metcalf [2] recently reported on an in vitro assay for culturing mixed colonies containing neutrophils, macrophages, eosinophils, megakaryocytes and erythrocytes. These mixed colonies were generated by single mouse fetal liver cells and, to a lesser extent, by mouse adult bone marrow cells after stimulation by medium conditioned by spleen cells in the presence of pokeweed mitogen. Interestingly, this conditioned medium contained no detectable erythropoietin. Adaptation o f this in vivo assay for human cells would provide an extremely useful assay for the study of human disease. Further studies presented at this meeting by Johnson and Metcalf ( E . H . p . 33) demonstrated that no examples of multiple hematopoietic differentiation were noted in which erythropoiesis was not observed when 7 day mixed colonies from 12 day CBA fetal liver cells were checked. In addition, in vivo spleen colonies produced by fetal liver cells from pooled mixed in vitro colonies had a higher erythroid to granulocytic ratio than obtained with normal fetal liver cells that had not first been cloned in agar culture medium. No lymphoid differentiation or B-lymphocyte colony forming cells were detected in the mixed colonies. It was suggested that the 12 day fetal cells which generated the mixed colonies in vitro belonged to a stem cell compartment which produces only nonlymphoid cells and which are relatively more differentiated than hematopoietic stem cells in terms of self-renewal capacity. * Hal E. Broxmeyer is a Scholar of the Leukemia Society of America. Abbreviations: CFU-c = colony forming cell in semi-solid medium; believed to be the granulocyte and monocyte-macrophage progenitor or committed stem cell. CSA, CSF = colony stimulating activity (factor); the molecules which trigger the proliferation and differentiation of CFU-c and which are continuously necessary for colony formation. BFU-e = burst forming cell; believed to be an erythroid progenitor cell. CFU-e = erythroid colony forming cell ; believed to be an erythroid precursor cell more mature than BFU-e. CFU-s = in vivo spleen colony forming cell: believed to be the hematopoietic pluripotential stem cell.




Attempts were made to isolate and characterize the CFU-s population using a light activated cell sorter, electrophoretic mobility and surface-receptors. Van den Engh and Visser (E.H.p. 71) observed that the CFU-s compartment has a uniform light scatter distribution indicating a single morphological entity. The cell diameter was estimated at 7.5/~m and it was suggested that the cell membrane and nucleus had a smoothly rounded shape. The advantage of this assay is that cells are not fixed and can be tested for their differentiation capacity. Bol, Doekes and Van den Engh (E.H.p. 31) found that CFU-s had an electrophoretic mobility higher than most of the nucleated cells and had a large amount of removable sialic acid groups at their cell membrane. CFU-s enrichment achieved by electrophoretic separation of neuraminidase treated cells (8 fold compared to unseparated cells) was higher in similar separation of untreated cells (4 fold). Price, Krogsrud, Stewart and Till ( E . H . p . 35) attempted to further isolate the heterogeneous group of CFU-s still apparent after physical cell enrichment procedures. Several surface antigens noted on mouse CFU-s and directly detected by enrichment with fluorescent cell sorting were a sperm-associated antigen cross-reactive with anti-brain sera, the 129 anti-F9 antigenic determinant, surface immunoglobulin and Thy 1 ; surface Ia was not seen. Cytoplasmic fluorescence polarization changes by 4 methyl histamine suggested CFU-s receptors for this compound. Efforts to understand the regulation of CFU-s proliferation centered on the use of agents which trigger CFU-s into cycle and on cells which seem to regulate CFU-s proliferation. Byron (E.H.p. 32) further elaborated on his former work demonstrating that testosterone triggers CFU-s from Go into S-phase [31 by investigating portions of the steroid molecule other than the C5 position. Batrachotoxin, which acts at the level of the cell membrane to increase the flow of sodium ions, was a potent inducer of DNA synthesis in CFU-s at 10-8 M concentrations. The cell cycle effects of testosterone and batrachotoxin were both antagonized by tetrodotoxin (10-7-10-6M and by procaine (10.6 M), agents which antagonize the action of batrachotoxin upon sodium channels. It was suggested that CFU-s cycle transition from Go into S-phase might be related to an alteration of transmembrane potentials. Porcellini, Grilli, Manna, Rizzoli and Shadduck ( E . H . p . 79) showed that various androgens had slightly different influences on granulopoiesis of CF1 female bone marrow cells placed into diffusion chambers implanted into irradiated mice. Testosterone propionate appeared to act at the level of the CFU-s, while calusterone, a weakly androgenic steroid, enhanced both stem cell proliferation and granulocyte differentiation. Wiktor-Jedrzejczak, Ahmed, Sharkis and Sell (E.H.p. 42) further characterized the surface phenotype and other properties of the anti-theta serum sensitive regulatory cell which appears to regulate the differentiation and proliferation of CFU-s and which is absent in W/W" mice but present in normal + / + littermate mice. W/W v anemic mice have an intrinsic stem cell defect which is reflected as a hypoplastic bone marrow, a severe macrocytic anemia and a reduced number of circulating granulocytes. The theta-sensitive regulatory cell had a surface phenotype that was Thy l% Ly 1+, Ly 2-, Ly 3-, Ia-, anti mouse thymic lymphocyte antigen- and was relatively sensitive to cortisone and relatively resistant to irradiation in vitro. Sharkis, Sensenbrenner, Ahmed, Stuart, Jedrzejczak and Sell ( E . H . p . 18) found that high specific activity 3HTdR treatment of bone marrow depleted of theta sensitive regulatory cells reduced CFU-s numbers by 75~, but + / + thymocytes protected CFU-s from the radiation effects by a mechanism which seemed to involve direct cell contact during exposure to the isotope. Visser, Van den Engh and Platenburg (E.H.p. 87) reported on some technical considerations involving the lodging of circulating CFU-s to the spleen. Several factors emerged. The seeding efficiency (f factor) of circulating CFU-s to the spleen was greater into colony-rich spleens and the contribution to the numbers of CFU-s by migration into colony-rich spleens seemed to be larger when the graft size was larger and the time after

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irradiation and reconstitution was longer. It was not established whether the CFU-s homed into the already established spleen colonies or elsewhere in the spleens. Blackburn and PaR (E.H.p. 90) studied the influence of murine fibroblast conditioned medium on the survival of CFU-s in vitro. Bone marrow "fibroblast" (adherent cells grown from marrow cells for 2 weeks or more) conditioned medium increased marrow CFU-s survival but bone, spleen and subcutaneous tissue did not. Embryo bone conditioned medium was better than adult bone marrow cell activity. The adult marrow cell conditioned medium increased spleen CFU-s survival better than marrow CFU-s survival and marrow conditioned medium increased cycling of CFU-s but increases in survival were not dependent on the increased cycling status. W/W Vmice have a defect in CFU-s and SI/SId mice have a defective microenvironment, but interestingly, conditioned medium from bone marrow fibroblasts of SI/S1d, W/W V,SI+/SI+ and W÷/W + mice all produced the active agent(s). II. CHARACTERIZATION OF GRANULOCYTE, EOSINOPHIL AND MACROPHAGE PROGENITOR CELLS Immunological attempts to separate and characterize granulocyte-macrophage progenitor cells (CFU-c) are relatively new. It has been established that using specific anti-sera in a complement cytotoxicity test that human Ia-like antigens characteristic of B lymphocytes are found on human CFU-c (Winchester et aL [4]; Cline and Billing [5]) and the human erythroid progenitor cells: BFU-e and CFU-e (Winchester et al. [6]). Studies such as these, presented at this meeting, have additionally suggested that human CFU-c express i and HLA antigenic determinants. O'Hara, Shumak and Price (E.H.p. 39) demonstrated that antisera to the blood group i antigen plus complement greatly reduced CFU-e and CFU-c numbers but that there appeared to be cycle selective expression of i antigenic determinants on CFU-c during S-phase. This was based on data demonstrating that the same kill of CFU-c was noted over a large dilution range of anti-i antisera and that no kill of anti-i plus complement was noted if the bone marrow cells were first treated with hydroxyurea, an S-phase inhibitory agent. Additionally, anti-i + complement also worked against marrow cells which, in suspension culture, may generate CFU-c. However, this population of "pre-CFU-c" is not usually in rapid cycle and cells from patients with neutropenia who have a larger proportion of these "pre-CFU = c" in S-phase were used to demonstrate that they may also contain i antigenic determinants. Fitchen and Cline (E.H.p. 47) used monospecific antisera against HLA-2 and -3 plus complement to show that they inhibit myeloid colony formation by marrow cells from individuals of the appropriate HLA type. Subpopulations of human CFU-c have been separated by velocity sedimentation (Johnson et al. [7]; Jacobsen et al. [8]) and have their own S-phase compartments as determined by high specific activity 3HTdR experiments. Dresch and Faille (E.H.p. 80) confirmed this using hydroxyurea and also noted that in human blood the majority of CFU-c were out of cycle and came from the fractions sedimenting at 5-7 mm/h, corresponding to the more slowly dividing cells in bone marrow and between the two peaks of high kill. In myelomonocytic leukemia nearly all the CFU-c were found in the 3.5-6 mm/h range, but developed by 7 days, contrary to normal marrow in which the more slowly sedimenting population forms colonies by 14 days. This may not be the case for CFU-c from all leukemic cell donors, since CFU-c from two patients with chronic myelogenous leukemia had the same sedimentation profile as normal cells (Broxmeyer et al. [9]). Verma, Spitzer, Zander, Dicke, Smith and McCredie (E.H.p. 81) suggested that numbers of circulating human CFU-c demonstrated diurnal variations but this concept awaits proof that it is not the endogenous circulating colony stimulatory activity (CSA) producing cells which are fluctuating during the day. In addition, it was determined that the majority of circulating human CFU-c were eosinophilic progenitor cells suggesting preferential



release of this population into the peripheral blood. Neutrophils and eosinophils are rarely found in the same colony derived from human marrow and blood cells, suggesting two different progenitor cells (reviewed in Broxmeyer and Moore [10]). However, Konwalinka, Glaser, Schmalzl, Michlmayr and Braunsteiner ( E . H . p . 48) did observe some mixed colonies and cells from a patient with eosinophilia gave predominantly mixed colonies when autologous serum was used suggesting that some CFU-c can give rise to neutrophil as well as eosinophil granulocytes. Moore has demonstrated the usefulness of the agar colony assay for CFU-c for bone marrow transplantation [11]. Pino and Santos (E.H.p. 42) gave evidence that estimation of CFU-c/kg given to bone marrow transplant patients offered a guide to establish the time of hematopoietic recovery. Thus, when 3415 CFU-c/kg were given to a patient, the total WBC was over 500/ram a on day 8 after the transplant. A second patient receiving 1587 CFU-c/kg demonstrated a total WBC over 500/mm 3 on day 11. When 400-500 CFU-c/kg were given the total WBC did not reach 500/mm 3 until at least day 17 and lower numbers of CFU-c were associated'with delayed engraftment and in one case with failure to engraft. It should be mentioned that bone marrow and blood cells contain many interacting cell populations and numbers of colonies noted are not always dependent on the number of CFU-c but are also dependent on the numbers of endogenous cells which elaborate stimulators and inhibitors of colony formation. Thus, cell separation procedures which enrich for CFU-c and deplete other cell types, and a potent source of CSA are needed to accurately determine actual numbers of CFU-c given to transplant patients. In addition, counting colonies on only one day underestimates the total CFU-c population size [8]. Characterization of murine progenitor cells was the topic of several investigations. Byrne, Heit and Kubanek (E.H.p. 80) suggested that there was a hierarchy of CFU-c which changed with time due to elaboration of different types of CSA. CFU-c were monitored after endotoxin administration to mice, separation of cells on the basis of buoyant density by isopycnic centrifugation in continuous albumin density gradients and stimulation by both mouse lung-conditioned medium and human urine extract. Low density CFU-c detected with mouse lung-conditioned medium were hypothesized to be the immediate progeny of CFU-s and high density CFU-c, detected by human urine CSA, were more differentiated and the progeny of low density CFU-c. Galla, Gallagher and Trentin ( E . H . p . 87) used various culture techniques and selectively absorbed rabbit anti-mouse brain serum in attempts to characterize the stem cells responsible for hemopoietic colony formation on intraperitoneal membranes; as originally described by Seki. The results suggested that they might be a mixture of CFU-s and CFU-c. Sawada, Kuznetsky and Adler (E.H.p. 88) felt that the majority of cells forming colonies on cellulose acetate membranes were antigenically distinct from CFU-s. McCarthy and MacVittie (E.H.p. 48) used velocity sedimentation to characterize the macrophage colony forming cells they originally described, and which appeared to be different than the macrophage colony forming cell from peritoneal exudate described by Lin and Stewart, in relation to the materials which stimulate the cells to form colonies. Their CFU-c population separated into two peaks at 4.1 and 6.5 mm/h. Both populations were found in marrow but only the 4.l mm/h peak was found in spleen and peripheral blood. The peak sedimentation value of the peritoneal exudate colony forming cells were around 6.9 mm/h. III. GRANULOCYTE-MACROPHAGE COLONY STIMULATORY FACTOR OR ACTIVITY (CSF, CSA) Granulocyte-macrophage colony stimulatory activity is still only a candidate granulopoietin as all evidence suggesting in vivo significance is at best only indirect (Broxmeyer and

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Moore [10]). Shadduck, Waheed, Porcellini and Rizzoli (E.H.p. 69) used two techniques to demonstrate the half life of a purified L-cell colony stimulating factor (CSF) in CF1, mice. l:5I-labeled CSF and a radioimmunoassay for CSF suggested a biphasic clearance rate with first disappearance in 30--42 min and second disappearance in 100--140 rain. Nephrectomy did not alter the initial decline in plasma activity but markedly prolonged the second phase. It was suggested that frequent and repeated administration of CSF may be necessary to demonstrate in vivo activity of CSF. Van Zant and Goldwasser (E.H.p. 33) extended their studies which suggest that the earliest granulocyte-macrophage progenitors and the earliest erythroid progenitors may share a common subpopulation responsive to both CSF and erythropoietin. This model is based on what appears to be competition for differentiation of murine progenitor cells based on the levels of CSF and erythropoietin added to the culture systems. In addition, the rate of differentiation into one or the other pathway may also be influenced by the presence and amounts of other inducers. For example, prostaglandin El, which suppresses CSF mediated proliferation of granulocyte-macrophage progenitors in vitro, reduced the competitive effect of CSF on erythropoietin stimulated 8 day hemoglobin synthesis. Effects on accessory cells are being studied to rule out actions on other cells which might negate competitive actions on a single progenitor cell. Hertogs and Pluznik (E.H.p. 49) reported on the relationship between the CSF enhancing and serum replacing activities of rat hemolysate. These activities by themselves will not stimulate CFU-c which are depleted of endogenous CSA producing cells but modify the action of CSF. Thus, rat hemolysate incubated for 30 rain at 70 °C lost its serum replacing activity but retained its CSF enhancing activity and incubation of rat hemolysate with 2.5 mg/ml trypsin for 30 min destroyed the replacing activity but did not influence the enhancing activity. There was no correlation between the amount of hemoglobin and serum replacing activity of hemolysates which shows characteristics of being a protein. Wagemaker, Brouwer, Bol and Visser (E.H.p. 31) characterized three different activities for mouse bone marrow cells obtained from human peripheral blood leukocytes. These activities induced DNA synthesis in CFU-s, induced CSF responsiveness in a low density subpopulation of mouse CFU-c and were required for the earliest erythroid progenitor cell (BFU-e) to express its potential to differentiate and proliferate. The three activities were not separated by gel filtration, ion exchange chromatography and affinity chromatography. The major band of the three activities after gel filtration had an apparent tool. wt of 17,000 but electrophoresis in 15 ~ poly-acryl-amide gel separated the 3 activities, suggesting biochemically related but distinct regulatory molecules. The final products were active at concentrations less than 5 ng. Human active CSA derives from human monocytes-macrophages, activated lymphocytes and endothelial cells. Quesenbery, Gimbrone and McDonald (E.H.p. 4) demonstrated that both granulocyte and bacterial products increased CSA production from human endothelial cells. These cells incubated for 24 h with 1 ~o granulocyte lysate (

Report on the 1978 annual meeting of the International Society of Experimental Hematology.

Leukemia Research Vol. 3. No. 3, p p . 109-116. Pergamon Press, Ltd. 1979. Printed in Great Britain CONGRESS ANALYTICAL R E P O R T REPORT ON THE 19...
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