(erythroid/neutrophil/basophil) hematopoietic progenitor

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1 Proc. Natl Acad. Sci. USA Vol. 80, pp , May 1983 Cell Biology Demonstration of permanent factor-dependent multipotential (erythroid/neutrophil/basophil) hematopoietic progenitor cell lines (self-renewal/stem cells/differentiation/bone marrow/growth factors) JOEL S. GREENBERGER*, MARY ANN SAKAKEENY*, R. KEITH HUMPHRIESt, CONNIE J. EAVESt, AND ROBERT J. ECKNER *Joint Center for Radiation Therapy, Department of Radiation Therapy, Sidney Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115; tclinical Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20205; tbritish Columbia Cancer Research Institute, Vancouver, British Columbia, V5Z 1L3, Canada; and Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts Communicated by Henry S. Kaplan, January 20, 1983 ABSTRACT Multipotential hematopoietic progenitor cell lines have been established from nonadherent cell populations removed from continuous mouse bone marrow cultures. Clonal sublines of lines B6SUtA or B6JUt derived from single cells formed mixed colonies containing erythroid cells, neutrophil-granulocytes, and basophil/mast cells in semisolid medium containing erythropoietin and conditioned medium from pokeweed mitogenstimulated spleen cells. Each of several subclones of cell line Ro cl formed colonies containing eosinophils, neutrophil-granulocytes, and basophil/mast cells in semisolid medium. Multipotentiality was maintained in vitro for over 21/2 years. In contrast, cell line 32D formed basophil/mast cell colonies with no detectable differentiation to other pathways. Multipotential cell lines did not produce detectable spleen colonies (CFUs) in vivo, nor did intravenous inoculation of up to 5 x 107 cells protect lethally irradiated mice from bone marrow failure. Newborn and adult mice inoculated with 5 X 107 cells showed no detectable leukemia or solid tumors after one year. Both multipotential and committed basophil/mast cell lines demonstrated absolute dependence upon a source of a growth factor(s) found in medium conditioned by WEHI- 3 cells. These cell lines should be of value in studies of the regulation of hematopoietic stem cell differentiation in vitro. The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact Hematopoietic stem cells, as defined by the spleen colonyforming unit (CFUs) assay (1), give rise to a variety of differentiated cell types. These may include B and T lymphocytes as well as erythroid cells, platelets, neutrophilic granulocytes, eosinophils, basophil/mast cells, and macrophages. Data supporting the common origin of lymphoid and myeloid lineages come primarily from two types of in vivo studies. In the first, radiation was used to induce unique chromosomal markers in donor marrow cells. Such cells were then transplanted into suitable recipients and subsequently found to have repopulated lymphoid as well as myeloid tissues with cells bearing the same marked karyotype (2, 3). The second line of evidence derived from the demonstration of the clonal nature of disease states that involve cells of several hematopoietic lineages, including lymphocytes as well as myeloid cells (4, 5). The concept of primitive but committed hematopoietic progenitor cells restricted to specific differentiation pathways is based on studies using in vitro colony assays. These have shown that colonies commonly contain cells of a single lineage even though different types of colonies may be present in the same cultures (6-8) and that these different types of colonies arise from progenitors with different properties (9, 10). In 1977, cultures containing mixed hematopoietic colonies of single-cell origin were reported (11). Such colonies contain cells of the erythroid lineage admixed with megakaryocytes, macrophages, and in some instances granulocytes, including eosinophils as well as neutrophils (11-13). We now report the characterization of permanent lines of factor-dependent and nonmalignant (14, 15) hematopoietic cells that differentiate along erythroid, neutrophil-granulocyte, and basophil/mast cell pathways after appropriate stimulation in vitro. MATERIALS AND METHODS Bone Marrow Cultures. Continuous mouse bone marrow cultures were established according to published procedures, using the contents of a femur and tibia inoculated into 25-cm2 plastic flasks (Corning) in 25% horse serum (Flow Laboratories) and 10,u M hydrocortisone (16). Cultures were established from B6.S, C57BL/6JUt, C3H/HeJ, CD-1 Swiss, N:NIH (Swiss), and BALB/c mice, medium was changed weekly, and all nonadherent cells were removed (17). Factor-dependent hematopoietic cell lines were derived and grown in McCoy's 5A modified medium containing 10% WEHI-3 cell conditioned medium (CM) (18) according to published methods (17). The exact molecule or molecules required for growth of these lines is not known; however, CM from pokeweed mitogen-stimulated spleen cells or the purified fraction of WEHI-3 CM termed interleukin 3 (IL-3) (19) contains the required factor(s). Cloning. Briefly, nonadherent cells were harvested from continuous mouse bone marrow cultures and were transferred in 4 ml to 6-cm plastic Falcon Petri dishes and passaged biweekly at 31 C. Cloning was carried out by transfer of serial 1:10 dilutions of cells into growth medium containing 0.8% methylcellulose. Single-cell-derived colonies at limiting dilution ( cells per ml of culture) were removed by sterile Pasteur pipette on day 7 and then expanded to cells. Recloning was then carried out on Terasaki microtiter plates (1-10 cells per ml, 1.0 ml per plate) (20). Only cells grown from a single cell progenitor were expanded as cloned lines. Microscopic, Karyotypic, Histochemical, and Immunologic Assays. Electron microscopy, histochemical assays for myeloperoxidase and esterase M (3-hydroxy-2-naphthoic acid 2-methoxyanilide chloroacetate substrate), benzidine stain for hemoglobin, toluidine blue assay for metachromasia, leukocyte alkaline phosphatase and lysozyme assays, and Wright/Giemsa Abbreviations: CM, conditioned medium; IL-2 and IL-3, interleukins 2 and 3; BFU, burst-forming unit; CFU, colony-forming unit; -e, erythroid; -c, culture; -s, spleen; -meta, metachromatic cells; GM-, granulocyte/macrophage; G-, granulocyte.

2 2932 Cell Biology: Greenberger et al. hematologic stain were as reported (21-23). Cells were tested for histamine synthesis (22), production of interleukin 2 (IL-2) (19) and IL-3 (19), Thyl.2, Lyl, Ly2, and Ly5 antigens and intracellular Ig as described (24). Karyotyping was as reported (14). Colony-Forming Assays. Colony assays for determination of BFUe, CFUe, GM-CFUc, G-CFUc, CFU-meta, and CFUs (by in vivo assay; BFU, burst-forming unit; CFU, colony-forming unit; -e, erythroid; -c, culture; GM-, granulocyte/macrophage; G-, granulocyte; -meta, metachromatic cells) were performed according to published procedures (25-27). RESULTS Factor-dependent cell lines were derived from retrovirus-infected and uninfected long-term bone marrow cultures of each of several mouse strains (28). As shown in Table 1, cell lines were derived from B6. S (B6SUtA cl 27) and C57BL/6JUt (B6JUt cl 7) as well as from C3H/Hej and CD-1 Swiss mouse strains. These had immature granulated-blast cell morphology, with small numbers of mature neutrophilic granulocytes apparent on Wright/Giemsa staining. After cloning in methylcellulose, each line was recloned by following growth of a single cell in Terasaki plates in WEHI-3 CM. Only subclones derived from a single cell that was visualized by inverted microscope were expanded and carried in vitro for 6 months, then tested in each assay described in the methods. As shown in Table 1, cell line B6SUtA cl 27 and B6JUtA cl 7 demonstrated histochemical properties of cells from at least three different hematopoietic pathways. Between 2% and 3% of cells in each of these lines were characterized as mast cell/ basophils with metachromasia positive by toluidine blue, and there was detectable histamine in the range of ng per 107 cells. The same cloned cell lines demonstrated evidence of neutrophil granulocyte differentiation with 2-3% myeloperoxidase positive by histochemistry. Each cloned line was also biologically characteristic of erythroid progenitor cells and formed 8-9 large erythroid colonies per 103 cells plated when assayed under conditions that support erythroid colony formation by fresh marrow BFUe (Fig. 1). These BFUe and other mixed colonies in the same plates contained cells positive in the benzidine histochemical reaction for hemoglobin (Fig. 1). The ultrastructural appearance of B6SUtA cl 27 cells maintained as a line was consistent with that of undifferentiated blasts; how- Proc. Natl. Acad. Sci. USA 80 (1983) ever, upon stimulation with erythropoietin and CM from pokeweed mitogen-stimulated spleen cells as a source of burst-promoting activity (25), cells with the characteristics of erythroblasts became apparent (Table 2). Another cell line, Ro cl 3-1, which contained similar numbers of metachromasia- and myeloperoxidase-positive cells, also demonstrated properties of eosinophils with detectable lysolecithinase synthesis and light microscopic appearance (Fig. 1). Unlike lines B6JUtA cl 7 and B6SUtA cl 27, Ro cl 3-1 failed to show erythropoietic activity when assayed for erythroid colonyforming capacity. The single cell origins of lines B6SUtA cl 27, B6JUtA cl 7, and Ro cl 3-1 subclone 24, were next meticulously confirmed by a further single cell recloning experiment. Ten of 36 subclones of B6SUtA were, like the original cloned line, able to form large erythropoietic colonies in vitro, and subclones of clone 27 continued to form mixed colonies (containing erythroid, neutrophil, and basophil/mast cells) and erythroid (BFUe), neutrophil-granulocyte (GM-CFUc), and basophil/mast cell (CFU-meta) (22) colonies after over 2 years passage in vitro (Table 3). In the 26 other subclones from the same line, capacity for erythroid colony formation was not demonstrable. The uncloned parent lines B6SUtA and B6JUtA, B6SUtA cl 27 (Table 3), and each of five single-cell-derived subelones of B6SUtA cl 27 (Table 3) were assayed at intervals throughout a period of several months to test the stability of their mixed erythroid/ neutrophil/basophil colony-forming capacity. All remained able to form readily detectable mixed macroscopic erythroid/neutrophil/basophil colonies, and during this period there was also no change in expression of granulopoietic differentiation potential (Table 3). A variation was observed in the frequency of mixed colonies and BFUe detected with cell line B6SUtA and its subclones during the 21/2 years of these studies, in part attributable to the use of different lots of fetal calf sera and sources of erythropoietin, and CM from pokeweed mitogen-stimulated spleen cells as a source of burst-promoting activity. Under the best conditions a frequency of 4-9/10' mixed colonies was observed (Tables 2 and 3). The frequency was at times about 1/10th. In multiple recloned sublines neutrophil-granulocyte and basophil/mast cell colonies were detected after 7 days in replicate cultures from the same assays that another 7 days later were found to contain large mixed and erythroid colonies (Fig. 1). Table 1. Morphological, biochemical, and cell surface properties of clonal hematopoietic progenitor cell lines Cell surface antigen, Morphologyt Histochemistry,t % cells positive % cells positive Cell line* Mouse strain (Wright/Giemsa) Mpo Lyz Est M Tol blue Thyl.1 Lyl Ly2 B6SUtA cl 27 B6.S BL, Pro 3 ± 0.1 <1 3 ± 1 3 ± <1 <1 B6JUt cl 7 C57BL/6JUt BL, Pro 2 ± 0.2 <1 2 ± ± 0.5 NT <1 <1 Rocl3-1 CD-1 Swiss BL, Eos, N, Pro 15 ± 2 6 ± 1 6 ± 1 8 ± <1 <1 32Dcl23 C3H/HeJ BL,Pro <1 <1 18 ± 1 47 ± 3 <1 100 <1 D9 cl-11 C57BL/6J BL, Pro <1 <1 <1 < <1 Mouse myeloma M-1 BALB/c BL, Pro <1 <1 <1 <1 NT NT NT T cl 9 erythroleukemia C3H/H3J BL <1 <1 <1 <1 NT NT NT Rfm AML Rfm/Un BL, Pro, Mtm 6 ± 1 18 ± 7 8 ± 1 <1 <1 <1 <1 * Cell lines were derived from corticosteroid-supplemented continuous mouse bone marrow culture as described (17, 29). Control cell line mouse myeloma, M-1 (30), erythroleukemia cell line T cl 9 (21), and Rfn mouse acute myeloid leukemia (AML) were as described (21). twright/giemsa-stained slides of each line were prepared as described (18) and at least 1,000 cells were scored. BL, blast; Pro, promyelocyte; Eos, eosinophil; N, neutrophil; Mtm, metamyelocyte. thistochemical methods for myeloperoxidase (Mpo), lysozyme (Lyz), esterase M (Est M), and toluidine blue (Tol blue) were as described. Results are mean ± SEM for at least 1,000 cells scored on triplicate preparations of each line tested at 1 year in culture (21). Methods for detection of Thyl.1, Lyl, and Ly2 by using monoclonal antibodies and a fluorescence-activated cell sorter have been reported (24).

3 Cell Biology: Greenberger et al Proc. Natl. Acad. Sci. USA 80 (1983) 2933 'V 0..A\.. FIG. 1. Macroscopic colonies visible in dishes containing Ep were bright red (A) and verified as containing hemoglobin-positive cells by benzidine staining and electron microscopy. Twenty to 50% of hemoglobin-containing colonies were of demonstrable mixed colony morphology containing erythroid cells, neutrophils, and basophil/mast cells. Individual colonies were tested in three histochemical assays: (B) Benzidine stain counterstained with Giemsa, showing positively staining erythroblasts (large solid arrows) and morphologically recognizable neutrophils (large open arrow) and metamyelocyte (small solid arrow). (x600.) (C) The neutrophilic cells in the same colony were histochemically positive for peroxidase. (x 1,800.) (D) Other mononuclear cells in the same B6SUtA cl 27 colony were positive for toluidine blue metachromasia (x 1,800.) (E) Wright/ Giemsa-stained appearance of a single-cell-derived mixed colony from line Ro cl 24 showing eosinophils and neutrophil-granulocytes. (x 1,200.) The histochemical evidence for multipotentiality of cell line B6SUtA cl 27 and its subelones was also maintained over the 2'/2 years of study. Myeloperoxidase, specific for the neutrophil-granulocyte pathway, and esterase M and toluidine blue metachromasia, found in neutrophil-granulocyte and mast cells but not in lymphocytes (21), were always detectable. The percent of cells scored as positive varied over the duration of study between 3 ± 1% (Table 1) and 91% in subelone 18 (Table 3), as is often observed with histochemical reactions with in vitro passaged cell lines (21). However, detectable numbers of B6SUtA cl 27 cells in each subelone were always positive in each of these histochemical assays and provided further strong evidence for multipotentiality. Each cell line was tested for production of IL-3 and IL-2 (19) and was negative. Each cell line was also tested for spleen colony-forming ability in irradiated syngeneic mice. Spleens were removed and fixed 8, 9, and even 14 days after injection, but no sign of spleen colony formation was observed (Table 2). When 107 or 5 x 107 cells of each line were inoculated intravenously into irradiated syngeneic mice there were no survivors after 30 days, although positive control mice receiving the same irradiation and 107 or 106 fresh syngeneic bone marrow cells were protected. Four clonal cell lines were tested for leukemogenicity in vivo. Two groups of 20 adult 30-g C57BL/6J mice were inoculated intravenously with 1 X 107 cells of line B6SUtA cl 27, or line B6JUt cl 7. Groups of 15 CD-1 Swiss mice and 20 C3H/HeJ mice, 30-g adults, were inoculated intravenously with 1 X 107 cells of lines Ro cl 24 and 32D cl 23, respectively. Mice were observed for 1 year for signs of leukemia or solid tumor formation. Peripheral blood counts and differential white cell counts were made monthly. In addition three litters of 1-day-old C57BL/ 6J mice (total of 18 mice) were injected intraperitoneally with 1 X 107 cells of line B6SUtA cl 27 and observed for 1 year. Cell line B6SUtA cl 27 was tested at three times after its establishment for tumorgenicity in adult mice (at 6 months, 1 year, and 18 months after cloning). No leukemias or solid tumors were observed in any of these groups of animals for 1 year. The karyotypes of B6SUtA cl 27 and B6JUt cl 7 were analyzed after the cells were in culture for 10 months. Examination of 50 metaphases from each line revealed 40 chromosomes in 40 of the metaphases of line B6SUtA cl 27 and 45 of the metaphases of line B6JUtA cl 7; other cells of each line were exactly tetraploid, with 80 chromosomes. These data are similar to those obtained with "normal" IL-2 dependent T>cell lines (32) and committed factor-dependent granulocyte lines (33). Cell lines B6SUtA cl 27, B6JUtA cl 7, 32D cl 23, and Ro cl 24 were tested for the release of infectious murine retroviruses and for viral gene expression by the following assays: Supernatant from cultures of 107 cells in 4.0 ml was tested for virusassociated reverse transcriptase activity (18) and for rescue of

4 2934 Cell Biology: Greenberger et al. Proc. Natl. Acad. Sci. USA 80 (1983) Table 2. Biologic properties of clonal hematopoietic progenitor cell lines Colonies per T-lymphocyte 10' cells Mixed. Basophil growth of 105 Pluripotent with no colonies Erythroid* Neutrophil mast cell cells in IL-2, stem cell growth per 103 BFUe per CFUe per GM-CFUc CFU-meta cells x 10' CFUs per Cell line* factor cellst 103 cells 106 cells per 103 cells per i03 cells at day cells B6SUtA cl ± 1 8 ± 2 <1 59 ± 6 17 ± 2 <0.01 <1 B6JUt cl ±1 9 ± 1 <1 68 ± 4 29 ± 2 <0.01 <1 Ro cl <1 <1 <1 47 ± 2 31 ± 5 <0.01 <1 32Dcl23 0 <1 <1 <1 <1 51 ± 2 <0.01 <1 D9 cl-11 0 <1 <1 <1 <1 <1 >15.7 <1 Fresh marrow C3H/HeJ <1 5 ± ± ,700 ± ± 0.6 NT NT 93 ± 4 CD-1 Swiss <1 NT 0.4 ± ,200 ± ± 0.6 NT NT 81 ± 2 C57BL/6J <1 4 ± ± 0.1 8,700 ± ± ± 2 NT NT NA cells from LTBMC day 60 C3H/HeJ <1 NT <0.1 < ± ± 6 < ± 3 CD-i Swiss <1 NT <0.1 < ± 0.3 NT < ± 4 C57BL/6J <1 NT <0.1 < ± 0.9 NT < ± 3 RfmAML >104 NT <0.1 <100 >10 <1 NT NT * Cell lines, fresh marrow single-cell suspensions, or nonadherent (NA) cells from long-term bone marrow culture (LTBMC) at day 60 were tested for colony formation in 0.8% methylcellulose-containing medium (Methocel, Dow) according to published methods (18). The methods for testing BFUe, CFUe, G-CFUc, and CFU-meta were as described (25). Results are the mean ± SEM of at least four plates per point. t Colonies were removed from representative BFUe assay plates at day 14 and stained histochemically with benzidine and counterstained with Giemsa. Individual colonies containing hemoglobin-containing erythroid cells and in addition neutrophilic, basophilic, or both granulocytes were scored as mixed colonies. t CFUe were scored as hemoglobin-containing erythroid colonies of >4 cells scored on days 2, 3, or 4 after plating. None were detected in cultures of cloned cell lines or nonadherent cells from long-term bone marrow culture. The data for fresh marrow are from scorings on day -4. The first detectable hemoglobin-containing colonies with the cloned cell lines were scored on day 8 and cannot be termed CFUe by classic definition (10), because CFUe are defined as cells forming >4 cell clusters in the presence of erythropoietin and scored prior to day 4. Hemoglobin-containing cell clusters were not detected in suspension cultures. Triplicate plates containing 105 cells of each line in 4.0 ml of McCoy's 5A medium were tested for growth in the presence of 10% IL-2 obtained from Harvey Cantor and Gary Nabel. The cloned T-cell line D9 cl-11 has been reported (20). the Kirsten murine sarcoma virus (KiMSV) genome from K-NRK, K-BALB, or K-NIH transformed nonproducer cells by titration of focus formation by filtered culture medium on logarithmicphase cultures of NRK, BALB/3T3, and NIH/3T3 cells (17). There was no evidence of virus release. Cell packs containing over 108 cells from each of the above four cloned lines were tested on two occasions for detectable levels of Rauscher murine leukemia virus gp7o and p30 proteins and for Friend spleen focus-forming virus-related gp55 glycoprotein by immunoprecipitation and autoradiography using goat antisera generously provided by Margarette Vogt (Salk Institute, La Jolla, CA). There was no detectable expression of any of these virus-associated proteins. DISCUSSION The existence of a multipotential but not totipotential hematopoietic stem cell is a well-established concept. The recent development of in vitro assays that support the growth of small mixed colonies in which extensive self-renewal of the cell of origin cannot be demonstrated might be viewed as a method of identifying such cells. Mixed colonies have been noted to contain various combinations of erythroid cells, megakaryocytes, neutrophilic granulocytes, monocytes, and macrophages and to require factors released by marrow or peripheral blood leukocytes for their growth (11-13, 34). More recently it has been shown that a significant proportion of mixed colonies of murine origin are derived from cells that under improved mixed colony assay conditions generate new cells capable of macroscopic spleen colony formation in irradiated mice (31) and new multipotential progenitors with undiminished proliferative capacity as measured by secondary colony size (25). In addition, it has recently been reported that at least some human progenitors of mixed colonies in vitro can be shown to yield T lymphocytes as well as myeloid progeny (35). Thus, colony assays do not readily lend themselves to the study of stages of differentiation intermediate between CFUs and their most primitive unipotent derivatives. The characteristics of two of the lines (B6SUtA and B6JUtA) reported here may provide a different approach. Both of these cloned multipotential cell lines have remained stable and nonmalignant with retention of multilineage differentiation capacity after continuous maintenance in vitro for over 2 years. The inability of these cell lines to reconstitute lethally irradiated mice or even to form visible spleen colonies in vivo after intravenous injection strongly argues for the loss of normally regulated self-renewal properties characteristic of fresh marrow stem cells. Their inability to form factor-independent colonies in vitro in the absence of their specific obligatory growth factor (29, 33) and their lack of detectable leukemogenicity in vivo strongly argue that these multipotential cell lines are not malignant in the classic Stable multipotential but nonmalignant hematopoietic sense. progenitor cell lines should prove valuable for study of hemopoietic cell differentiation. We thank Dr. Stuart A. Aaronson for immunoglobulin assays and for many helpful discussions, Dr. Peter Weller for lysolecithinase assays, Dr. James Ihle for IL-2 and IL-3 synthesis assays and for purified IL- 3 and IL-2, and Ms. Paula Marks, Ms. Donna Reid, and Mr. Thomas This work was supported by National Novak for technical assistance. Cancer Institute Research Grants CA25412 and CA26785, American Cancer Society Grant CH-171, and the National Cancer Institute of Canada. C.J. E. is a Research Associate of the National Cancer Institute of Canada.

5 Cell Biology: Greenberger et al Proc. Natl. Acad. Sci. USA 80 (1983) 2935 Table 3. Biology of clonal sublines of multipotential hematopoietic progenitor cells Colony formation in vitro* Mixed Histochemistryrt Histcheisty~t% %cells cels positive osiive colonies per BFUe per GM-CFUc per CFU-meta per Cell line* Morphologyt Est M Mpo Lyz Tol blue 103 cells 103 cells 104 cells 104 cells B6SUtA cl 27 BL < ± 1 8 ± ± ± 1 Subclone5 BL < ±1 4±1 121± 8 31±1 6 BL < ± 5 6 ± ± 7 27 ± 1 18 BL < ± 1 7 ± 1 76 ± 1 4 ± 1 19 BL NT 1.0 5±1 6±1 113± 2 18±1 23 BL NT NT NT 3 ± 1 90 ± 3 NT Rocl24 BL,Pro, Eos,N 71 NT 40 6 <0.1 < ± 6 18 ± 6 Subclone 4 BL, Pro, Eos, N <0.1 < ± 4 43 ± 1 8 BL,Pro,Eos,N <0.1 < ± 4 8 ± 1 26 BL,Pro, Eos, N NT NT NT NT 6 ± 1 29 BL,Pro,Eos,N NT 18 NT NT 113 ± 2 NT 31 BL,Pro,Eos, N 70 NT NT NT NT 13 ± 1 32D cl 23 BL, Pro 25 <0.1 < <0.1 <0.1 <1 40 ± 4 Subclone 9 BL, Pro 80 <0.1 < <0.1 <0.1 <1 19 ± 2 11 BL, Pro 71 <0.1 < <0.1 <0.1 <1 37 ± 3 15 BL, Pro 63 <0.1 <0.1 9 <0.1 <0.1 <1 41 ± 1 19 BLPro NT <0.1 <0.1 NT <0.1 <0.1 <1 39.± 4 23 BLPro 71 <0.1 <0.1 4 NT NT <1 18 ± 1 B6JUtcl7 BL <1 6 5 ± 1 19 ± ± ± 5 Subclone8 BL <1 NT 6 ± 1 8 ± ± 8 41 ± 2 11 BL <1 17 NT 4±1 93± 6 41±2 * Each clonal line was subcloned by Terasaki plate analysis and subclones were expanded to 107 cells before analysis. t Morphology and histochemistry were analyzed as described in the legend to Table 1. t Each offour plates containing cells per ml was scored for the number ofindividual hemoglobin-containing colonies at day 12 that contained erythroid, neutrophil, and basophil cells when removed from culture and stained with Wright/Giemsa; 12-day BFUe (31), 7-day G-CFUc (28), and 7-day CFU-meta (22); toluidine blue-positive mast cell/basophils according to published methods. 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