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Home , Basophil, Metamyelocyte, Neutrophil, Progenitor cell, Promyelocyte

Proc. Natl Acad. Sci. USA Vol. 80, pp. 2931-2935, May 1983 Biology

Demonstration of permanent factor-dependent multipotential (erythroid//) hematopoietic lines (self-renewal/stem cells/differentiation//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 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 02115 Communicated by Henry S. Kaplan, January 20, 1983

ABSTRACT Multipotential hematopoietic progenitor cell lines tures containing mixed hematopoietic colonies of single-cell or- have been established from nonadherent cell populations re- igin were reported (11). Such colonies contain cells of the moved from continuous mouse bone marrow cultures. Clonal sub- erythroid lineage admixed with , , lines of lines B6SUtA or B6JUt derived from single cells formed and in some instances , including as mixed colonies containing erythroid cells, neutrophil-granulo- well as (11-13). We now report the characterization cytes, and basophil/mast cells in semisolid medium containing of permanent lines of factor-dependent and nonmalignant (14, erythropoietin and conditioned medium from pokeweed mitogen- 15) hematopoietic cells that differentiate along erythroid, neu- stimulated spleen cells. Each of several subclones of cell line Ro trophil-, and cell after cl formed colonies containing eosinophils, neutrophil-granulo- basophil/mast pathways ap- cytes, and basophil/mast cells in semisolid medium. Multipoten- propriate stimulation in vitro. tiality was maintained in vitro for over 21/2 years. In contrast, cell MATERIALS AND METHODS line 32D formed basophil/ colonies with no detectable differentiation to other pathways. Multipotential cell lines did not Bone Marrow Cultures. Continuous mouse bone marrow produce detectable spleen colonies (CFUs) in vivo, nor did intra- cultures were established according to published procedures, venous inoculation of up to 5 x 107 cells protect lethally irradiated using the contents of a femur and tibia inoculated into 25-cm2 mice from bone marrow failure. Newborn and adult mice inoc- plastic flasks (Corning) in 25% horse serum (Flow Laboratories) ulated with 5 X 107 cells showed no detectable or solid and 10 ,uM hydrocortisone (16). Cultures were established from tumors after one year. Both multipotential and committed baso- B6.S, C57BL/6JUt, C3H/HeJ, CD-1 Swiss, N:NIH (Swiss), phil/mast cell lines demonstrated absolute dependence upon a and BALB/c mice, medium was changed weekly, and all non- source of a growth factor(s) found in medium conditioned by WEHI- adherent cells were removed (17). Factor-dependent hemato- 3 cells. These cell lines should be of value in studies of the reg- poietic cell lines were derived and grown in McCoy's 5A mod- ulation of hematopoietic differentiation in vitro. ified medium containing 10% WEHI-3 cell conditioned medium (CM) (18) according to published methods (17). The exact mol- Hematopoietic stem cells, as defined by the spleen colony- ecule or molecules required for growth of these lines is not forming unit (CFUs) assay (1), give rise to a variety of differ- known; however, CM from pokeweed mitogen-stimulated spleen entiated cell types. These may include B and T as cells or the purified fraction of WEHI-3 CM termed interleu- well as erythroid cells, , neutrophilic granulocytes, kin 3 (IL-3) (19) contains the required factor(s). eosinophils, basophil/mast cells, and macrophages. Data sup- Cloning. Briefly, nonadherent cells were harvested from con- porting the common origin of lymphoid and myeloid lineages tinuous mouse bone marrow cultures and were transferred in come primarily from two types of in vivo studies. In the first, 4 ml to 6-cm plastic Falcon Petri dishes and passaged biweekly radiation was used to induce unique chromosomal markers in at 31°C. Cloning was carried out by transfer of serial 1:10 di- donor marrow cells. Such cells were then transplanted into lutions of cells into growth medium containing 0.8% methyl- suitable recipients and subsequently found to have repopulated cellulose. Single-cell-derived colonies at limiting dilution (10- lymphoid as well as myeloid tissues with cells bearing the same 100 cells per ml of culture) were removed by sterile Pasteur marked karyotype (2, 3). The second line of evidence derived pipette on day 7 and then expanded to 107-108 cells. Recloning from the demonstration of the clonal nature of disease states was then carried out on Terasaki microtiter plates (1-10 cells that involve cells of several hematopoietic lineages, including per ml, 1.0 ml per plate) (20). Only cells grown from a single lymphocytes as well as myeloid cells (4, 5). cell progenitor were expanded as cloned lines. The concept of primitive but committed hematopoietic pro- Microscopic, Karyotypic, Histochemical, and Immunologic genitor cells restricted to specific differentiation pathways is Assays. Electron microscopy, histochemical assays for myelo- based on studies using in vitro colony assays. These have shown peroxidase and esterase M (3-hydroxy-2-naphthoic acid 2-meth- that colonies commonly contain cells of a single lineage even oxyanilide chloroacetate substrate), benzidine stain for hemo- though different types of colonies may be present in the same globin, toluidine blue assay for metachromasia, leukocyte al- cultures (6-8) and that these different types of colonies arise kaline phosphatase and assays, and Wright/Giemsa from progenitors with different properties (9, 10). In 1977, cul- Abbreviations: CM, conditioned medium; IL-2 and IL-3, The publication costs ofthis article were defrayed in part by page charge 2 and 3; BFU, burst-forming unit; CFU, colony-forming unit; -e, ery- payment. This article must therefore be hereby marked "advertise- throid; -c, culture; -s, spleen; -meta, metachromatic cells; GM-, gran- ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. ulocyte/; G-, granulocyte. 2931 Downloaded by guest on September 30, 2021 2932 : Greenberger et al. Proc. Natl. Acad. Sci. USA 80 (1983)

hematologic stain were as reported (21-23). Cells were tested ever, upon stimulation with erythropoietin and CM from poke- for synthesis (22), production of 2 (IL-2) weed mitogen-stimulated spleen cells as a source of burst-pro- (19) and IL-3 (19), Thyl.2, Lyl, Ly2, and Ly5 and in- moting activity (25), cells with the characteristics of erythroblasts tracellular Ig as described (24). Karyotyping was as reported became apparent (Table 2). (14). Another cell line, Ro cl 3-1, which contained similar numbers Colony-Forming Assays. Colony assays for determination of of metachromasia- and -positive cells, also BFUe, CFUe, GM-CFUc, G-CFUc, CFU-meta, and CFUs (by demonstrated properties of eosinophils with detectable lyso- in vivo assay; BFU, burst-forming unit; CFU, colony-forming lecithinase synthesis and light microscopic appearance (Fig. 1). unit; -e, erythroid; -c, culture; GM-, granulocyte/macrophage; Unlike lines B6JUtA cl 7 and B6SUtA cl 27, Ro cl 3-1 failed to G-, granulocyte; -meta, metachromatic cells) were performed show erythropoietic activity when assayed for erythroid colony- according to published procedures (25-27). forming capacity. The single cell origins of lines B6SUtA cl 27, B6JUtA cl 7, RESULTS and Ro cl 3-1 subclone 24, were next meticulously confirmed Factor-dependent cell lines were derived from retrovirus-in- by a further single cell recloning experiment. Ten of 36 sub- fected and uninfected long-term bone marrow cultures of each clones of B6SUtA were, like the original cloned line, able to of several mouse strains (28). As shown in Table 1, cell lines form large erythropoietic colonies in vitro, and subclones of were derived from B6. S (B6SUtA cl 27) and C57BL/6JUt (B6JUt 27 continued to form mixed colonies (containing ery- cl 7) as well as from C3H/Hej and CD-1 Swiss mouse strains. throid, neutrophil, and basophil/mast cells) and erythroid These had immature granulated-blast cell morphology, with small (BFUe), neutrophil-granulocyte (GM-CFUc), and basophil/mast numbers of mature neutrophilic granulocytes apparent on cell (CFU-meta) (22) colonies after over 2 years passage in vitro Wright/Giemsa . After cloning in methylcellulose, each (Table 3). In the 26 other subclones from the same line, ca- line was recloned by following growth of a single cell in Terasa- pacity for erythroid colony formation was not demonstrable. The ki plates in WEHI-3 CM. Only subclones derived from a single uncloned parent lines B6SUtA and B6JUtA, B6SUtA cl 27 (Ta- cell that was visualized by inverted microscope were expanded ble 3), and each of five single-cell-derived subelones of B6SUtA and carried in vitro for 6 months, then tested in each assay de- cl 27 (Table 3) were assayed at intervals throughout a period of scribed in the methods. several months to test the stability of their mixed erythroid/ As shown in Table 1, cell line B6SUtA cl 27 and B6JUtA cl neutrophil/basophil colony-forming capacity. All remained able 7 demonstrated histochemical properties of cells from at least to form readily detectable mixed macroscopic erythroid/neu- three different hematopoietic pathways. Between 2% and 3% trophil/basophil colonies, and during this period there was also of cells in each of these lines were characterized as mast cell/ no change in expression of granulopoietic differentiation po- with metachromasia positive by toluidine blue, and tential (Table 3). there was detectable histamine in the range of 10-30 ng per 107 A variation was observed in the frequency of mixed colonies cells. The same cloned cell lines demonstrated evidence of and BFUe detected with cell line B6SUtA and its subclones neutrophil granulocyte differentiation with 2-3% myeloperox- during the 21/2 years of these studies, in part attributable to the idase positive by histochemistry. Each cloned line was also bi- use of different lots of fetal calf sera and sources of erythro- ologically characteristic of erythroid progenitor cells and formed poietin, and CM from pokeweed mitogen-stimulated spleen cells 8-9 large erythroid colonies per 103 cells plated when assayed as a source of burst-promoting activity. Under the best con- under conditions that support erythroid colony formation by ditions a frequency of 4-9/10' mixed colonies was observed fresh marrow BFUe (Fig. 1). These BFUe and other mixed col- (Tables 2 and 3). The frequency was at times about 1/10th. In onies in the same plates contained cells positive in the benzi- multiple recloned sublines neutrophil-granulocyte and baso- dine histochemical reaction for hemoglobin (Fig. 1). The ul- phil/mast cell colonies were detected after 7 days in replicate trastructural appearance of B6SUtA cl 27 cells maintained as a cultures from the same assays that another 7 days later were line was consistent with that of undifferentiated blasts; how- 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 ,§ 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 ± 1 100 <1 <1 B6JUt cl 7 C57BL/6JUt BL, Pro 2 ± 0.2 <1 2 ± 0.5 2 ± 0.5 NT <1 <1 Rocl3-1 CD-1 Swiss BL, Eos, N, Pro 15 ± 2 6 ± 1 6 ± 1 8 ± 1 100 <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 <1 100 100 <1 Mouse myeloma M-1 BALB/c BL, Pro <1 <1 <1 <1 NT NT NT T cl 9 erythro- leukemia 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 (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, ; Eos, ; N, neutrophil; Mtm, . 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 and a fluorescence-activated cell sorter have been reported (24). NT, not tested. Downloaded by guest on September 30, 2021 Cell Biology: Greenberger et aL Proc. Natl. Acad. Sci. USA 80 (1983) 2933

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FIG. 1. Macroscopic colonies visible in dishes containing Ep were bright red (A) and verified as containing hemoglobin-positive cells by ben- zidine staining and electron microscopy. Twenty to 50% of hemoglobin-containing colonies were of demonstrable mixed colony morphology con- taining 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 per- oxidase. (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 B6JUt cl 7. Groups of 15 CD-1 Swiss mice and 20 C3H/HeJ B6SUtA cl 27 and its subelones was also maintained over the mice, 30-g adults, were inoculated intravenously with 1 X 107 2'/2 years of study. Myeloperoxidase, specific for the neutro- cells of lines Ro cl 24 and 32D cl 23, respectively. Mice were phil-granulocyte pathway, and esterase M and toluidine blue observed for 1 year for signs of leukemia or solid tumor for- metachromasia, found in neutrophil-granulocyte and mast cells mation. Peripheral blood counts and differential white cell counts but not in lymphocytes (21), were always detectable. The per- were made monthly. In addition three litters of 1-day-old C57BL/ cent of cells scored as positive varied over the duration of study 6J mice (total of 18 mice) were injected intraperitoneally with between 3 ± 1% (Table 1) and 91% in subelone 18 (Table 3), 1 X 107 cells of line B6SUtA cl 27 and observed for 1 year. Cell as is often observed with histochemical reactions with in vitro line B6SUtA cl 27 was tested at three times after its establish- passaged cell lines (21). However, detectable numbers of B6SUtA ment for tumorgenicity in adult mice (at 6 months, 1 year, and cl 27 cells in each subelone were always positive in each of these 18 months after cloning). No or solid tumors were histochemical assays and provided further strong evidence for observed in any of these groups of animals for 1 year. multipotentiality. Each cell line was tested for production of The karyotypes of B6SUtA cl 27 and B6JUt cl 7 were ana- IL-3 and IL-2 (19) and was negative. lyzed after the cells were in culture for 10 months. Examination Each cell line was also tested for spleen colony-forming abil- of 50 metaphases from each line revealed 40 chromosomes in ity in irradiated syngeneic mice. Spleens were removed and 40 of the metaphases of line B6SUtA cl 27 and 45 of the meta- fixed 8, 9, and even 14 days after injection, but no sign of spleen phases of line B6JUtA cl 7; other cells of each line were exactly colony formation was observed (Table 2). When 107 or 5 x 107 tetraploid, with 80 chromosomes. These data are similar to those cells of each line were inoculated intravenously into irradiated obtained with "normal" IL-2 dependent T>cell lines (32) and syngeneic mice there were no survivors after 30 days, although committed factor-dependent granulocyte lines (33). positive control mice receiving the same irradiation and 107 or Cell lines B6SUtA cl 27, B6JUtA cl 7, 32D cl 23, and Ro cl 106 fresh syngeneic bone marrow cells were protected. 24 were tested for the release of infectious murine retroviruses Four clonal cell lines were tested for leukemogenicity in vivo. and for viral gene expression by the following assays: Super- Two groups of 20 adult 30-g C57BL/6J mice were inoculated natant from cultures of 107 cells in 4.0 ml was tested for virus- intravenously with 1 X 107 cells of line B6SUtA cl 27, or line associated reverse transcriptase activity (18) and for rescue of Downloaded by guest on September 30, 2021 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- 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 103cells 106cells per 103 cells per i03 cells at day 7 106cells B6SUtA cl 27 0 4 ± 1 8 ± 2 <1 59 ± 6 17 ± 2 <0.01 <1 B6JUt cl 7 0 6 ±1 9 ± 1 <1 68 ± 4 29 ± 2 <0.01 <1 Ro cl 3-1 0 <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 ± 1 0.6 ± 0.2 11,700 ± 600 8.9 ± 0.6 NT NT 93 ± 4 CD-1 Swiss <1 NT 0.4 ± 0.1 12,200 ± 500 3.3 ± 0.6 NT NT 81 ± 2 C57BL/6J <1 4 ± 1 0.3 ± 0.1 8,700 ± 600 5.9 ± 0.7 8 ± 2 NT NT NA cells from LTBMC day 60 C3H/HeJ <1 NT <0.1 <100 4.7 ± 0.2 17 ± 6 <0.01 27 ± 3 CD-i Swiss <1 NT <0.1 <100 5.9 ± 0.3 NT <0.01 18 ± 4 C57BL/6J <1 NT <0.1 <100 6.8 ± 0.9 NT <0.01 9 ± 3 RfmAML >104 NT <0.1 <100 >10 <1 NT NT NT, not tested. * 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, , 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, been reported that at least some human progenitors of mixed K-BALB, or K-NIH transformed nonproducer cells by titration colonies in vitro can be shown to yield T lymphocytes as well of focus formation by filtered culture medium on logarithmic- as myeloid progeny (35). phase cultures of NRK, BALB/3T3, and NIH/3T3 cells (17). Thus, colony assays do not readily lend themselves to the There was no evidence of virus release. study of stages of differentiation intermediate between CFUs Cell packs containing over 108 cells from each of the above and their most primitive unipotent derivatives. The character- four cloned lines were tested on two occasions for detectable istics of two of the lines (B6SUtA and B6JUtA) reported here levels of Rauscher murine leukemia virus gp7O and p30 pro- may provide a different approach. Both of these cloned mul- teins and for Friend spleen focus-forming virus-related gp55 tipotential cell lines have remained stable and nonmalignant glycoprotein by immunoprecipitation and autoradiography us- with retention of multilineage differentiation capacity after ing goat antisera generously provided by Margarette Vogt (Salk continuous maintenance in vitro for over 2 years. The inability Institute, La Jolla, CA). There was no detectable expression of of these cell lines to reconstitute lethally irradiated mice or even any of these virus-associated . to form visible spleen colonies in vivo after intravenous injec- tion strongly argues for the loss of normally regulated self-re- DISCUSSION newal properties characteristic of fresh marrow stem cells. Their to form colonies in vitro in the ab- of a but not hema- inability factor-independent The existence multipotential totipotential sence of their factor and their topoietic stem cell is a well-established concept. The recent de- specific obligatory growth (29, 33) that the of small lack of detectable leukemogenicity in vivo strongly argue that velopment of in vitro assays support growth are not in the classic mixed colonies in which extensive self-renewal of the cell of these multipotential cell lines malignant a of sense. Stable multipotential but nonmalignant hematopoietic origin cannot be demonstrated might be viewed as method progenitor cell lines should prove valuable for study of hemo- identifying such cells. Mixed colonies have been noted to con- cell differentiation. tain various combinations of erythroid cells, megakaryocytes, poietic and and to neutrophilic granulocytes, , macrophages A. Aaronson for and for or blood leu- We thank Dr. Stuart immunoglobulin assays require factors released by marrow peripheral Dr. Peter Weller for More it has been many helpful discussions, lysolecithinase assays, kocytes for their growth (11-13, 34). recently Dr. James Ihle for IL-2 and IL-3 synthesis assays and for purified IL- shown that a significant proportion of mixed colonies of murine 3 and IL-2, and Ms. Paula Marks, Ms. Donna Reid, and Mr. Thomas origin are derived from cells that under improved mixed colony Novak for technical assistance. This work was supported by National assay conditions generate new cells capable of macroscopic spleen Cancer Institute Research Grants CA25412 and CA26785, American colony formation in irradiated mice (31) and new multipotential Cancer Society Grant CH-171, and the National Cancer Institute of progenitors with undiminished proliferative capacity as mea- Canada. C.J. E. is a Research Associate of the National Cancer Institute sured by secondary colony size (25). In addition, it has recently of Canada. Downloaded by guest on September 30, 2021 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 HistochemistryrtHistcheisty~t%%cellscels positiveosiive 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 90 31 <1 0.9 4 ± 1 8 ± 2 119 ± 16 18 ± 1 Subclone5 BL 87 27 <1 1.1 6±1 4±1 121± 8 31±1 6 BL 79 43 <1 0.3 5 ± 5 6 ± 1 102 ± 7 27 ± 1 18 BL 91 51 <1 4.0 6 ± 1 7 ± 1 76 ± 1 4 ± 1 19 BL 84 18 NT 1.0 5±1 6±1 113± 2 18±1 23 BL 90 41 NT NT NT 3 ± 1 90 ± 3 NT Rocl24 BL,Pro, Eos,N 71 NT 40 6 <0.1 <0.1 141 ± 6 18 ± 6 Subclone 4 BL, Pro, Eos, N 90 41 18 1.0 <0.1 <0.1 113 ± 4 43 ± 1 8 BL,Pro,Eos,N 64 50 71 4 <0.1 <0.1 87 ± 4 8 ± 1 26 BL,Pro, Eos, N 90 49 60 NT NT NT NT 6 ± 1 29 BL,Pro,Eos,N 83 30 NT 18 NT NT 113 ± 2 NT 31 BL,Pro,Eos, N 70 NT 14 12 NT NT NT 13 ± 1 32D cl 23 BL, Pro 25 <0.1 <0.1 39 <0.1 <0.1 <1 40 ± 4 Subclone 9 BL, Pro 80 <0.1 <0.1 21 <0.1 <0.1 <1 19 ± 2 11 BL, Pro 71 <0.1 <0.1 18 <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 81 17 <1 6 5 ± 1 19 ± 1 114 ± 16 93 ± 5 Subclone8 BL 91 41 <1 NT 6 ± 1 8 ± 1 153 ± 8 41 ± 2 11 BL 83 15 <1 17 NT 4±1 93± 6 41±2 NT, not tested. * 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 offourplates containing 103-104 cells perml was scoredforthe number ofindividual hemoglobin-containing colonies atday 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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