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Blood, 15 February 2007, Vol. 109, No. 4, pp. 1460-1471. Prepublished online as a Blood First Edition Paper on October 12, 2006; DOI 10.1182/blood-2006-07-030726.
HEMATOPOIESIS The hypomorphic Gata1low mutation alters the proliferation/differentiation potential of the common megakaryocytic-erythroid progenitor1 Department of Hematology, Oncology, and Molecular Medicine, Istituto Superiore Sanità, Rome, Italy; 2 Department of Biomorphology, University G. D'Annunzio, Chieti, Italy; 3 Department of Cell Biology and Neurosciences, Istituto Superiore Sanità, Rome, Italy; 4 Department of Hematology and Oncology, University of Florence, Italy; 5 Department of Pediatric Oncology, Children's Hospital, Dana Farber Cancer Institute, Harvard Medical School and Howard Hughes Medical Institute, Boston, MA; 6 Department of Medicine and Myeloproliferative Disease–Research Consortium (Pathology), University of Illinois at Chicago
Recent evidence suggests that mutations in the Gata1 gene may alter the proliferation/differentiation potential of hemopoietic progenitors. By single-cell cloning and sequential replating experiments of prospectively isolated progenitor cells, we demonstrate here that the hypomorphic Gata1low mutation increases the proliferation potential of a unique class of progenitor cells, similar in phenotype to adult common erythroid/megakaryocytic progenitors (MEPs), but with the "unique" capacity to generate erythroblasts, megakaryocytes, and mast cells in vitro. Conversely, progenitor cells phenotypically similar to mast cell progenitors (MCPs) are not detectable in the marrow from these mutants. At the single-cell level, about 11% of Gata1low progenitor cells, including MEPs, generate cells that will continue to proliferate in cultures for up to 4 months. In agreement with these results, trilineage (erythroid, megakaryocytic, and mastocytic) cell lines are consistently isolated from bone marrow and spleen cells of Gata1low mice. These results confirm the crucial role played by Gata1 in hematopoietic commitment and identify, as a new target for the Gata1 action, the restriction point at which common myeloid progenitors become either MEPs or MCPs.
Among the GATA family of transcription factors,1 Gata1 exerts a specific role in the control of erythroid,2 megakaryocytic,3,4 eosinophil,5 and mast6 cell differentiation. Genetic alterations of this gene, however, are not only associated with X-linked inherited erythroid or megakaryocytic disorders, but are also found in acquired myeloproliferative disorders. Each mutation is associated with a specific abnormality: point mutations that abrogate the ability of the amino-terminal zinc finger domain of the protein to bind either DNA or Fog1, a partner of Gata1, are found in inherited disorders.7–10 On the other hand, frame shift and splice mutations encoding GATA1s, a protein lacking the amino-terminal domain, are associated not only with impaired inherited erythropoiesis,11,12 but are also found in patients with megakaryocytic leukemia in Down syndrome,13,14 in newborns with transient myeloproliferative syndromes,15 and in one adult patient with megakaryocytic leukemia.16 These observations suggest that, in addition to its effect on terminal differentiation, Gata1 might control the biologic properties of hematopoietic progenitor cells, predisposing them to accumulate secondary mutations in a multistep leukemogenic process. However, direct proof for a possible function of Gata1 in progenitor cells has not been provided as yet.
We had previously described that hematopoietic tissues from mice carrying the hypomorphic Gata1low mutation contain high numbers ( The aim of this study was to clarify the role of Gata1 in hematopoietic commitment by identifying the antigenic profile and proliferation potential of the Gata1low progenitors giving rise to trilineage colonies. First, we compared the number and function of mast cells generated in bone marrow-derived mast cell cultures (BMMCs) seeded with marrow from Gata1low and wild-type (positive controls) mice. Heterozygous W/Wv mice (ie, expressing the W, truncated, and Wv, kinase defective, form of c-Kit25), that do not express mast cells in vivo but whose marrow generates defective mast cells in vitro, were used as negative controls. Next, we examined the frequency of CMPs, MEPs, and MCPs in marrow and spleen from wild-type and Gata1low littermates. The different populations were also isolated and their differentiation and proliferation potential characterized under conditions of limiting dilution followed by single cell recloning. Our results confirm that mast cell differentiation is defective in Gata1low mice (decreased differentiation and increased proliferation). The defect includes the ability to generate, with high frequency, factor-dependent trilineage cell lines. In Gata1low mice, the frequency of cells with the antigenic profile of CMPs, MEPs, and GMPs was normal in marrow and markedly increased in spleen, whereas those with the MCP profile were not detectable. However, mutant cells isolated according to the MEP phenotype, had the "unique" property to generate mast cells and their precursors in 7 days of culture, in addition to erythroblasts and megakaryocytes. Furthermore, the progeny of about 10% of mutant MEPs could be propagated in culture, as single cells, with 95% efficiency, for up to 4 months. In comparison, the progeny of wild-type MEPs became extinguished in 7 to 14 days. These results indicate that cells with the phenotype corresponding to myeloid, but not those corresponding to mastocytic, progenitors are detectable in tissues from Gata1low mice. However, in these mice, the mast cell-generating activity is abnormally acquired by MEPs, which, therefore are antigenically, but not functionally, equivalent to wild-type MEPs. These results indicate that the Gata1low mutation also targets cells at the restriction point between CMPs and either MCPs or MEPs.
Mice Mice carrying the hypomorphic Gata1low mutations (ie, deletion of the first enhancer and of the distal promoter of the gene)26 were bred at the Istituto Superiore di Sanità. Transgenic µLCR-hGATA1 males27,28 (carrying the hGATA1 cDNA under the control of a µLCR linked to the ß-globin promoter), were crossed with homozygous Gata1low/low females. Because Gata1 is on the X chromosome whereas µLCR-hGATA1 is autosomal, 50% of the male Gata1low/0 offspring will carry the transgene. Offspring were genotyped by polymerase chain reaction (PCR) at birth, as described,29 and µLCR-hGATA1–Gata1low/0 males used as negative controls. WBB6FAW/Wv mice were purchased from Jackson Laboratories (Bar Harbor, ME). Mice were housed under good animal care practice conditions and all the experiments were performed with 3- to 4-month old males, under protocols approved by the institutional animal care committee. Prospective isolation of hematopoietic progenitors
Myeloid (CMP and MEP) and mast cell (MCP) progenitors were purified according to modifications30 of procedures described by Akashi et al18 and Chen et al.21 Briefly, mononuclear cell suspensions were labeled with the biotinylated rat antimouse antibodies CD3, CD45R/B220, CD19, CD127 (IL-7R Flow cytometry
Cells were suspended in Ca++Mg++-free phosphate-buffered saline (PBS) containing 0.5% (vol/vol) bovine serum albumin (BSA; Sigma) and 2 mM EDTA. Mast cells were recognized by labeling with PE-CD117, FITC-CD34, FITC-T1/ST2, and FITC–anti-Fc Liquid cultures of prospectively isolated progenitor cells MEPs, CMPs, and MCPs (1 x 103 cells/mL) were cultured for 6 to 21 days in Iscove modified Dulbecco medium (IMDM; Gibco, Invitrogen, Carlsband, CA) supplemented with fetal calf serum (FCS, 10% vol/vol; Sigma), 7.5 x 10–5 M ß-mercaptoethanol (Sigma), penicillin, and streptomycin sulfate (50 U/mL, Gibco), and glutamine (2 mM, Gibco). Single-cell cloning experiments were performed by limiting dilution, that is, by resuspending the cells as 3 cells/mL and culturing 100 µL of this solution in each of a 96-well plate.31 Cultures were stimulated either with rat SCF (100 ng/mL; Amgen, Thousand Oaks, CA) and murine IL-3 (10 ng/mL, PeproTech, London, United Kingdom)32,33 or with a cocktail containing rat SCF (10 ng/mL), murine IL-3 (10 ng/mL), and GM-CSF (10 ng/mL; PeproTech) and human FLT3-ligand (10 ng/mL; PeproTech), IL-11 (10 ng/mL; PeproTech), thrombopoietin (TPO, 50 ng/mL; PeproTech), and erythropoietin (EPO, 3 U/mL; Hoffman-La Roche, Basel, Switzerland).34 The cultures were incubated at 37°C in a humidified incubator containing 5% CO2 in air and cell growth and phenotype analyzed 6 to 7, 14, and 21 days later. The proliferation potential of the progenitor cell progeny was evaluated by randomly selecting 10 wells and by culturing their content under conditions of limiting dilution. The wells were scored 7 days later for sign of cell proliferation and subcloned again as long as growth was detected. BMMCs Marrow and spleen cells (1 x 105 cells/mL) from wild-type, Gata1low, and W/Wv mice were cultured in IMDM supplemented with FBS (30% vol/vol), BSA (1.5% wt/vol), glutamine (2 mM), penicillin and streptomycin sulfate (50 U/mL), SCF (100 ng/mL), and IL-3 (10 ng/mL), as described.6,32,33 Every 3 to 4 days, cells were counted and the cultures replenished with fresh medium to maintain cell density at 2.5 x 105/mL. Culture of the SN cell lines The SN cell lines were obtained from Gata1low BMMCs and passed twice a week in IMDM supplemented with FBS (20% vol/vol), BSA (1.5% wt/vol), glutamine (2 mM), penicillin and streptomycin sulfate (50 U/mL), SCF (100 ng/mL), and IL-3 (10 ng/mL). Immunohistochemistry Cytocentrifuged cell preparations (3 x 103-104 cells/smear) were stained either with May-Grünwald Giemsa (Sigma), benzidine, acetyl cholinesterase, or Alcian blue, according to standard protocols.6 RNA isolation and RT-PCR analysis
Total RNA was prepared with TRIzol (Gibco), using glycogen (20 µg/L; Hoffman-LaRoche), as carrier. For semiquantitative analysis, RNA (1 µg) was reverse transcribed with random hexamers and Moloney murine leukemia virus reverse transcriptase (RT; Perkin-Elmer, Downers Grove, IL) and cDNA was amplified with the specific sense and antisense primers described in Table S1 (available on the Blood website; see the Supplemental Materials link at the top of the online article). The reaction was performed in 100 µL 10 mM Tris-HCl, pH 8.3, containing MgCl2 (2 mM), dNTP (200 µM each), 0.1 µCi (0.037 MBq) [ Serotonin-release assay Cells (1 x 106/mL) were incubated with 5-hydroxy[3H] tryptamine trifluoroacetate (3H-serotonin, 2 µCi/mL [0.074 MBq]); 84.0 Ci/mmol, Amersham) for 6 hours at 37°C, washed twice, and incubated again, at a concentration of 20 x 106/mL, for 1 hour on ice either with 10 µg/mL monoclonal mouse anti–DNP-IgE (clone SPE-7, Sigma) or medium alone. After washing, cells were divided into aliquots (0.4 x 106/50 µL) and stimulated, for 15 minutes at 37°C, with either medium alone, 2 µg/mL monoclonal rat anti–mouse IgE (R35-72, PharMingen), 1 µg/mL DNP-human serum albumin (Sigma), or 1 µg/mL ionomycin (Sigma). Reactions were terminated with 50 µL cold Hanks balanced salt solution (Sigma), cells removed by centrifugation and levels of [3H]serotonin in supernatants measured with the Packard 1600TR liquid scintillator counter (Perkin Elmer). Total 3H-serotonin incorporation was determined by lysing unstimulated cells with 1% Triton X-100 (Sigma). The amount of 3H-serotonin released by the cells was calculated as counts per minute in the supernatant ÷ counts per minute of total incorporation x 100 (see also Rodewald et al22). Statistical analysis Statistical analysis was performed by analysis of variance (ANOVA test) using Origin 6.1 software for Windows (Microcal Software, Northampton, MA).
Hematopoietic tissues from Gata1low mice contain cells with the antigenic profile of myeloid progenitors but lack those with the mastocytic progenitor profile Normal progenitor cell subclasses were defined according to the antigenic profiles summarized in Figure 1A. Cells with MCP, MEP and CMP phenotype were detected, at the expected frequencies, in marrow from wild-type animals (Figure 1B; Table 1). The fact that these phenotypes discriminate different populations was confirmed by the observation that the sorted cells retained their phenotype on reanalyses (> 80% purity in all cases; Figure 1B). Lin–c-Kit+ cells from marrow (and spleen) of Gata1low mice were prospectively divided by CD34-CD16/CD32 staining into populations similar to those observed in wild-type tissues (Figure 1B; Table 1) that, once sorted, retained their phenotype on reanalysis (> 88% purity). In contrast, T1/ST2 staining failed to label Lin–c-Kit+ marrow (and spleen) cells from Gata1low mice (Figure 1B; Table 1). To prove that T1/ST2+ cells were indeed absent in Gata1low tissues, Lin–c-Kit+ cells (myeloid progenitors [MPs]) were artificially divided into those in the third lower (MPlow) and third higher (MPhigh) portion of the T1/ST2 profile (Figure 1B). The profile of the 2 populations overlapped on reanalysis (< 60% pure), confirming that T1/ST2+ cells are rare among Gata1low Lin–c-Kit+ cells.
The levels of Gata1 expressed by progenitor cells prospectively isolated from Gata1low and wild-type marrow are compared in Figure 1C. The wild-type cells expressing the highest and lowest Gata1 levels were MEPs (2–  Ct = 0.1 ± 0.05) and CMPs (2–Ct = 0.014 ± 0.006, 1 log lower), respectively. CMPs and MEPs isolated from Gata1low mutants expressed levels of Gata1 lower than those expressed by the corresponding normal cells. Surprisingly, the Gata1low cells in which Gata1 expression was reduced the most were represented by CMPs (2–Ct = 0.10 ± 0.01 x 10–2,  1-log reduction) and not MEPs (2–Ct = 0.023 ± 0.001, 4-fold reduction; Figure 1C). In addition, the expression of Fog1 was similar in wild-type CMPs and MEPs (2–Ct = 0.10-0.11, in both) and decreased by 50% in wild-type MCPs (2–Ct = 0.06 ± 0.01, P < .05). Also Gata1low CMPs and MEPs expressed similar Fog1 levels, but the values were significantly higher than those expressed by wild-type cells (2–Ct = 0.13-0.14, P < .05). In conclusion, whereas MCPs were barely detectable, CMPs and MEPs were identified in hematopoietic tissues from Gata1low mice and expressed levels of Gata1 about 0.5 to 1-log lower and of Fog1 about 50% higher than the corresponding wild-type cells. The altered proliferation and differentiation observed in BMMCs from Gata1low mice is permissive to the establishment of factor-dependent cell lines In BMMCs seeded with either wild-type or W/Wv cells, the cell number increased regularly (by 1 log) during the first 3 weeks to remain constant at later time points (Figure 2A). In contrast, in BMMCs from Gata1low mice, the cell number increased regularly up to the fourth to fifth week and was significantly higher than in wild-type and W/Wv BMMCs after 25 to 30 days (Figure 2A).
In wild-type and W/Wv BMMCs, few (5%-10%) cells expressed the mature c-KithighFc  RI+ mast cell phenotype by day 7 (Figure 2B-C) but almost all (90%-100%) of them were c-KithighFcRI+ by day 21 to 26. In contrast, in BMMCs from Gata1low mice, although many (35%) of the cells were c-Kithigh already by day 7, many (20%) of the c-Kit+ cells remained FcRI– until day 21 to 26 (Figure 2B-C). Differences were observed in level of antigen expression between cells obtained after 26 days in BMMCs from different mouse groups. As expected,32 W/Wv BMMCs expressed less c-Kit and FcRI than the corresponding wild-type cells (mean fluorescence intensity/cell: c-Kit = 31 ± 1 versus 192 ± 22, P < .01, and FcRI = 7 ± 1 versus 18 ± 1, P < .05, respectively). On the other hand, Gata1low BMMCs expressed levels of c-Kit similar to those expressed by normal cells (mean fluorescence intensity/cell = 265 ± 17) but levels of FcRI receptor similar to those expressed by W/Wv cells (mean fluorescence intensity/cell = 8 ± 1). Expression profiling confirmed the immature nature of day 26 Gata-1low BMMCs. In fact, they expressed consistently more Gata1, Gata2, MITF, and MMCP-6 (early marker38) but less MC-CPA (a serosal maturation marker39) than the corresponding wild-type cells (Figure 2D). Interestingly, these cells expressed NFE2, a gene involved in the control of megakaryocytic-erythroid, rather than mastocytic, differentiation40 (Figure 2D). BMMCs from all the mice groups investigated were capable of incorporating and releasing serotonin after IgE- IgE stimulation already by day 7 (Figure 2E). In all the cases, however, maximal levels of serotonin were released by cells obtained by day 21. Those released by Gata1low cells remained significantly lower than those released by the other 2 cell types at all time points (Figure 2E). The proliferation observed after the first 3 weeks in Gata1low BMMCs prompted us to ask whether these cells had become cell lines. To test this hypothesis, Gata1low, and wild-type as control, BMMCs were regularly replenished with fresh medium and growth factors for 2 additional months. Whereas wild-type cultures became extinguished within 1 month, Gata1low cells were maintained and, by the end of the 2 months, cultured as single cells in 96-well plates. One week later, cell proliferation was observed in about 30% of the wells and the cells in 10, randomly selected, wells transferred into flasks. These cultures, once replenished with fresh medium and growth factors, could be regularly passed for 6 additional months. At this time point (9 months from the beginning of the Gata1low BMMCs), the cells in each culture were considered as clonal cell lines. Because similar results were obtained in 2 additional experiments, a total of 30 individual lines were established over the course of the study. Ten of 30 Gata1low BMMC lines were characterized in terms of morphology, growth factor requirement, and antigenic profile, and 3 of them were also analyzed by expression profiling and serotonin-releasing activity. Because results obtained with different clones were similar, only those obtained with the representative SN1 cell line are presented (Figure 3). SN1 cells were large with few cytoplasmic granules reactive with Alcian blue (5%-10%), acetyl cholinesterase A (5%; Figure 3A), and benzidine (0.1%; not shown).
These cells failed to survive in the absence of growth factors (Figure 3B). Few of them (20%-30%) survived in the presence of either SCF, IL-3, or TPO alone. For optimal proliferation, they required the presence of both SCF and IL-3. In their presence, a doubling time of about 24 hours was observed (Figure 3B).
SN1 cells expressed 10 times more surface c-Kit, similar levels of CD34 and Sca1, and lower levels of Fc In conclusion, BMMCs seeded with marrow (and spleen, not shown) cells from Gata1low mice are characterized by increased proliferation and decreased cell differentiation and consistently generate 3-lineage growth factor-dependent cell lines. Gata1low MEPs generate mast cells, and their precursors, in BMMC cultures
The fact that mast cells, although defective, were present in skin and peritoneum of Gata1low mice, as well as in BMMCs seeded with their marrow (Migliaccio et al6 and this report), indicated that the mastocytic activity of Gata1low stem cells was not impaired. The fact that MCPs were not detectable in Gata1low tissues implies, then, that a cell different from an MCP acquired MCP function in these mutants. To identify which of the Gata1low progenitor cells acquired mast cell-generating activity, cells prospectively isolated from tissues of wild-type and Gata1low littermates were cultured in BMMCs and the cultures analyzed 7 days later for presence of mast cells (c-KithighFc
Numerous cells ( 300-500-fold increase with respect to day 0) were generated in 7 days of culture by all of the prospectively isolated progenitor cells (wild-type and Gata1low alike), with the exception of wild-type MPs that grew poorly (Figure 4A). As expected, wild-type MEPs and CMPs generated mostly erythroid and megakaryocytic cells (Table S2 and Suda et al,17 Akashi et al,18 and Nakorn et al34), and few (1%-2%) mast cells and their precursors. These cells represented, instead, more than 7% to 8% of the progeny of wild-type MCPs (Figure 4B; Table S2; Akashi et al18). In contrast, MEPs and CMPs isolated from Gata1low animals consistently generated many (> 20%) c-KithighFcRI+ and c-Kithigh/T1/ST2high cells, in addition to erythroblasts and megakaryocytes (Figure 4B; Table S2).
The mastocytic nature of cells present among the progeny of Gata1low MEPs was confirmed by functional assay (c-KithighT1/ST2high cells; Table S3) and expression profiling (c-KithighFc
On the other hand, c-KithighFc To further confirm that Gata1low MEPs, but not wild-type MEPs, expressed mast cell-generating activity, the progeny of both cells was cultured for additional 14 to 21 days (Figure S1). High numbers of mast cells were consistently detected in cultures seeded with Gata1low MEPs but not in those seeded with the corresponding wild-type cells. In conclusion, Gata1low MEPs generate numerous mature and precursor mast cells in BMMC cultures. Altered differentiation/proliferation potential of single Gata1low MEPs To confirm that Gata1low MEPs generate mast cells and to determine the extent of the abnormal proliferation of their progeny, prospectively isolated Gata1low, and wild-type as controls, MEPs were cultured as single cells, using the limiting dilution protocol. The morphology and recloning potential of their progeny was then evaluated at weekly intervals (Figure 5A).
Approximately 15% of single wild-type MCPs proliferated by day 7, giving rise to about 6 x 103 cells by day 14 of culture (Figure 5B). In all the cases, the progeny had mast cell morphology (Figure 5C). Also 15% of single Gata1low MEPs proliferated under these conditions, generating about 6 x 103 to 105 cells by day 7 to 14 (Figure 5B). The morphology of the Gata1low MEP-derived progeny included mast cells, in addition to megakaryocytes and erythroblasts, by day 7 (Figure 5C). Starting from day 14, the progeny of selected wells was harvested, and recultured again under conditions of limiting dilution at weekly intervals. The replating efficiency of wild-type MCP-derived cells declined over time from 5% at day 14 to undetectable levels at day 21. These cultures were discontinued. In contrast, the number and replating efficiency of the Gata1low MEP-derived progeny increased over time. By the third to fourth passage, each Gata1low cell generated about 8 to 10 x 105 cells/wk, with a cloning efficiency of about 30%. When corrected for the limiting dilution factor (0.3 cells/well), a cloning efficiency of 30% indicates that all the cells had proliferated (Figure 5D; Table S4). The progeny of Gata1low MEPs could be propagated as single cells for at least 4 additional months. The design of the replating experiments allows tracking of the proliferation potential of the progeny of individual MEPs. The tracking indicates that about 11% of single Gata1low MEPs have the potential to generate cells capable of growth with 100% efficiency after 4 passages (Table S4). Ectopic expression of hGATA1 restores the functions of Gata1low MEPs
To prove that the differentiation/proliferation alterations expressed by Gata1low MEPs are a direct consequence of reduced Gata1 expression, we analyzed the biologic properties of MEPs purified from marrow of µLCRhGATA1 Gata1low/0 males (Figure 6). MEPs purified from µLCRhGATA1/Gata1low/0 mice expressed, as those purified from their Gata1low/0 littermates, low levels of mGata1 (Figure 6). These cells, however, also expressed hGATA1 (Figure 6). Once again, MEPs from Gata1low littermates generated many (
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Abstract
Introduction
III/II; PharMingen), and streptavidin-Cy7-PE (Caltag Laboratories, Burlingame, CA). The second aliquot was used for purification of MCPs by staining with FITC-T1/ST2 (BD Biosciences, St. Paul, MN), PE-CD117, and streptavidin-Cy7-PE. Cells labeled with fluorochrome-conjugated isotype antibodies (PharMingen) were used to gate nonspecific signals; dead cells were excluded by propidium iodide staining (5 µg/mL, Sigma, St Louis, MO). Cell analysis and sorting were performed with the FACSAria (Becton Dickinson, San Jose, CA) equipped with 3 air-cooled and solid state lasers (488 nm, 633 nm, and 407 nm). 
), and W/Wv (tight hatched bars) mice. Cells were analyzed either at day 7, 21, or 26 of culture, as indicated on the x-axes. Results are presented as the mean (± SD) of at least 3 independent experiments per group of mice. Values observed in Gata1low BMMCs and statistically different from those obtained in the corresponding cultures with wild-type and W/Wv cells are indicated with a single asterisk (P < .05) and double asterisks (P < .01), respectively. (D). Semiquantitative RT-PCR analysis for the expression of mast cell specific genes (MMCP-6, MC-CPA, and MITF) as well as of Gata1, Gata2, and NFE2 in cells obtained after 26 days in BMMCs seeded with marrow from wild-type, Gata1low, or W/Wv mice, as indicated. The Gata1low c-Kithigh cells were divided into immature (Fc
) and Gata1low MEP (
). Results are presented as mean (± SD) of those obtained in 4 independent experiments per animal group (see also Table S4). (C) May-Grünwald staining of the progeny observed over time in cultures of a representative wild-type MCP and a representative Gata1low MEP, as indicated. At day 7, the progeny of wild-type MCP have an homogeneous mastocytic morphology, whereas megakaryocytes (Mk), erythroblasts (Ery), and mast cells (MC) are present among the progeny of Gata1low MEPs. From 14 days on, only cells with mast cell morphology were detectable in the cultures. The results are representative of those obtained with at least 10 individual wells per experimental point; b.d. = below detectable levels. Original magnification, x 40. Images were obtained using a Zeiss Axiostat Plus microscope equipped with a Zeiss CP-ACHORMAT 40x/0.65 objective lens (Zeiss, Arese, Italy). Photographs were taken with an Olympus Camedia c-5060 digital camera and were acquired with Camedia Master 4.1 software (Opera Zerbo, Milan, Italy). Subsequent image processing was performed with Adobe Photoshop 6.0 software. (D) Total cell number per well and replating efficiency (ie, number of single cells that proliferated in following passages) in single-cell cultures of the progeny of wild-type MCPs (
13%) mast cells (c-KithighFc