Abstract
Previous studies have shown that mouse bone marrow cells can produce mast cells when stimulated in vitro by stem cell factor (SCF) and interleukin-3 (IL-3). Experiments to define the marrow cells able to generate mast cells showed that the most active subpopulations were the Kit+ Sca1– progenitor cell fraction and the more ancestral Kit+ Sca1+ blast colony-forming cell fraction. In clonal cultures, up to 64% of blast colony-forming cells were able to generate mast cells when stimulated by SCF and IL-3, and, of these, the most active were those in the CD34– Flt3R– long-term repopulating cell fraction. Basophils, identified by the monoclonal antibody mMCP-8 to mouse mast cell serine protease-8, were also produced by 50% of blast colony-forming cells with a strong concordance in the production of both cell types by individual blast colony-forming cells. Enriched populations of marrow-derived basophils were shown to generate variable numbers of mast cells after a further incubation with SCF and IL-3. The data extend the repertoire of lineage-committed cells able to be produced by multipotential hematopoietic blast colony-forming cells and show that basophils and mast cells can have common ancestral cells and that basophils can probably generate mast cells at least under defined in vitro conditions.
Mast cells are of major biological importance as key cells in the initiation of many inflammatory or allergic responses because of the numerous bioactive agents in their cytoplasmic granules (1).
Following the purification of the hematopoietic regulator interleukin-3 (IL-3) (2), it was documented that IL-3 stimulation of murine bone marrow cells in vitro could lead to the formation of mast cells (3–5). Puzzlingly, mast cells do not occur in vivo in murine bone marrow and IL-3 production has never been documented to occur in vivo in normal mice (6). Despite this, murine lymphoid cells readily produce IL-3 in vitro when stimulated by mitogens or alloantigens (6). Mast cells do develop in the marrow of mice transplanted with marrow cells or leukemic cells producing excessive amounts of IL-3 (7, 8). Stem cell factor (SCF) was subsequently characterized and shown also to be able to stimulate mast cell production in vitro by marrow cells (9). More significantly, SCF has also been shown to be necessary in vivo for the production of mature tissue-type mast cells (10).
Mast cells generated in vitro from mouse bone marrow are immature but mature to become tissue mast cells after locating in appropriate tissues (11). Although the bone marrow is the logical source of new mast cell production and committed mast cell precursors have been identified in the marrow (12), it is not well documented which less mature cells in the marrow generate such committed mast cell precursors. Candidates for the most immature cell type initiating mast cell production are the multipotential hematopoietic stem cell, the colony-forming unit–spleen (CFU-S), and the blast colony-forming cell. In this regard, CFU-S have been shown to produce progeny that contain cells able to form mast cells in vivo (13).
The most immature hematopoietic cells able to be cultured clonally in vitro, i.e., the blast colony-forming cells in murine marrow and spleen, are likely to be the de facto stem cells maintaining basal levels of blood cell formation (14). These blast colony-forming cells can self-generate, form CFU-S, and produce T and B lymphocytes, dendritic cells, immature erythroid precursors, and extensive numbers of committed progenitor cells in the granulocyte, macrophage, eosinophil, and megakaryocytic lineages (14, 15). To possibly extend the repertoire of cells able to be produced by blast colony-forming cells, the present experiments were undertaken to determine whether these cells could also generate mast cells and basophils. To set such data in context, the mast cell-generating capacity of other precursor cells in the marrow was also investigated.
Basophils are present in the bone marrow and have cytoplasmic granules similar to, but smaller and sparser, than those in mast cells (1). Clearly, basophils and mast cells are closely related, but the origin of basophils in relation to the development of mast cells has not been well characterized (16). Basophils appear to have nonredundant functions in vivo (17–19), but common progenitor cells for basophils and mast cells have been described (20). However, in P1 runt-related transcription factor-1 (Runx1)-deficient mice, basophils are severely depleted, but mast cell numbers are normal (21). In the present experiments, the development of basophils from blast colony-forming cells was also monitored to clarify their relationship to mast cells.
Results
Identification of Mast Cells and Basophils.
In cultures of marrow cells with SCF+IL-3 or IL-3 alone, most mast cells were mononuclear cells with bulky cytoplasm and abundant metachromatic granules (Fig. 1A). After 3 wk in culture with these stimuli, all basophils staining with the monoclonal antibody [mouse mast cell serine protease-8 (mMCP-8)] were mononuclear cells with bulky cytoplasm of variable size (Fig. 1B).
Fig. 1.
Blast colony-forming cells generate mast cells and basophils. Eleven-day blast colonies were cultured for 3 wk with SCF+IL-3. (A) A blast colony that generated mast cells (May-Grünwald Giemsa stain). (B) Another blast colony that had formed a subpopulation of basophils (arrows) (mMCP-8 monoclonal antibody, counterstained with hematoxylin).
The mMCP-8 antibody has been reported to be specific for basophils and not to react with mast cells (22, 23). This was verified in the present experiments by numerous instances where the clonal progeny of individual blast colony-forming cells contained one cell type but not the other (see examples in Fig. 2).
Fig. 2.
Individual blast colony-forming cells generate both mast cells and basophils. Analysis of populations cultured from 28 sequential 10-d blast colonies; 17 blast colonies formed mast cells, and, of these, 13 also formed basophils. Note the overall close concordance between the two cell types produced by individual colonies but the lower number of basophils than cells identified as mast cells.
However, in the present study, a histochemical survey of mouse tissues showed that the majority of bone marrow polymorphs and eosinophils also stained positively with this antibody. Cell types that were negative included T and B lymphocytes, nucleated red cells, monocytes, and macrophages. Based on this information, care was taken to exclude granulocytes from counts of basophil numbers. In fact, granulocytes were present in only a few of the cultured populations and were frequently unstained by the antibody.
In some experiments, basophils were enriched in marrow populations by incubation for 1 wk with SCF plus IL-3 (16, 24). FACS fractionation of this population for basophils was performed using selection for CD49b+, Kit−, FcγR3+, IgE+ cells (21) and included, in addition, Gr1− selection to exclude granulocytes. The cells in this presumptively pure basophil population were 100% positive for the mMCP-8 antibody (Fig. 3B) but exhibited a variable nuclear morphology (Fig. 3A). Some had a round or oval nucleus and others a U-shaped or ring-shaped nucleus. Only about one-third of the cells exhibited metachromatic granules, and these granules were small and sparse in number.
Fig. 3.
Highly purified basophils generate mast cells in vitro. After culture for 1 wk with SCF plus IL-3, CD49b+, Kit−, FcγR3+, IgE+, and Gr1− marrow cells were 100% positive for the basophil antibody mMCP-8 (B) but had a varied nuclear morphology when Giemsa-stained (A). After 2 more weeks of culture with SCF plus IL-3, the cells enlarged. Most cells remained positive with the basophil antibody (D), but a significant proportion of the cells had the morphology of mast cells (C). Cells in C and D are from the same well and represent cells with dual characteristics. All photomicrographs of cytocentrifuged cells are at the same magnification.
Generation of Mast Cells in Vitro.
To verify the adequacy of the culture protocol to be used, 104 C57BL marrow cells were cultured for 3 wk in 1-mL wells with either IL-3 alone or IL-3+SCF. Of 24 wells stimulated by IL-3, 22 contained mast cells with a mean percentage of mast cells of 31% ± 27%. Of 24 wells stimulated by IL-3+SCF, all contained mast cells with a mean percentage of mast cells of 62% ± 38%. On this basis, the combination stimulus of IL-3+SCF was used in most subsequent experiments.
To document the primacy of bone marrow as a source of mast cell production, at least in vitro, the performance of 104 cells per well was compared for replicate cultures of bone marrow cells, peritoneal cells, spleen cells, mesenteric lymph node cells, and thymus cells. After 3 wk of incubation with SCF+IL-3, the percentages of wells containing mast cells were the following: bone marrow cells, 100%; peritoneal cells, 83%; spleen cells, 20%; mesenteric lymph node cells, 0%; and thymus cells, 0%. Peritoneal cell populations in vivo contain 1–5% mast cells (25), and it was notable that the mast cells in wells of cultured peritoneal cells were few in number and appeared unhealthy, suggesting that the stimuli were allowing some cell survival but not new cell formation. Although mouse bone marrow does not contain mast cells, the results suggested that bone marrow cells were the major source of newly formed mast cells in vivo.
To identify marrow subpopulations capable of generating mast cells in vitro, varying numbers of subfractionated bone marrow cells were cultured for 3 wk. As shown in Table 1, the data indicated that most cells generating mast cells were lineage– and, of these cells, the most frequent mast cell-generating cells were either Kit+ Sca1– or Kit+ Sca1+, the two fractions containing, respectively, most lineage-committed progenitor cells and most blast colony-forming cells (15).
Table 1.
Bone marrow subpopulations generate mast cells
| Marrow subpopulation | No. of cells per well | % mast cell-containing wells |
| Lineage+ | 10,000 | 8 |
| Lineage– | 10,000 | 100 |
| Lin– Kit+ Sca1– | 500 | 100 |
| Lin– Kit– Sca1– | 500 | 0 |
| Lin– Kit– Sca1+ | 500 | 17 |
| Lin– Kit+ Sca1+ | 500 | 100 |
A total of 12 replicate wells in two separate experiments were used per fraction. In some cases, negative wells contained no living cells.
To explore whether granulocyte–macrophage colonies contained cells able to generate mast cells, 7-d granulocyte and/or macrophage colonies were stimulated to develop using GM-CSF or macrophage colony-stimulating factor (M-CSF) (6) (stimuli unable to stimulate mast cell proliferation) or IL-3 or SCF+IL-6 (the latter two stimuli are able to stimulate mast cell survival and/or proliferation) (6). In the case of the IL-3–stimulated cultures, to avoid possible sampling of mast cell colonies, care was taken to remove and culture only colonies with the obvious single center morphology of granulocytic colonies. None of these 7-d colonies, when removed and cultured in wells containing IL-3 plus SCF, was able to generate mast cells with the exception of two granulocytic colonies from cultures stimulated by SCF+IL-6 where contamination with blast colony cells was possible. In addition, in cultures using GM-CSF or M-CSF, intercolony agar in volumes corresponding to those removed during colony transfer were also removed and assayed for their capacity to generate mast cells but all failed to do so.
Attention was then concentrated on the multicentric blast colonies already known to produce committed progenitor cells in multiple lineages (14, 15). As in previous studies, the stimulus used in the primary cultures was 100 ng SCF plus 100 ng IL-6 to generate the least ambiguous cultures containing only multicentric blast colonies or readily distinguishable granulocytic colonies (14). Previous studies had shown that such blast colonies contained higher numbers of blast colony-forming cells and megakaryocyte-committed progenitors if the culture period was extended from 7 to 11 d (14). Although some blast colonies of all ages were capable of generating mast cells in secondary liquid cultures, the frequency of mast cell-generating colonies rose with colony age: day 3, 1 of 12 (8%); day 4, 4 of 38 (11%); day 5, 3 of 20 (15%); day 7, 22 of 79 (28%); and day 10–11, 20 of 31 (64%). Blast colonies were clearly heterogeneous in their capacity to produce mast cells and this heterogeneity was also apparent in the number of mast cells present in positive wells. As shown by the typical data from day 10 colonies in Fig. 2, the percentage of mast cells in positive liquid cultures usually ranged from 1% to 70% but, less frequently, highly cellular wells were generated in which close to 100% of cells had the morphology of mast cells. The latter wells suggested that a small subset of blast colony-forming cells might be programmed to generate exceptional numbers of mast cells.
Parallel agar cultures of portions of the blast colonies assayed for their ability to generate mast cells failed to document a correlation between an ability to form granulocytic or macrophage progenitors and the ability to form mast cells.
The variability of mast cell-forming capacity between blast colonies raised the possibility that mast cell-generating cells might not be genuine members of the blast colony but be contaminating cells trapped in, or attracted to, the expanding blast colonies. Cytological analysis eliminated the possibility that day 7 or day 10 blast colonies might themselves contain Giemsa-staining mast cells. The possibility that blast colonies might contain contaminating unidentified mast cell-generating cells was made very unlikely by two procedures. First, assays on colony-free agar adjacent to blast colonies, but using volumes of medium similar to those used in removal blast colonies, failed to detect mast cell-forming activity in 56 attempts using day 7 or day 11 cultures. Second, suspensions of individual day 10 blast colonies were divided into 24 aliquots and then each aliquot was tested for mast cell-generating capacity. Six colonies were tested in this manner, and the positive aliquots were, respectively, the following: 12 of 24; 16 of 24; 24 of 24; 23 of 23; 24 of 24; and 14 of 24 fractions. If mast cell-generating capacity had been due to contaminating cells, the data indicated that an improbable number of contaminants would have had to have entered each colony, which is a very unlikely possibility, particularly as direct assays on medium adjacent to blast colonies failed to detect mast cell-forming cells.
Phenotype of Marrow Cells Forming Blast Colony-Forming Cells Able to Generate Mast Cells.
Multicentric blast colony-forming cells are located mainly in the lineage− Sca1+ Kit+ (LSK) fraction of bone marrow cells (15), the same fraction with a consistent capacity to generate mast cells (Table 1). LSK populations can be further subdivided using CD34 and Flt-3R markers to generate CD34– Flt-3R– long-term repopulating stem cells (LT-HSC), CD34+ Flt-3R– short-term repopulating stem cells, and CD34+ Flt-3R+ multipotential progenitor cells. As shown in Table 2, mast cells were able to be generated by blast colonies from all three LSK subfractions but, with continued incubation to day 10–11, blast colonies formed by LT-HSC fractions were superior in their ability to generate mast cells.
Table 2.
Blast colonies grown from subsets of marrow LSK fractions generate mast cells
| Colonies generating mast cells |
||
| Fraction generating blast colonies | Day 7–8 | Day 10–11 |
| LSK | 8/22 (36%) | 7/22 (32%) |
| MPP | 4/22 (18%) | 7/28 (25%) |
| ST-HSC | 4/25 (16%) | 22/58 (38%) |
| LT-HSC | 5/29 (17%) | 35/61 (57%) |
Data for day 7–8 colonies pooled from two experiments and data for day 10–11 colonies pooled from four experiments are presented. MPP, CD34+ Flt3R+ multipotential progenitor cells; ST-HSC, CD34+ Flt3R− short-term repopulating stem cells.
Phenotype of Mast Cell-Forming Cells Within Blast Colonies.
Pools of day 11 blast colonies were resuspended, filtered free of agar, and then subjected to FACS fractionation. As shown in Table 3, Kit+ Sca1– cells had no capacity to generate mast cells. Conversely, Kit– Sca1+ cells clearly had the highest capacity to generate mast cells. These data are quite contrary to the data in Table 1 on the phenotype of mast cell-forming cells in whole bone marrow and further support the conclusion that the mast cell-forming cells in blast colonies are genuine members of these clones and are not contaminating marrow cells from the adjacent medium.
Table 3.
Subsets of blast colony cells vary in their capacity to produce mast cells and basophils
| Fraction | No. of cells cultured per well | No. of assay wells | % wells with mast cells | % wells with basophils |
| Kit+ Sca1− | 2,000 | 12 | 0 | 18 |
| Kit+ Sca1+ | 580 | 12 | 17 | 17 |
| Kit− Sca1− | 2,000 | 23 | 13 | 22 |
| Kit− Sca1+ | 2,000 | 25 | 68 | 82 |
Fractionated blast colony cells were cultured for 3 wk with SCF plus IL-3. Pooled data from two separate experiments are presented. In some cases, negative wells contained no living cells.
Production of Basophils.
As shown by the typical examples in Fig. 2, when individual 10-d blast cell colonies were cultured with SCF+IL-3 for 3 wk, basophils, identified with the specific monoclonal antibody mMCP-8 (24), developed in up to 50% of the cultures. In general, there was a good concordance in individual colonies between mast cell-forming capacity and basophil-forming capacity, but the frequency of basophils in the progeny of positive colonies was almost always lower than that of mast cells. Furthermore, there were colonies producing very high numbers of mast cells with no detectable basophils, eliminating the possibility that the monoclonal antibody might, in fact, be cross-reactive with mast cells. The data from these clonal cultures documented that up to 50% of blast colony-forming cells could generate both mast cell and basophil progeny, meaning that these two cell types frequently share the same ancestral cell. This extends the earlier data of Arinobu et al. (20) indicating a common cellular origin of both cell populations. Because basophils also contain small numbers of metachromatic granules, their presence in colony progeny means that counts of metachromatic granule-positive cells in Giemsa preparations at times would overestimate mast cell numbers, but the frequency difference between the two cell types indicated that, regardless of this, both cell types were present in the progeny of these colonies.
As shown in Table 3, FACS fractionation of blast cell populations also revealed a close concordance between the ability to form mast cells and basophils, with Kit− Sca1+ cells most often producing both cell types.
The lower content of granules in basophils compared with mast cells suggested that basophils may be a less mature form of mast cell and that some at least might convert to mast cells on continued incubation in vitro. Marrow cells were cultured with 0.5 ng/mL SCF plus 0.5 ng/mL IL-3 for 1 wk to develop an increased population of basophils (16, 24).
From this population, FACS sorting produced a small population of cells that were CD49b+, Kit–, FcγR3+, IgE+, and Gr1–. These cells were 100% positive when stained by the basophil monoclonal antibody (α–anti-mMCP-8) (Fig. 3B) but had a variable morphology in Giemsa-stained preparations (Fig. 3A) with only 33% of cells having metachromatic granules. No cells had the morphology of mast cells. Twelve aliquots of 5 × 103 cells were cultured for 2 more weeks with SCF+IL-3. After this time, average cell counts were 3.5 ± 2.3 × 103 cells per well. As shown in Table 4, the composition of wells was quite variable, but 78% ± 34% of the cells were enlarged mononuclear cells that were strongly positive with the mMCP-8 antibody (Fig. 3D) whereas 30% ± 31% of the cells had the unambiguous morphology of mast cells (Fig. 3C). In Table 4, there are clear examples where cells must have exhibited both characteristics (Fig. 3 C and D), which is a situation not seen in vivo. This result was confirmed in several replicate experiments. Although these experiments used cell populations that could not be absolutely proved to be pure basophils, the data very strongly suggest that basophils can generate mast cells at least in vitro, even though some of the resulting mast cells exhibited anomalous dual characteristics.
Table 4.
Purified basophils generate mast cells in vitro
| Well no. | % cells positive with α–mMCP-8 after culture | % mast cells after culture |
| 1 | 100 | 8 |
| 2 | 9 | 20 |
| 3 | 100 | 35 |
| 4 | 100 | 17 |
| 5 | 99 | 100 |
| 6 | 100 | 15 |
| 7 | 34 | 4 |
| 8 | 19 | 2 |
| 9 | 95 | 23 |
| 10 | 89 | 88 |
| 11 | 90 | 12 |
| 12 | 96 | 32 |
Each well contained 5 × 103 CD49b+, Kit–, FCγR+, IgE+, and Gr1− cells, 100% of which positively stained with anti–mMCP-8. The cells were then stimulated for 2 wk in liquid culture by SCF+IL-3. After this incubation, the mean cell counts in the wells were 3.5 ± 2.3 × 103.
Discussion
The present experiments have documented the ability of blast colony-forming cells to generate progeny able to generate mast cells in vitro when stimulated by SCF and IL-3. This is in contrast to the facts that no morphologically identifiable mast cells are present either in murine bone marrow (6) or in blast colonies and that the stimulus used for generating blast colonies included SCF, a factor with known mast cell-stimulating activity in vitro and in vivo (9, 10).
Regrettably, multicentric blast colonies can only be stimulated to develop using combinations including IL-3 or SCF, both of which are agents with direct mast cell-stimulating activity. It was not possible therefore to determine whether the use of these stimuli induced mast cell-forming commitment in some cells in the developing blast colonies or whether this commitment arose by some other mechanism.
The present studies showed that, in tissue culture, bone marrow cells were the primary source of mast cells. Analysis of the capacity of FACS-fractionated marrow to produce these cells showed that most were made by Kit+ Sca1− or Kit+ Sca1+ cells in agreement with the known phenotype of other lineage-committed progenitor cells. However, attention was concentrated on the ancestors of such lineage-committed progenitor cells—the blast colony-forming cells. As previously documented, heterogeneity of blast colony-forming cells was again evident in the present study because only two-thirds of blast colony-forming cells were capable of generating mast cells.
The possibility that blast colonies might have accidentally incorporated nonrelated mast cell-forming cells was made improbable by assaying surrounding agar medium and subdividing individual colonies to document the capacity of each subfraction to produce mast cells. The proposition was made further unlikely by FACS analysis of blast colonies that showed that the cells generating mast cells or basophils were predominantly Kit− cells, the opposite of the situation in marrow populations where mast cell-forming cells were Kit+.
The data from recultured blast colonies indicated that mast cells combined with basophils were formed by only ∼50% of blast colony-forming cells. Notably, however, with such positive colonies, there was a strong concordance in the generation of both cell types although in differing frequencies. This indicated a particularly close lineal relationship between the two cell types in addition to sharing common ancestral blast colony-forming cells.
Because of the similar special granules in both cells, it was tempting to postulate that some of the more lightly granulated basophils might be able to mature to form tissue mast cells in a process comparable, for example, with the manner in which blood monocytes can generate peritoneal macrophages (26).
Basophils have been noted to survive less well in culture than in mast cells (16), but the reason for their early disappearance was not examined. Furthermore, little is known of the fate of basophils in vivo. The present data may provide a clue to explain, in part, the fate of basophils by documenting that at least some basophils can mature to, or generate, mast cells. However, as indicated by the P1 Runx-1–deficient mice where mast cell numbers are normal but basophils are few (21), not all mast cells need to have basophil precursors. Indeed, the present data showed that many blast colony-forming cells can produce mast cells without accompanying basophils.
The fact that basophils have some nonredundant functions (17–19) in no way diminishes the present evidence that basophils and mast cells are a closely linked dual population showing common ancestral blast colony-forming cells. A formal possibility remains that additional unique pathways may exist for the selective production of some mast cells and basophils.
Materials and Methods
Mice.
Mice used were 6- to 8-wk-old C57BL/6 mice of both sexes. The mice had been born and raised under pathogen-free conditions.
Culture Detection of Mast Cells and Basophils.
Test cell populations were cultured in 1-mL wells containing Dulbecco’s modified Eagle’s medium (DMEM) and 10% (vol/vol) FCS in a fully humidified atmosphere of 10% (vol/vol) CO2 in air. In most experiments, the stimulus used for mast cell formation was the superior combination of a final concentration of 10 ng/mL IL-3 and 100 ng/mL SCF, although some experiments were performed using only IL-3 stimulation.
Culture wells were routinely sampled after 3 wk of incubation, and duplicate cytocentrifuge preparations were stained with: (i) May-Grünwald Giemsa and (ii) the basophil-specific mMCP-8 monoclonal antibody (Biolegend) (24). Cultures were recorded as positive for mast cells or basophils if 1% or more of the harvested cells were positive.
Generation of Blast Colonies.
Cultures of 25,000 C57BL bone marrow cells were stimulated to generate multicentric blast colonies using 100 ng/mL SCF plus 100 ng/mL interleukin-6 in agar cultures of DMEM containing 10% FCS and incubated in 10% CO2 in air (14, 15). After 4–14 d of incubation, individual blast colonies were removed under direct vision at ×35 magnification using a fine Pasteur pipette and then resuspended and added to 1-mL vol of medium for liquid culture. Alternatively, pools of individually harvested blast colonies were resuspended in DMEM, filtered to remove agar fragments, and then subjected to antibody labeling and FACS fractionation.
Cell Sorting and Staining.
For agar culture experiments using 7- to 12-wk C57BL bone marrow, single suspension bone marrow cells were stained with biotin-conjugated rat anti-mouse antibodies specific for lineage markers CD4 (GK1.5), CD8 (53-6.7), B220 (682), Gr-1 (8C5), TER119, and Mac1 (M1/70) and were lineage-depleted using antibiotin microbeads in an LS MACS column (Miltenyi Biotec). The lineage-negative population was fractionated using fluorochrome-conjugated antibodies against cKit (2B8), Sca1 (D7), CD34 (RAM34), and Flt3R (CD135 A2F10.1), and the CD34− and CD34+ LSK fractions were sorted by flow cytometry on a FACSAria (BD Biosciences) for primary cultures.
Basophils were sorted using cKit (2B8)-PE.Cy7 (clone 2B8; BD Pharmingen), CD49b (DX5)-PE (clone DX5; eBioscience), IgE-FITC (clone 23G3; eBioscience) Gr1-APC (clone RB6-8C5; BD Pharmingen), and FcγR3 (clone 2.4G2; BD Pharmingen).
Cytocentrifuge preparations were made in duplicate, one slide being stained with May-Grünwald Giemsa and the other being fixed with 4% paraformaldehyde, stained with the specific anti-mouse monoclonal antibody mMCP-8 (24) (Biolegend), and counterstained with hematoxylin.
Acknowledgments
This work was supported by the Carden Fellowship Fund of the Cancer Council, Victoria; the National Health and the Medical Research Council, Canberra (Programs 461219 and 1016647); the National Health and Medical Research Council (Australia) Independent Research Institutes Infrastructure Support Scheme Grant 361646; and a Victorian State Government Operational Infrastructure Support grant. A.P.N. was a recipient of an Australian Postgraduate Award.
Footnotes
The authors declare no conflict of interest.
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