Generation of mast cells from mouse fetus: analysis of differentiation and functionality, and transcriptome profiling using next generation sequencer

Abstract

While gene knockout technology can reveal the roles of proteins in cellular functions, including in mast cells, fetal death due to gene manipulation frequently interrupts experimental analysis. We generated mast cells from mouse fetal liver (FLMC), and compared the fundamental functions of FLMC with those of bone marrow-derived mouse mast cells (BMMC). Under electron microscopy, numerous small and electron-dense granules were observed in FLMC. In FLMC, the expression levels of a subunit of the FcεRI receptor and degranulation by IgE cross-linking were comparable with BMMC. By flow cytometry we observed surface expression of c-Kit prior to that of FcεRI on FLMC, although on BMMC the expression of c-Kit came after FcεRI. The surface expression levels of Sca-1 and c-Kit, a marker of putative mast cell precursors, were slightly different between bone marrow cells and fetal liver cells, suggesting that differentiation stage or cell type are not necessarily equivalent between both lineages. Moreover, this indicates that phenotypically similar mast cells may not have undergone an identical process of differentiation. By comprehensive analysis using the next generation sequencer, the same frequency of gene expression was observed for 98.6% of all transcripts in both cell types. These results indicate that FLMC could represent a new and useful tool for exploring mast cell differentiation, and may help to elucidate the roles of individual proteins in the function of mast cells where gene manipulation can induce embryonic lethality in the mid to late stages of pregnancy.

Figure 1. Surface expression of Fc epsilon RI and c-Kit on FLMC and BMMC.

BMMC and FLMC were stained with FITC-conjugated anti-FcεRI Ab and PE-conjugated anti c-Kit Ab, and analyzed by flow cytometry. The live cells, with a rate of over 95% mast cells, were gated and 14,000 cells/sample were analyzed, and the expression of both c-Kit and FcεRI was observed. The surface expression of c-Kit occurred prior to the surface expression of FcεRI on FLMCs, although the surface expression of c-Kit on BMMC came later than FcεRI expression. At 6 weeks culture, the proportion of FcεRI-c-Kit double-positive cells was about 90% in FMLC (A). The results are representative of three independent experiments (A). The number of FcεRI-c-Kit double-positive cells increased sharply from day 14 in both BMMC and FLMC. However, the rate of increase was greater in BMMC than in FLMC. The bar graph expresses mean ± S.E of three independent experiments (B).

Figure 2. Comparison between characteristics of putative mast cell progenitors in BMMC and FLMC.

The cells were freshly isolated from bone marrow or fetal liver, and suspended in culture medium as described in Materials and Methods. The numbers of cells were counted using a counting chamber (A). These cells were labeled with antibodies against lineage markers (CD5, B220, CD11b, Gr-1, 7–4, Ter-119), and fractionated with a magnetic cell sorting kit. The effluents were collected as lineage negative (lin^-) cells, and the numbers of lin^- cells were counted using a counting chamber (B). The lin- cells were then stained with both PE-conjugated anti-c-Kit antibody and FITC-conjugated anti-Sca-1 antibody, or their respective isotype control IgG. 31.9% of c-Kit^dim-Sca-1^- cells were observed in lin^- cells from bone marrow (D), and 47% of c-Kit^dim-Sca-1^- cells were observed in lin^- cells from fetal liver (F). The isotype control-stained lin^- cells from bone marrow and from fetal liver are indicated in panels (C) and (E). The bar graphs express mean ± S.E of three independent experiments, and the histograms are representative of three independent experiments.

Figure 3. Comparison of properties of FLMC with BMMC.

Mouse mast cell protease (mMCP) mRNA expression was assessed by RT-PCR (A). All but mMCP-1 mRNA expression was detected both on FLMC and BMMC. Chymase and tryptase expression was detected by Western blotting (B). Both mast cell types express tryptase although chymase was observed in neither FLMC nor BMMC. The results are representative of three independent experiments. Histamine content was measured by HPLC (C). Histamine content was almost the same in both FLMC and BMMC, and the amount of histamine contained in both FLMC and BMMC was about 0.8 pg/cell. Data represent the mean ± SEM of three to five experiments. The components of FcεRI in FLMC were compared with BMMC (D). As with BMMC, FcεRI on FLMC had both a beta chain and a gamma chain. The results are representative of three independent experiments. FLMC and BMMC were stained by both safranin O and alcian blue (E). The granules in mast cells were densely stained by alcian blue; these granules were lightly but not densely stained by safranin O compared with mast cells isolated from peritoneal fluid of C57BL/6 mice. The representative photos are 5-week cultured. The results are representative of three independent experiments.

Figure 4. TEM of FLMC and BMMC.

FLMC and BMMC were observed by transmission electron microscopy. The black arrow indicates mast cell granules and the white arrow indicates mitochondria. Both mast cells had a lot of microvilli on surface of the cells. The nucleus in both cells resembled the shape of that in mononuclear cells, but not in polymorphonuclear leucocytes. The granules in FLMC and BMMC were both slightly smaller and the electron density was not so high; these characteristics appeared to be similar to that of mucosal-type mast cells. The electron density of the granules in FLMC was lower than in BMMC. The photographs are representative of fifty or more photographs of BMMC or FLMC.

Figure 5. Degranulation ratio, histamine release and cytokine generation from FLMC and BMMC.

Both FLMC and BMMC were stimulated by DNP-HSA following sensitization of anti-DNP IgE as described in Materials and Methods. Degranulation caused by FcεRI cross-linking was measured by β-hexosaminidase assay (A). Histamine release by FcεRI stimulation was measured by HPLC (B). Degranulation ratio and histamine release were almost the same in both FLMC and BMMC. Cytokine generation after the stimulation was measured by flow cytomix (C–L). Although IL-6 production (G) in FLMC was significantly larger than that in BMMC, little IL-17 (I) and IFN-γ (J) was generated in FLMC. Production of the other cytokines showed no significant difference between FLMC and BMMC. *: p<0.05, **: p<0.01 vs. no added IgE of each cells. #: p<0.05, ##: p<0.01 vs. IgE stimulated BMMC. Data represent the mean ± SEM of three to six experiments.

Figure 6. Comparison of genes expressed by FLMC and BMMC.

The expression levels of 28,800 pairs of identical genes were compared. The Y axis represents transcript level on FLMC, and the X axis indicates those on BMMC. The square value of Pearson product-moment correlation coefficient was 0.969, indicating that these expression levels showed a strong correlation.

Figure 7. mRNA expression comparison between BMMC and FLMC.

mRNA expression showing statistically significant differences between BMMC and FLMC are displayed as a heat map colored according to the expression level.