Cultures of Human Skin Mast Cells, an Attractive In Vitro Model for Studies of Human Mast Cell Biology

Abstract

Studies of mast cell biology are dependent on relevant and validated in vitro models. Here, we present detailed information concerning the phenotype of both freshly isolated human skin mast cells (MCs) and of in vitro cultures of these cells that were obtained by analyzing their total transcriptome. Transcript levels of MC-related granule proteins and transcription factors were found to be remarkably stable over a 3-week culture period. Relatively modest changes were also seen for important cell surface receptors including the high-affinity receptor for IgE, FCER1A, the low-affinity receptor for IgG, FCGR2A, and the receptor for stem cell factor, KIT. FCGR2A was the only Fc receptor for IgG expressed by these cells. The IgE receptor increased by 2-5-fold and an approximately 10-fold reduction in the expression of FCGR2A was observed most likely due to the cytokines, SCF and IL-4, used for expanding the cells. Comparisons of the present transcriptome against previously reported transcriptomes of mouse peritoneal MCs and mouse bone marrow-derived MCs (BMMCs) revealed both similarities and major differences. Strikingly, cathepsin G was the most highly expressed granule protease in human skin MCs, in contrast to the almost total absence of this protease in both mouse MCs. Transcript levels for the majority of cell surface receptors were also very low compared to the granule proteases in both mouse and human MCs, with a difference of almost two orders of magnitude. An almost total absence of T-cell granzymes was observed in human skin MCs, indicating that granzymes have no or only a minor role in human MC biology. Ex vivo skin MCs expressed high levels of selective immediate early genes and transcripts of heat shock proteins. In validation experiments, we determined that this expression was an inherent property of the cells and not the result of the isolation process. Three to four weeks in culture results in an induction of cell growth-related genes accompanying their expansion by 6-10-fold, which increases the number of cells for in vitro experiments. Collectively, we show that cultured human skin MCs resemble their ex vivo equivalents in many respects and are a more relevant in vitro model compared to mouse BMMCs for studies of MC biology, in particular human MC biology.

Keywords: Fc receptors; IgE; cathepsin G; chymase; granule proteases; heparin; leukotrienes; mast cells; prostaglandins; tryptase.

Figure 1

A schematic presentation of the experiment.

Figure 2

Expression of IEGs/HSPs quickly returns to baseline after FcεRI stimulation but not after purification of skin MCs. (A) Cultured skin-derived MCs were stimulated by FcεRI aggregation for the indicated times and expression of the respective genes was quantified by RT-qPCR and normalized to unstimulated control. N ≥ 7. (B) Skin MCs were kept in standard medium, 10%FCS, and no growth factors at 37 °C for 1 and 2 d following isolation, as described in methods. Cultured MCs were used after around 3 weeks in culture. Gene expression was studied as in (A). n ≥ 4. Normalized against 4 HKGs. The p-values are given directly in the figure.

Figure 3

Conditions applied during isolation do not (efficiently) induce expression of IEGs/HSPs. Cultured skin MCs were exposed to digestive enzymes with/without additional shaking or kept in normal culture conditions (“cultured”). Expression of the respective genes in cultured skin MCs (exposed/non-exposed) and in freshly isolated skin MCs (ex vivo) was quantified by RT-qPCR and normalized to the control (black column). N = 2.

Figure 4

A summary of the major granule proteases of this transcriptional analysis of human skin MCs. EV stands for ex vivo or freshly isolated cells, CF for cultured female cells, and CM for cultured male cells.

Figure 5

A summary of a few key genes of this transcriptional analysis of human skin MCs including Fc and MRGPRX2 receptors, growth factor receptors, histamine and leukotriene synthesis enzymes and transcription factors. EV stands for ex vivo or freshly isolated cells, CF for cultured female cells, and CM for cultured male cells.