2019 Volume 44 Issue 11 Pages 789-797
Mast cells are key players in the inflammatory response with an important role in allergic reactions and are therefore useful for assessing the risk of anaphylaxis. However, they are difficult to isolate due to their low abundance and wide distribution. To overcome this, we generated and characterized mast cell-like cells derived from human induced pluripotent stem (hiPS) cells. These hiPS cell-derived mast cells (hiPS-MCs) were generated using recombinant human bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor 165 (VEGF), stem cell factor (SCF), interleukin-4 (IL-4), interleukin-6 (IL-6), and interleukin-9 (IL-9) in a StemPro-34 medium. The hiPS-MCs exhibited the morphological characteristics of human mast cells, expressing high affinity-IgE receptor (FcεRI) and mast cell markers such as tryptase, chymase, and CD117. In addition, FcεRI stimulation with agonistic anti-IgE functionally increased the expression of activation markers CD63 and CD203c, as well as the amount of released histamine. We think the hiPS-MCs generated in this study will be useful for assessing the pharmacology and toxicity of anti-allergy medicines.
Mast cells have many granules rich in histamine and heparin, and express high affinity-IgE receptors (FcεRI) on their cell surfaces which mediate their immunological activity (Metzger, 1992; Wernersson and Pejler, 2014). When antigens bind to IgE molecules on the surface of mast cells, linkages between IgE receptors are formed and granules or chemical mediators are released (Galli and Tsai, 2012; Metcalfe et al., 1997). This mechanism is fundamental to immediate-type allergic reactions and asthma (Amin, 2012; Pawankar, 1999; Hu et al., 2018; Platts-Mills, 2001).
Anaphylaxis is a severe and life-threatening hypersensitivity reaction, the incidence of which has been continuously rising worldwide (Ben-Shoshan and Clarke, 2011). Any medication or biological agent can potentially trigger anaphylaxis, even well-known drugs like intramuscular penicillin—which continues to be used for rheumatic fever—and non-steroidal anti-inflammatory drugs (NSAIDs) can trigger it (Bhattacharya, 2010; Renaudin et al., 2013; Simons et al., 2011). Asthma is a chronic inflammatory disease of the airways strongly associated with elevated serum IgE (Sears et al., 1991; Burrows et al., 1995). Because mast cells activated by IgE release chemical mediators that include histamine, they have been recognized as effector cells for early and late asthmatic reactions. In addition, it is reported that mast cells migrate into other structures like airway epithelium, mucous glands, and airway smooth muscle in patients with asthma (Reuter et al., 2010).
To assess drug-induced anaphylaxis, diagnose allergic disease, and predict the pharmacology and toxicity of anti-allergic medicines, a histamine release test (HRT) using peripheral blood basophils and a basophil activation test (BAT) were developed (Pineda et al., 2015; Kim et al., 2016; Kneidinger et al., 2008). However, these tests have a false-negative risk due to the type of drug or drug metabolite, and approximately 15% of basophils were found to unresponsive to IgE-mediated stimulation (Nguyen et al., 1990; Kim et al., 2016; Pineda et al., 2015). Therefore, to better predict the pharmacology and toxicity of anti-allergic medicines, it is important conduct investigations using biologically active human mast cells in the pre-clinical stage (Holgate, 2007). However, mast cells are difficult to isolate due to their low abundance and wide distribution in a variety of tissues (Janssens et al., 2005). Because it is so difficult to obtain human mast cells with high purity in sufficient numbers, a lot of studies rely on rodent mast cells such as rat peritoneal mast cells or mouse bone marrow-derived cultured mast cells (Pearce and Thompson, 1986; Mousli et al., 1989; Eklund et al., 1994), human cord blood-derived mast cells (Braselmann et al., 2006), mouse (Sugiyama et al., 2008; Tsai et al., 2000, 2002) and human (Kovarova et al., 2010) embryonic stem (ES) cell-derived mast cells, and mouse induced pluripotent stem (miPS) cell-derived mast cells (Yamaguchi et al., 2013). However, in most cases, there are some issues. In the case of rodent cells, they are sometimes not suitable because mast cells are heterogeneous and there are species differences in cell function (Moon et al., 2010). In the case of human cord blood-derived mast cells, they are sometimes not ideal because of low sensitivity to activation signals (Andersen et al., 2008). To resolve these issues, the generation of mast cells from human induced pluripotent stem (hiPS) cells has investigated (Igarashi et al., 2018). Although we can get the cells maintained a certain quality repeatedly in the case of the generation from hiPS cells, there are still some issues in terms of sensitivity.
In this study, we generated mast cell-like cells from hiPS cells by the method different from previous studies. Mast cell-like cells generated in this study were characteristically similar to human mast cells and were more sensitive than mast cells from routine sources, thus we think these cells will be useful for the diagnosis of allergic disease and anaphylaxis, as well as the in vitro evaluation of anti-allergic medicines.
The present study complied with the “Ethical Guidelines for Research that Uses Human-derived Test Material” promulgated in Chugai Pharmaceutical Co., Ltd. and was approved by the company’s Research Ethics Committee.
Cell cultureIn this study, we used the hiPS cell line 201B7 from Kyoto University (Takahashi et al., 2007), which was generated by retroviral transduction of human fibroblasts with Oct3/4, Sox2, Klf4, and c-Myc. The undifferentiated hiPS cell colonies were cultured with mitomycin C–inactivated SNL 76/7 (SNL) feeder cells (Cell Biolabs, Inc., San Diego, CA, USA) in Primate ES cell medium (ReproCELL Inc., Kanagawa, Japan) supplemented with 4 ng/mL of basic fibroblast growth factor (Wako Pure Chemical Industries, Ltd., Osaka, Japan). The medium was replaced with a fresh one every day and passaged every 7 days.
Differentiating hiPS cells into mast cellsAt first, Matrigel (MG), growth-factor reduced (BD Biosciences, San Jose, CA, USA) and diluted 40-fold with MEM Alpha 1x + Glutamax I (Life Technologies [Thermo Fisher Scientific] Inc., Waltham, MA, USA) was added in an amount of 2 mL to each 60-mm dish and left at 37°C for 12 to 72 hr under 5% CO2 conditions to prepare a MG dish. Gelatin from porcine skin (Sigma-Aldrich Corp., Taufkirchen, Germany) diluted to 0.1% with distilled water was prepared in a solid form by warming, added in an amount of 2 mL to each 60-mm dish, and left at 37°C for 30 to 180 min under 5% CO2 conditions to prepare a gelatin-coated dish. The procedure for inducing the differentiation of hiPS cells into mast cells is composed of three steps.
Step 1: For undifferentiated colonies of hiPS cells, 2 U/mL of neutral protease, grade I (Roche Applied Science, Indianapolis, IN, USA), was added to each dish, and the feeder cells were removed first from the dishes. Next, a colony of the undifferentiated hiPS cells was removed from each dish using a scraper, suspended in MEM Alpha 1x + Glutamax I supplemented with 20% fetal bovine serum, embryonic stem cell-qualified (FBS) (Life Technologies [Thermo Fisher Scientific] Inc.), 2 mM L-glutamine (Life Technologies [Thermo Fisher Scientific] Inc.), 50 U/mL penicillin and 50 μg/mL streptomycin (Life Technologies [Thermo Fisher Scientific] Inc.), and 55 μM 2-mercaptoethanol (Life Technologies [Thermo Fisher Scientific] Inc.); this was seeded in each gelatin-coated dish from which the supernatant had been removed, and cultured at 37°C for 1 hr under 5% CO2 conditions so that the feeder cells were attached to the dish bottom and separated from unattached hiPS cell colonies. The unattached hiPS colonies were suspended in Primate ES cell medium supplemented with 1% insulin-transferrin-selenium-X 100X (ITS; Life Technologies [Thermo Fisher Scientific] Inc.), inoculated to each MG, and cultured at 37°C for 1 day under 5% CO2 conditions.
Step 2: After removing all of the medium from the dish, StemPro-34 (SP34) medium (Life Technologies [Thermo Fisher Scientific] Inc.) supplemented with 1% ITS and 50 ng/mL recombinant human bone morphogenetic protein 4 (rhBMP4; HumanZyme, Inc., Chicago, MI, USA) was added to each dish, and cultured at 37°C for 4 days under 5% CO2 conditions. After removing all of the medium, SP34 medium supplemented with 1% ITS, 40 ng/mL recombinant human vascular endothelial growth factor 165 (rhVEGF; R&D Systems, Inc., Minneapolis, MN, USA), and 50 ng/mL recombinant human stem cell factor (rhSCF; R&D Systems, Inc.) was added to each dish, and cultured at 37°C for 2 days under 5% CO2 conditions.
Step 3: To generate mast cells, adherent cells in the dishes from step 2 were cultured in SP34 medium containing 1% ITS, 50 ng/mL rhSCF, 50 ng/mL recombinant human interleukin-3 (rhIL-3; HumanZyme, Inc.), and 50 ng/mL recombinant human interleukin-6 (rhIL-6; HumanZyme, Inc.) for 30-40 days under 5% CO2 conditions during which the culture solution was replaced with a fresh one every 3 to 4 days. After removing all of the medium, SP34 medium supplemented with 1% ITS, 50 ng/mL rhSCF, 50 ng/mL rhIL-3, 50 ng/mL rhIL-6, and 10 ng/mL recombinant human interleukin-9 (rhIL-9; HumanZyme, Inc.) was added to each dish, and cultured at 37°C under 5% CO2 conditions during which the culture solution was replaced with a fresh one and non-adherent cells were restored every 3 to 4 days. Non-adherent cells were harvested from the dish at a frequency of once every 7 to 14 days and used as hiPS cell-derived mast cells (hiPS-MCs).
Maturation of hiPS cell-derived mast cellsThe harvested hiPS-MCs were suspended in SP34 medium supplemented with 1% ITS, 50 ng/mL rhSCF, 10 ng/mL rhIL-6, 10 ng/mL recombinant human interleukin-4 (rhlL-4; HumanZyme, Inc.), and 0.2 μg/mL human IgE myeloma (hIgE; Merck Millipore Corp., Darmstadt, Germany) seeded in a 6-well plate, and cultured at 37°C under 5% CO2 conditions during which the culture solution was replaced with a fresh one every 3 to 4 days. Non-adherent cells were harvested from the plate at a frequency of once every 7 to 14 days and used as matured hiPS-MCs.
May-Grunwald-Giemsa stainingCytospin smear preparations of hiPS-MCs were made with Cytospin 4 Cytocentrifuge (Thermo Fisher Scientific Inc.). May-Grunwald-Giemsa staining was performed automatically using SP-10 (Sysmex, Hyogo, Japan).
Immunohistochemistry stainingCytospin smear preparations of hiPS-MCs were made in the same way as in May-Grunwald-Giemsa staining. For detection of mast cell markers tryptase and chymase, the preparations were blocked in 2.5% hose serum (Vector Laboratories, Burlingame, CA, USA) for 25 min, incubated with mouse anti-tryptase and anti-chymase antibodies (Both from Merck Millipore Corp.) overnight at 4°C, washed, and incubated with horseradish peroxidase-labeled (HRP) anti-mouse IgG antibody (Vector Laboratories) for 40 min. Peroxidase staining was done using 3,3’-Diaminobenzidine (DAB) and H2O2.
Flow cytometryThe following anti-human antibodies were used: CD63-FITC, CD117-PE, CD123-PerCP Cy5.5, CD203c-BV421 (all from BD Biosciences); FcεR-PE-Cy7 (BioLegend, San Diego, CA, USA). Tryptase and chymase (both from Abcam, Cambridge, MA, USA) were labeled with the Alexa Flour 488 or Alexa Flour 647 Antibody Labeling Kit (Thermo Fisher Scientific Inc.). As isotype-matched controls, rabbit IgG and mouse IgG (both from Abcam) were used and were also labeled with the Alexa Flour 488 or Alexa Flour 647 Antibody Labeling Kit. The cell samples were stained with fluorochrome-conjugated antibodies (CD63-FITC, CD117-PE, CD123-PerCP Cy5.5, CD203c-BV421, or FcεR-PE-Cy7) for 30 min and washed twice with phosphate-buffered saline supplemented with 0.5% bovine serum albumin (BSA; Rockland Immunochemicals, Gilbertsville, PA, USA). Then, cell samples were fixed using Transcription Factor Fixation/Permeabilization (Invitrogen [Thermo Fisher Scientific] Inc., Waltham, MA, USA) and washed three times. After that, cell samples were stained with the labeled antibodies we prepared (tryptase and chymase) and washed three times with Perm buffer and phosphate-buffered saline supplemented with 0.5% BSA. The stained cell samples were analyzed on a flow cytometer (FACSCanto II, BD Biosciences).
Histamine release assayMatured hiPS-MCs were pre-incubated with anti-IgE (0-100 ng/mL) at 37°C for 10 min. The cells were removed from the supernatant by centrifugation, and histamine in the supernatant was measured using a histamine EIA kit (SPI-BIO [Bertin Bioreagent], Bretonneux, France). To determine total amounts of cellular histamine contents, hiPS-MCs were lysed in PBS containing 0.01% Triton-X, and the histamine was measured using a histamine EIA kit.
A scheme for obtaining mast cell-like cells by differentiating hiPS cells is shown in Fig. 1. This method generated hiPS-MCs with the characteristics of mast cells after 90 days of differentiation. Matured hiPS-MCs were obtained by adding several cytokines—SCF, IL-6, IL-4 and IgE—to hiPS-MCs. Under our culture conditions, hiPSC-MCs lines could be maintained for at least 2 months without losing their MC phenotype and function. Approximately 4.0 × 106 mast cells/60-mm diameter culture dish could be obtained in two months.
The scheme for deriving mast-like cells and matured mast-like cells from hiPS cells, 201B7.
At first, we identified the characteristics of hiPS-MCs using May-Grunwald-Giemsa staining and immunohistochemistry. May-Grunwald-Giemsa staining of the hiPS-MCs revealed that generated mast cells gave rise to a phenotype uniform with basophilic granule-containing cells (Fig. 2A). In addition, expression of tryptase and chymase, which are general mast cell markers, was identified by immunohistochemistry staining (Fig. 2B).
Morphological analysis of hiPS-MCs. (A) Smears stained with May-Grunwald Giemsa stain in hiPS-MCs. Rough basophilic granule-containing cells. (B) Immunohistochemical detection of tryptase- and chymase-positive cells in hiPS-MCs.
We next performed flow cytometric analysis to examine the surface expression of mast cell markers on matured hiPS-MCs. The differentiated cells expressed tryptase, chymase, CD117 and FcεRI (Fig. 3). However, CD123, a general marker of basophils or macrophages not expressed in mast cells, was also not expressed on the differentiated cells (Fig. 3), which identified them as mast cells. Mast cell marker-positive cells reached 80-90% of the total nonadherent cells at day 40.
Surface expression profiling of matured hiPS-Mcs. (A) hiPSMCs were stained with Alexa Flour 488-labeled anti-tryptase (red line in upper left histogram) and Alexa Flour 647-labeled anti-chymase (red line in upper right histogram). Rabbit and mouse IgG were stained as isotype-matched controls (blue line in upper histogram). Black line shows unstained control. The expression of tryptase and chymase was observed in matured hiPS-MCs. (B) hiPS-MCs were stained with PE-labeled CD117 (red line in bottom left histogram), PE-Cy7-labeled FcεRI (red line in bottom center histogram), and PerCP Cy5.5-labeled CD123 (red line in bottom right histogram). Black line shows unstained control. The expression of CD117 and FcεR was observed, but not CD123.
To analyze the function of matured hiPSC-MCs, we examined their responsiveness to IgE-FcεRI stimulation with an agonistic anti-IgE using flow cytometry and CD63 and CD203c as mast cell activation markers. We observed increases in the mean fluorescence intensity of CD63 and CD203c (Fig. 4).
Effect of agonistic anti-IgE stimulation on CD63 (red line in left histogram) and CD203c (red line in right histogram). Black line shows unstained control. Increase of expression of CD63 and CD203c was observed.
We next investigated the reaction sensitivity of matured hiPS-MCs. Agonistic anti-IgE increased the expression of CD63 and CD203c at 0.001 μg/mL (Fig. 5A). Furthermore, anti-IgE mediated histamine releases were observed at the same concentration as activation markers (Fig. 5B), and expression of activation markers and amount of released histamine were increased by anti-IgE stimulation in a concentration-dependent manner.
Anti-IgE concentration-dependent increases in expression of activation markers and amount of histamine released. (A) Anti-IgE stimulation increased CD63 (red line in left histogram) and CD203c (red line in right histogram) expression at 0.001 μg/mL. Black line shows unstained control. (B) Anti-IgE stimulation also increased amount of histamine released at 0.001 μg/mL.
In this study, we established a mast cell differentiation procedure consisting of mesoderm induction, mast cell lineage differentiation, and the maturation of mast cells from hiPS cells. We confirmed that they exhibited the morphological characteristics of human mast cells. In addition, we confirmed that they express tryptase, chymase, CD117, and FcεRI, and that IgE-FcεRI stimulation with an agonistic anti-IgE increased the expression of activation marker and amount of released histamine. Our results show these hiPS-MCs exhibited not only the morphology and phenotypes of mast cells, but also their functional characteristics.
Two types of mast cells have been reported in human tissues (Wernersson and Pejler, 2014; Irani et al., 1986). Mast cells on mucosal surfaces contain tryptase but not chymase, and they are located in places such as the intestinal mucosa and the lung alveolar wall. In contrast, mast cells in connective tissue contain tryptase and chymase and are mainly located in the intestinal submucosa and the skin (Irani et al., 1989). Our results show both tryptase and chymase expression, suggesting that the hiPS-MCs generated in this study can be classified as the connective tissue type.
In our culture condition, we obtained a of total 4.0 × 106 mast cells (1.0 × 106 mast cells per 2-weeks) from one 60-mm diameter culture dish possessing mast cell phenotype and function. Mast cells are often used as activation assays to predict the pharmacology and toxicity of medicines and to diagnose allergic diseases and anaphylaxis (Bahri et al., 2018; Weaver et al., 2015). In general, these assays require 5000 to 10000 cells per test (Kuehn et al., 2010). This makes it difficult to assess many allergens at the same time since only around 105 to 106 stem cells per batch can be isolated from cord blood cells or peripheral blood cells (Kato and Radbruch, 1993), and it is difficult to sufficiently expand the isolated cells. In our procedure, we could perform around 100 tests harvesting from single culture dish at one time (we can perform a total of 400 tests), which is sufficient for mast cell activation assays. Thus, by increasing the culture size or repeating the derivation from hiPS cells, we attained hiPS-MCs effective for predicting the pharmacology and toxicity of anti-allergy medicines, and for the consistent diagnosis of allergic disease and anaphylaxis.
In this study, we found that matured hiPS-MCs are activated by FcεRI stimulation with an agonistic anti-IgE through the crosslinking of IgE bound to FcεRI. In contrast, we confirmed that hiPS-MCs are not activated by anti-IgE if the hiPS-MCs are matured without IgE in the culture medium during maturation period (data not shown). This result show that the FcεRI stimulation of hiPS-MCs induced a reaction based on the IgE-FcεRI interaction. IgE in the culture medium during maturation period influences the release of the histamine by anti-IgE and the density of FcεRI, and there are correlated with IgE concentration (Frandsen et al., 2013). In addition, surface FcεRI expression level is correlated with incubation time of IgE in the medium (Yamaguchi et al., 1999). Since the reaction of hiPS-MCs is similar to that of mast cells derived from cord or peripheral blood (Hoffmann et al., 2012), hiPS-MCs can be used in assays to assess the activation of mast cells.
Agonistic anti-IgE increased the expression of activation markers such as CD63 and CD203c at 1 ng/mL, and concurrently mediated histamine release at the same concentration. It has been reported that mast cells from routine sources, including human mast cell lines and cord blood-derived mast cells, are activated from around 1 μg/mL anti-IgE (Saleh et al., 2014; Yamaguchi et al., 1999) and mast cells from hiPS cells are activated from around 0.3 μg/mL (Igarashi et al., 2018). Similarly, human basophils are activated from around 0.3 μg/mL anti-IgE (Chirumbolo et al., 2008). In comparison, hiPS-MCs were approximately 300-1000 times more sensitive to activation and histamine release. Therefore, hiPS-MCs can be used to detect allergen-specific biologically active IgE even in subjects with low levels of a specific IgE. To prove high sensitivity or usefulness of hiPS-MCs generated in this study, further investigations including comparison of functions with routine sources are needed
Mast cells have various physiological functions, including vasodilation, angiogenesis, bacterial, and parasite elimination although IgE-mediated allergic reactions through the FcεRI receptor are the main mechanism of mast cells (Amin, 2012; Krystel-Whittemore et al., 2016). To regulate the functions of many organs and tissues, mast cells generate and release a lot of molecules, such as histamine, proteases, leukotrienes, heparin, and many cytokines, chemokines, and growth factors. The functions of hiPS-MCs we investigated in this study were focused on a part of the function of human mast cells. Therefore, further studies such as cytokine production are required in order to identify the overall functions of hiPS-MCs,
In conclusion, we successfully developed a differentiation procedure for generating mature mast cells from hiPS cells. The hiPS-MCs generated in this study shared many of the same characteristics as human mast cells—especially in activation by FcεRI stimulation—and were more sensitive than mast cells from routine sources. We think the hiPS-MCs generated in this study will be useful for assessing the pharmacology and toxicity of anti-allergy medicines.
We would like to thank both Toshiko Hara and Jumpei Kiyokawa for kindly maintaining the hiPS cells.
Conflict of interestThe authors declare that there is no conflict of interest.
