Correspondence: Tatsuya Ikuno (E-mail: ikuno.tatsuya89@chugai-pharm.co.jp)

Human induced pluripotent stem cell-derived mast cells useful for in vitro mast cell activation assay exhibiting

phenotypes and morphological characteristics of human mast cells

Tatsuya Ikuno, Shunsuke Ito and Tomoaki Inoue

Research Division, Chugai Pharmaceutical Co., Ltd., 1-135 Komakado, Gotemba, Shizuoka, 412-8513, Japan

(Received April 22, 2019; Accepted August 1, 2019)

ABSTRACT — Mast cells are key players in the inflammatory response with an important role in aller-gic 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 character-ized mast cell-like cells derived from human induced pluripotent stem (hiPS) cells. These hiPS cell-de-rived 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 mor-phological 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 phar-macology and toxicity of anti-allergy medicines. Key words: Human induced pluripotent stem cells, Human mast cells, High affinitiy-IgE receptor,

Tryptase, Chymase, Histamine

INTRODUCTION

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 hypersen-sitivity reaction, the incidence of which has been con-tinuously rising worldwide (Ben-Shoshan and Clarke, 2011). Any medication or biological agent can poten-tially trigger anaphylaxis, even well-known drugs like intramuscular penicillin—which continues to be used

for rheumatic fever—and non-steroidal anti-inflamma-tory drugs (NSAIDs) can trigger it (Bhattacharya, 2010; Renaudin et al., 2013; Simons et al., 2011). Asthma is a chronic inflammatory disease of the airways strong-ly associated with elevated serum IgE (Sears et al., 1991; Burrows et al., 1995). Because mast cells activat-ed by IgE release chemical mediators that include hista-mine, they have been recognized as effector cells for ear-ly 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 aller-gic disease, and predict the pharmacology and toxicity of anti-allergic medicines, a histamine release test (HRT) using peripheral blood basophils and a basophil activa-tion 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

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or drug metabolite, and approximately 15% of basophils were found to unresponsive to IgE-mediated stimula-tion (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 con-duct 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 abun-dance and wide distribution in a variety of tissues (Jans-sens 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 peri-toneal mast cells or mouse bone marrow-derived cul-tured 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 character-istically similar to human mast cells and were more sen-sitive than mast cells from routine sources, thus we think these cells will be useful for the diagnosis of allergic dis-ease and anaphylaxis, as well as the in vitro evaluation of anti-allergic medicines.

MATERIALS AND METHODS

The present study complied with the “Ethical Guide-lines 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 fibrob-lasts with Oct3/4, Sox2, Klf4, and c-Myc. The undiffer-entiated 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 pre-pare a MG dish. Gelatin from porcine skin (Sigma-Aldrich Corp., Taufkirchen, Germany) diluted to 0.1% with dis-tilled 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 com-posed of three steps.

Step 1: For undifferentiated colonies of hiPS cells, 2 U/mL of neutral protease, grade I (Roche Applied Sci-ence, Indianapolis, IN, USA), was added to each dish, and the feeder cells were removed first from the dish-es. 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 strep-tomycin (Life Technologies [Thermo Fisher Scientif-ic] Inc.), and 55 μM 2-mercaptoethanol (Life Technolo-gies [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% insu-lin-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

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dish, StemPro-34 (SP34) medium (Life Technologies [Thermo Fisher Scientific] Inc.) supplemented with 1% ITS and 50 ng/mL recombinant human bone morpho-genetic protein 4 (rhBMP4; HumanZyme, Inc., Chica-go, MI, USA) was added to each dish, and cultured at 37°C for 4 days under 5% CO2 conditions. After remov-ing all of the medium, SP34 medium supplemented with 1% ITS, 40 ng/mL recombinant human vascular endothe-lial 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 dish-es 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 recom-binant 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 har-vested 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 inter-leukin-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 cul-tured 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 stain-ing. For detection of mast cell markers tryptase and chy-mase, the preparations were blocked in 2.5% hose serum (Vector Laboratories, Burlingame, CA, USA) for 25 min, incubated with mouse anti-tryptase and anti-chy-mase antibodies (Both from Merck Millipore Corp.) over-night at 4°C, washed, and incubated with horseradish per-oxidase-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 sam-ples 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 Immunochem-icals, 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 sam-ples were stained with the labeled antibodies we pre-pared (tryptase and chymase) and washed three times with Perm buffer and phosphate-buffered saline sup-plemented 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 his-tamine in the supernatant was measured using a hista-mine 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 hista-mine EIA kit.

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RESULTS

Generation of mast cells from hiPS cellsA scheme for obtaining mast cell-like cells by differen-

tiating hiPS cells is shown in Fig. 1. This method generat-ed 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.

At first, we identified the characteristics of hiPS-MCs using May-Grunwald-Giemsa staining and immunohisto-chemistry. May-Grunwald-Giemsa staining of the hiPS-MCs revealed that generated mast cells gave rise to a phe-notype uniform with basophilic granule-containing cells (Fig. 2A). In addition, expression of tryptase and chy-mase, which are general mast cell markers, was identified by immunohistochemistry staining (Fig. 2B).

Expression of mast cell markers on matured hiPS cell-derived mast cells

We next performed flow cytometric analysis to exam-ine the surface expression of mast cell markers on

Fig. 1. The scheme for deriving mast-like cells and matured mast-like cells from hiPS cells, 201B7.

Fig. 2. 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.

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matured hiPS-MCs. The differentiated cells expressed tryptase, chymase, CD117 and FcεRI (Fig. 3). Howev-er, 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.

Activation by FcεRI stimulation on matured hiPS cell-derived mast cells

To analyze the function of matured hiPSC-MCs, we examined their responsiveness to IgE-FcεRI stimula-tion 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).

Increases in the expression of activation markers and the amount of histamine release depending on anti-IgE concentration

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). Fur-thermore, 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.

DISCUSSION

In this study, we established a mast cell differentia-tion 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, chy-mase, 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 mor-phology and phenotypes of mast cells, but also their func-

Fig. 3. 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.

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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.

Fig. 5. 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.

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tional 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 intes-tinal 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 tryp-tase and chymase expression, suggesting that the hiPS-MCs generated in this study can be classified as the con-nective 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 anaph-ylaxis (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 aller-gens 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 activa-tion assays. Thus, by increasing the culture size or repeat-ing 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 activat-ed 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 incuba-tion 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 acti-vation 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 generat-ed in this study, further investigations including compari-son of functions with routine sources are needed

Mast cells have various physiological functions, including vasodilation, angiogenesis, bacterial, and para-site 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 differen-tiation 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-aller-gy medicines.

ACKNOWLEDGMENTS

We would like to thank both Toshiko Hara and Jumpei Kiyokawa for kindly maintaining the hiPS cells.

Conflict of interest---- The authors declare that there is no conflict of interest.

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Human iPS cell-derived mast cells useful for activation assay