Development Of Human Mast Cells Research Articles

Wnt-3a Induces Cytokine Release in Human Mast Cells

Mast cells are well known for their detrimental effects in allergies and asthma, and Wnt signaling has recently been implicated in asthma and other airway diseases. However, it is not known if or how Wnts affect human mast cells. Since Wnt expression is elevated in individuals with asthma and is linked to a Th2 profile, we hypothesized that mast cells could be affected by Wnts in the context of asthma. We therefore sought to investigate the role of Wnt signaling in human mast cell development and activation. We first examined the expression of the 10 main Wnt receptors, Frizzled 1–10 (FZD1–10), and found expression of several FZDs in human mast cells. Treatment with purified recombinant Wnt-3a or Wnt-5a did not affect the proliferation or maturation of CD34+ progenitors into mast cells, as indicated by cellular expression of CD117 and FcεRI, activation by FcεRI crosslinking, and histamine and tryptase release. Furthermore, Wnt treatment did not change the phenotype from MCT to MCTC, since MrgX2 expression, compound 48/80-mediated activation, and carboxypeptidase A3 content were not affected. However, Wnt-3a activated WNT/β-catenin signaling in mature human mast cells, as revealed by stabilization of β-catenin, upregulation of IL-8 and CCL8 mRNA expression, and release of IL-8 protein. Thus, our data suggest that Wnt-3a activation of mast cells could contribute to the recruitment of immune cells in conditions associated with increased Wnt-3a expression, such as asthma.

Human Mast Cell Development from Hematopoietic Stem Cells in a Connective Tissue-Equivalent Model.

Mast cells (MCs) play critical roles in the pathogenesis of IgE- and non-IgE-mediated immune responses, as well as host defense against parasites, bacteria, and viruses. Due to the effect of extracellular matrix components on tissue morphogenesis and cell behavior, utilizing a tissue model that mimics MC microenvironmental conditions in vivo has greater relevance for in vitro studies. For this work, MCs were developed within a connective tissue-equivalent model and cell function was examined in response to an allergen. MCs are located in proximity to fibroblasts and endothelial cells (ECs) that play a role in MC development and maturity. Accordingly, MC progenitors isolated from human peripheral blood were co-cultured with human primary fibroblasts in a 3D collagen matrix to represent the connective tissue. The matrix was coated with type IV collagen and fibronectin before seeding with primary human ECs, representing the capillary wall. The stem cell-derived cells demonstrated MC characteristics, including typical MC morphology, and the expression of cytoplasmic granules and phenotypic markers. Also, the generated cells released histamine in IgE-mediated reactions, showing typical MC functional phenotype in an immediate-type allergenic response. The created tissue model is applicable to a variety of research studies and allergy testing. Impact Statement Mast cells (MCs) are key effector and immunoregulatory cells in immune disorders; however, their role is not fully understood. Few studies have investigated human ex vivo MCs in culture, due to the difficulties in isolating large numbers. Our study demonstrates, for the first time, the generation of cells exhibiting MC phenotypic and functional characteristics from hematopoietic stem cells within a connective tissue-equivalent model with ancillary cells. Utilizing the 3D matrix-embedded cells can advance our understanding of MC biological profile and immunoregulatory roles. The tissue model can also be used for studying the mechanism of allergic diseases and other inflammatory disorders.

Development of Human Mast Cells from Hematopoietic Stem Cells within a 3D Collagen Matrix: Effect of Stem Cell Media on Mast Cell Generation.

Mast cells (MCs) arise from hematopoietic stem cells (HSCs) that mature within vascularized tissues. Fibroblasts and endothelial cells (ECs) play a role in the maturation of HSCs in the tissues. Due to difficulties in isolating MCs from tissues, large numbers of committed MC precursors can be generated in 2D culture systems with the use of differentiation factors. Since MCs are tissue-resident cells, the development of a 3D tissue-engineered model with ancillary cells that more closely mimics the 3D in vivo microenvironment has greater relevance for MC studies. The goals of this study were to show that MCs can be derived from HSCs within a 3D matrix and to determine a media to support MCs, fibroblasts, and ECs. The results show that HSCs within a collagen matrix cultured in StemSpan media with serum added at the last week yielded a greater number of c-kit+ cells and a greater amount of histamine granules compared to other media tested. Media supplemented with serum were necessary for EC survival, while fibroblasts survived irrespective of serum with higher cell yields in StemSpan. This work demonstrates the development of functional MCs within a 3D collagen matrix using a stem cell media that supports fibroblast and ECs.

Carbonic anhydrase enzymes regulate mast cell-mediated inflammation

Abstract Type 2 cytokine responses are necessary for the development of protective immunity to helminth parasites but also cause the inflammation associated with allergies and asthma. Recent studies have found that peripheral hematopoietic progenitor cells contribute to type 2 cytokine-mediated inflammation through their enhanced ability to develop into mast cells. Here we show that carbonic anhydrase (Car) enzymes are upregulated in type 2-associated progenitor cells and demonstrate that Car enzyme inhibition is sufficient to prevent murine mast cell responses and inflammation following Trichinella spiralis infection or the induction of food allergy-like disease. Further, we employed CRISPR/Cas9 technology and illustrate that genetically editing Car1 is sufficient to selectively reduce mast cell development. Finally, we demonstrate that Car enzymes can be targeted to prevent human mast cell development. Collectively these studies identify a previously unrecognized role for Car enzymes in regulating mast cell lineage commitment and suggest that Car enzyme inhibitors may possess therapeutic potential that can be employed to treat mast cell-mediated inflammation.

Development of peripheral blood-derived human mast cells from patients with chronic spontaneous urticaria : comparison with normal controls

Development of peripheral blood-derived human mast cells from patients with chronic spontaneous urticaria : comparison with normal controls

Carbonic anhydrase enzymes regulate mast cell-mediated inflammation.

Type 2 cytokine responses are necessary for the development of protective immunity to helminth parasites but also cause the inflammation associated with allergies and asthma. Recent studies have found that peripheral hematopoietic progenitor cells contribute to type 2 cytokine-mediated inflammation through their enhanced ability to develop into mast cells. In this study, we show that carbonic anhydrase (Car) enzymes are up-regulated in type 2-associated progenitor cells and demonstrate that Car enzyme inhibition is sufficient to prevent mouse mast cell responses and inflammation after Trichinella spiralis infection or the induction of food allergy-like disease. Further, we used CRISPR/Cas9 technology and illustrate that genetically editing Car1 is sufficient to selectively reduce mast cell development. Finally, we demonstrate that Car enzymes can be targeted to prevent human mast cell development. Collectively, these experiments identify a previously unrecognized role for Car enzymes in regulating mast cell lineage commitment and suggest that Car enzyme inhibitors may possess therapeutic potential that can be used to treat mast cell-mediated inflammation.

Carbonic anhydrase enzymes regulate mast cell-mediated immunity to Trichinella spiralis.

Abstract Type 2 cytokine responses are necessary for the development of protective immunity to helminth parasites but also cause the detrimental inflammation associated with allergies and asthma. Recent studies have found that peripheral hematopoietic progenitor cell populations contribute to type 2 cytokine-mediated inflammation through their enhanced ability to develop into mast cells. Here we identify that carbonic anhydrase (Car) enzymes are upregulated in type 2-asscoaited progenitor cells and demonstrate that Car enzyme inhibition was sufficient to prevent murine mast cell development, type 2 cytokine-mediated inflammation and protective immunity to Trichinella spiralis. Moreover, we show that Car enzyme inhibition was also sufficient to prevent intestinal mast cell responses in a murine model of food allergy-like disease. Finally, we performed translational studies and demonstrated that Car enzymes can be targeted with an FDA-approved inhibitor to prevent human mast cell development. Collectively these studies identify a previously unrecognized role for Car enzymes in regulating mast cell lineage commitment and suggest that FDA-approved Car enzyme inhibitors may possess additional off-label therapeutic potential that can be employed to treat mast cell-mediated inflammation.

Transcriptional regulation of mast cell and basophil lineage commitment

Basophils and mast cells have long been known to play critical roles in allergic disease and in immunity against parasitic infection. Accumulated evidence also supports that basophils and mast cells have important roles in immune regulations, host defense against bacteria and viruses, and autoimmune diseases. However, origin and molecular regulation of basophil and mast cell differentiation remain incompletely understood. In this review, we focus on recent advances in the understanding of origin and molecular regulation of mouse and human basophil and mast cell development. A more complete understanding of how basophils and mast cells develop at the molecular level will lead to development of interventions that are more effective in achieving long-term success.

Long-term treatment with imatinib results in profound mast cell deficiency in Ph+ chronic myeloid leukemia.

Although mast cells (MC) play an important role in allergic reactions, their physiologic role remains unknown. In mice, several models of MC-deficiency have been developed. However, no comparable human model is available. We examined the in vitro- and in vivo effects of the KIT-targeting drug imatinib on growth and development of human MC. Imatinib was found to inhibit stem cell factor (SCF)-induced differentiation of MC in long-term suspension cultures (IC50: 0.01 µM). Correspondingly, long-term treatment of chronic myeloid leukemia (CML) patients with imatinib (400 mg/day) resulted in a marked decrease in MC. In patients with continuous complete molecular response during therapy, bone marrow MC decreased to less than 5% of pre-treatment values, and also serum tryptase concentrations decreased significantly (pre-treatment: 32.0 ± 11.1 ng/ml; post-therapy: 3.4 ± 1.8, p<0.01). Other myeloid lineages, known to develop independently of KIT, were not affected by imatinib-therapy. Imatinib also produced a substantial decrease in MC-development in mice. However, no clinical syndrome attributable to drug-induced MC-deficiency was recorded in our CML patients. Together, imatinib suppresses MC production in vitro and in vivo. However, drug-induced MC depletion is not accompanied by adverse clinical events, suggesting that MC are less relevant to homeostasis in healthy tissues than we assumed so far.

Mechanisms controlling mast cell and basophil lineage decisions.

Basophils and mast cells have long been known to play critical roles in allergic disease and host defense against parasitic infections. Recent recognition of these effector cells in immune regulations, host defense against bacteria and virus, and autoimmune diseases entices increased interest in studying these cells. However, origin and molecular regulation of basophil and mast cell differentiation remain incompletely understood. In this review, we focus on recent advances of the understanding the origin and molecular regulation of mouse basophil and mast cell development. We also summarize progress in the understanding of the origin and molecular regulation of human basophil and mast cell development. A more complete understanding of molecular regulation of basophils and mast cells will lead to the development of interventions that are more effective in achieving long-term success.

Mastocytosis associated with a rare germline KIT K509I mutation displays a well-differentiated mast cell phenotype

Mastocytosis associated with germline KIT activating mutations is exceedingly rare. We report the unique clinicopathologic features of a patient with systemic mastocytosis caused by a de novo germline KIT K509I mutation. We sought to investigate the effect of the germline KIT K509I mutation on human mast cell development and function. Primary human mast cells derived from CD34(+) peripheral blood progenitors were examined for growth, development, survival, and IgE-mediated activation. In addition, a mast cell transduction system that stably expressed the KIT K509I mutation was established. KIT K509I biopsied mast cells were round, CD25(-), and well differentiated. KIT K509I progenitors cultured in stem cell factor (SCF) demonstrated a 10-fold expansion compared with progenitors from healthy subjects and developed into mature hypergranular mast cells with enhanced antigen-mediated degranulation. KIT K509I progenitors cultured in the absence of SCF survived but lacked expansion and developed into hypogranular mast cells. A KIT K509I mast cell transduction system revealed SCF-independent survival to be reliant on the preferential splicing of KIT at the adjacent exonic junction. Germline KIT mutations associated with mastocytosis drive a well-differentiated mast cell phenotype distinct to that of somatic KIT D816V disease, the oncogenic potential of which might be influenced by SCF and selective KIT splicing.

Imatinib Inhibits SCF-Induced Development Of Human Mast Cells In Vitro and Induces Profound and Selective Mast Cell Deficiency In Patients With Ph+ CML

Although mast cells (MC) play an important role as effector cells of allergic reactions and may contribute to vascular repair processes after a thromboembolic event, their physiological role in healthy tissues is currently unknown. In mice, several ´MC-deficiency models´ have been developed. In these models, MC-deficiency is usually triggered by a lack of KIT or lack of a functional KIT-ligand, stem cell factor (SCF). Currently, no comparable human model is available. We examined the in vitro and in vivo effects of the KIT-targeting drug imatinib on growth and differentiation of MC. Imatinib was found to inhibit SCF-induced development of human MC from cord blood-derived progenitors in an in vitro long-term culture assay (100 ng/ml SCF, day 28). The effects of imatinib on MC differentiation in this assay were dose-dependent (IC50: 0.01 µM). Correspondingly, efficacious long-term treatment of CML patients with imatinib (400 mg daily) resulted in a profound reduction of MC numbers. Whereas no substantial decrease in the numbers of MC was seen within the first year of treatment, the numbers of tryptase+ and KIT+ MC in the bone marrow (BM) decreased to less than 5% of pre-treatment values in 23 patients who were in continuous complete molecular response (CMR) for at least 2 years during imatinib therapy (numbers of tryptase+ BM cells: pre-treatment values: 21.1±13.7% of nucleated BM cells vs CMR-values in the same patients: 0.5±0.6% vs normal BM: 3.8±1.6%; p<0.001). Tryptase mRNA levels and KIT mRNA levels in the BM also decreased significantly during treatment with imatinib in these patients (p<0.01). Moreover, during treatment with imatinib, serum tryptase concentrations decreased to low or even undetectable levels (before therapy: 32.0±11.1 ng/ml vs CMR: 3.4±1.8 ng/ml, p<0.01). Other myeloid lineages in the BM, known to develop independently of KIT, were not affected by imatinib therapy. Remarkably, imatinib-induced MC deficiency was not accompanied by any specific symptoms or side effects (no increased rate of bacterial infections, thromboembolic events or secondary neoplastic lesions). In a next step, we examined MC numbers in C57BL/6J mice and cellular histamine levels in BALB/c mice treated with imatinib. In these experiments, we found that treatment with imatinib (60 mg/kg/day vs control saline by intraperitoneal=i.p. injection over 42 days; 4 mice per group) leads to a significant decrease in the numbers of MC (skin MC numbers per high power field in control mice: 36.7±10.1 versus imatinib-treated mice: 15.7±4.6, p<0.001). MC numbers and cellular histamine levels in peritoneal samples of BALB/c mice treated with imatinib (50 mg/kg/day i.p. vs control saline; 3 mice each) also decreased substantially during treatment with imatinib (histamine levels on day 24: imatinib: 9.2±1.4 ng/106 cells vs control: 75.7±2.1 ng/106cells, p<0.001). In conclusion, our data show that imatinib produces a profound MC deficiency in mice and men, without causing a particular clinical condition or syndrome. Based on these observations, we hypothesize that MC may be less relevant in physiologic processes and tissue homeostasis in healthy organs than has so far been assumed. Part of the work, including data on mast cells in BM sections, has been presented at EHA 2013. However, in our current ASH abstract, we have included additional data on mast cell numbers and histamine levels in 2 different mouse models. These mouse data have not been presented at EHA or any other congress before. Disclosures:Valent:Novartis: Consultancy, Honoraria, Research Funding.

Establishment of a Human Allergy Model Using Human IL-3/GM-CSF–Transgenic NOG Mice

The development of animal models that mimic human allergic responses is crucial to study the pathophysiology of disease and to generate new therapeutic methodologies. Humanized mice reconstituted with human immune systems are essential to study human immune reactions in vivo and are expected to be useful for studying human allergies. However, application of this technology to the study of human allergies has been limited, largely because of the poor development of human myeloid cells, especially granulocytes and mast cells, which are responsible for mediating allergic diseases, in conventional humanized mice. In this study, we developed a novel transgenic (Tg) strain, NOD/Shi-scid-IL2rγ(null) (NOG), bearing human IL-3 and GM-CSF genes (NOG IL-3/GM-Tg). In this strain, a large number of human myeloid cells of various lineages developed after transplantation of human CD34⁺ hematopoietic stem cells. Notably, mature basophils and mast cells expressing FcεRI were markedly increased. These humanized NOG IL-3/GM-Tg mice developed passive cutaneous anaphylaxis reactions when administered anti-4-hydroxy-3-nitrophenylacetyl IgE Abs and 4-hydroxy-3-nitrophenylacetyl. More importantly, a combination of serum from Japanese cedar pollinosis patients and cedar pollen extract also elicited strong passive cutaneous anaphylaxis responses in mice. Thus, to our knowledge, our NOG IL-3/GM-Tg mice are the first humanized mouse model to enable the study of human allergic responses in vivo and are excellent tools for preclinical studies of allergic diseases.

Lab Reports And Cat Scans: Can Veterinary Oncology Guide Our Way To New Treatments For Human Cancers?

Future Medicinal ChemistryVol. 4, No. 11 CommentaryLab reports and cat scans: can veterinary oncology guide our way to new treatments for human cancers?Rachel AirleyRachel AirleyDivision of Pharmacy & Pharmaceutical Sciences, School of Applied Sciences, University of Huddersfield, Huddersfield, West Yorkshire, UK. Search for more papers by this authorEmail the corresponding author at r.airley@hud.ac.ukPublished Online:2 Aug 2012https://doi.org/10.4155/fmc.12.81AboutSectionsView ArticleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit View articleKeywords: cancerclinical trialscomparative oncologysynergytyrosine kinase inhibitorveterinaryReferences1 Martinez MN. Welcome to the animal pharmaceuticals special focus. Future Med. Chem.3(7),845–846 (2011).Link, CAS, Google Scholar2 Cardiff RD, Ward JM, Barthold SW. ‘One medicine – one pathology’: are veterinary and human pathology prepared?. Lab. 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PLoS ONE6(11),e27018 (2011).Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByDevelopment and verification of LEP 300 kilovoltage radiotherapy system for companion animals18 August 2022 | Journal of the Korean Physical Society, Vol. 81, No. 11β‑elemene enhances anticancer bone neoplasms efficacy of paclitaxel through regulation of GPR124 in bone neoplasms cells6 June 2018 | Oncology LettersTyrosine kinase inhibitors (TKIs) in human and pet tumours with special reference to breast cancer: A comparative reviewCritical Reviews in Oncology/Hematology, Vol. 88, No. 2 Vol. 4, No. 11 Follow us on social media for the latest updates Metrics Downloaded 311 times History Published online 2 August 2012 Published in print July 2012 Information© Future Science LtdKeywordscancerclinical trialscomparative oncologysynergytyrosine kinase inhibitorveterinaryFinancial & competing interests disclosureThe author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.PDF download

Development of Mature and Functional Human Myeloid Subsets in Hematopoietic Stem Cell-Engrafted NOD/SCID/IL2rγKO Mice

Although physiological development of human lymphoid subsets has become well documented in humanized mice, in vivo development of human myeloid subsets in a xenotransplantation setting has remained unevaluated. Therefore, we investigated in vivo differentiation and function of human myeloid subsets in NOD/SCID/IL2rγ(null) (NSG) mouse recipients transplanted with purified lineage(-)CD34(+)CD38(-) cord blood hematopoietic stem cells. At 4-6 mo posttransplantation, we identified the development of human neutrophils, basophils, mast cells, monocytes, and conventional and plasmacytoid dendritic cells in the recipient hematopoietic organs. The tissue distribution and morphology of these human myeloid cells were similar to those identified in humans. After cytokine stimulation in vitro, phosphorylation of STAT molecules was observed in neutrophils and monocytes. In vivo administration of human G-CSF resulted in the recruitment of human myeloid cells into the recipient circulation. Flow cytometry and confocal imaging demonstrated that human bone marrow monocytes and alveolar macrophages in the recipients displayed intact phagocytic function. Human bone marrow-derived monocytes/macrophages were further confirmed to exhibit phagocytosis and killing of Salmonella typhimurium upon IFN-γ stimulation. These findings demonstrate the development of mature and functionally intact human myeloid subsets in vivo in the NSG recipients. In vivo human myelopoiesis established in the NSG humanized mouse system may facilitate the investigation of human myeloid cell biology including in vivo analyses of infectious diseases and therapeutic interventions.

Effects of the KIT K509I Extracellular Activating Mutation on Human Mast Cell Homeostasis

KIT is a tyrosine kinase receptor which binds stem cell factor (SCF) and is critical to human mast cell (HuMC) development and survival. Recently we identified a patient with systemic mastocytosis harboring a rare heterozygous germline KIT K509I mutation. K509I is located in the 5th immunoglobulin region and such mutations are thought to enhance KIT homodimerization and activation. With this in mind, the effect of the K509I mutation on HuMC development was investigated.

Membrane-Bound Human Stem Cell Factor Promotes Differentiation of Human Granulocytes and Mast Cells In Vivo,

Abstract 3419Recently, advances in xenograft models for human hemamtopoietic stem cells (HSCs), or the humanized mice, have begun to allow investigators to examine the differentiation of human hematopoietic and immune cells in vivo. However, lymphoid-skewed human hematopoietic development in the mouse bone marrow is one of the remaining limitations in the humanized mouse models. The inefficient human myeloid development could at least partly be attributed to the mouse microenvironment not fully supporting differentiation and maturation of human myeloid lineage. To overcome this problem, we focused on the role of membrane-bound human stem cell factor in supporting the maintenance of human HSCs and inducing the development of human myeloid cells and created human stem cell factor transgenic NOD/SCID/IL2rgKO (hSCF Tg NSG) mice. Transplantation of 5000–50000 cord blood-derived Lin-CD34+CD38- cells resulted in significantly higher engraftment of human CD45+ leukocytes at 3–6 months post-transplantation in the bone marrow, spleen, and peripheral blood of hSCF Tg NSG recipients compared with those of non-transgenic NSG recipients. The enhanced human CD45+ engraftment was most prominent in the bone marrow (hSCF Tg recipients: 98.0 +/− 1.3%, n= 15, non-Tg NSG controls: 75.3 +/− 7.3%, n=7). In the bone marrow, the frequency of human CD33+ myeloid cells within the total human CD45+ population was significantly higher in the hSCF Tg NSG recipients than in the non-Tg NSG recipients and constituted the majority of human hematopoietic cells (hSCF Tg recipients: 54.6 +/− 4.5%, n=15 and non-Tg NSG controls: 29.3 +/− 4.0%, n=7). Flow cytometric analysis demonstrated that the majority of engrafted human myeloid cells in the hSCF Tg recipient bone marrow were side-scatter high, HLA-DR negative granulocytes. Reflecting the effect of human SCF on the development of human mast cells, human c-Kit+CD203c+ mast cells were identified in the bone marrow, spleen, and gastrointestinal tracts of the hSCF Tg NSG recipients. Altogether, the in vivo humanized mouse model demonstrates the essential role of membrane-bound SCF in human myeloid development. The hSCF Tg NSG humanized mice may facilitate the in vivo investigation of human HSCs, myeloid progenitors and mature myeloid lineage. Disclosures:No relevant conflicts of interest to declare.

Immunologic Studies in Humans.

Immunologic Studies in Humans.

Generation, Isolation, and Maintenance of Human Mast Cells and Mast Cell Lines Derived from Peripheral Blood or Cord Blood

Antigen-mediated mast cell activation is a pivotal step in the initiation of allergic disorders including anaphylaxis and atopy. To date, studies aimed at investigating the mechanisms regulating these responses, and studies designed to identify potential ways to prevent them, have primarily been conducted in rodent mast cells. However, to understand how these responses pertain to human disease, and to investigate and develop novel therapies for the treatment of human mast cell-driven disease, human mast cell models may have greater relevance. Recently, a number of systems have been developed to allow investigators to readily obtain sufficient quantities of human mast cells to conduct these studies. These mast cells release the appropriate suite of inflammatory mediators in response to known mast cell activators including antigen. These systems have also been employed to examine the signaling events regulating these responses. Proof of principle studies has also demonstrated utility of these systems for the identification of potential inhibitors of mast cell activation and growth. In this unit, techniques for the development and culture of human mast cells from their progenitors and the culture of human mast cell lines are described. The relative merits and drawbacks of each model are also described.

Human embryonic stem cells: a source of mast cells for the study of allergic and inflammatory diseases

AbstractHuman mast cells are tissue resident cells with a principal role in allergic disorders. Cross-linking of the high-affinity receptor for immunoglobulin E (FcϵRI) results in release of inflammatory mediators initiating the clinical symptoms of allergy and anaphylaxis. Much of our knowledge regarding the mechanisms of mast cell activation comes from studies of mouse bone marrow–derived mast cells. However, clear differences have been identified between human and mouse mast cells. Studies of human mast cells are hampered by the limited sources available for their isolation, the resistance of these cells to genetic manipulation, and differences between cultures established from different persons. To address this limitation, we developed a simple coculture-free method for obtaining mast cells from human embryonic stem cells (hES). These hES-derived mast cells respond to antigen by releasing mast cell mediators. Moreover, the cells can be generated in numbers sufficient for studies of the pathways involved in their effector functions. Genetically modified mast cells, such as GFP-expressing cells, can be obtained by introduction and selection for modification in hES cells before differentiation. This direct coculture-free differentiation of hES cells represents a new and unique model to analyze the function and development of human mast cells.