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

Abstract

Human mast cells are tissue resident cells with a principal role in allergic disorders. Cross-linking of the high-affinity receptor for immunoglobulin E (FcepsilonRI) 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.

Figure 1

Differentiation of hES cells to hematopoietic progenitors. (A) hES cells were removed from mouse embryonic fibroblasts by treating with collagenase IV and cocultured for 10 days with a monolayer of OP9 in α-minimum essential medium containing 15% fetal calf serum. (B) FACS of a single-cell suspension after 12 days of hES cell coculture with OP9 cells; staining for CD34 and CD43. (C) EBs. hES cells were removed from mouse embryonic fibroblasts by treating with dispase and cultured in EB-differentiating medium for 4 days. (D) Phase-contrast image of adherent EBs differentiated in IL-6, IL-3, SCF, and Fms-like tyrosine kinase 3 ligand for 3 weeks with hematopoietic progenitors releasing to medium (small arrows indicate progenitors; and large arrow, differentiated EBs). (E) Phase-contrast image of homogeneous population of hematopoietic progenitors collected from medium 4 weeks after EB differentiation. (F) Forward-side scatter plot of hematopoietic progenitors. (G) CD34 surface expression on hematopoietic progenitors. Histogram shows antibody staining (in dark) relative to isotype-matched control (transparent). (H) CD45 and CD43 surface expression. Microscopic figures (A,C-E) were captured using an Olympus IX81 inverted fluorescence microscope with 4× (A,C,D) and 10× (E) objectives with a Hamamatsu ORCA RC camera, operated by Velocity software (PerkinElmer Life and Analytical Sciences). Bars in insets represent 100 μm. Data shown are representative from at least 3 experiments. (I) Schema showing coculture-free differentiation of human mast cells from hES cells.

Figure 2

Phenotype of ES cell–derived mast cells. (A) Human mast cells differentiated from hES cells by EB formation were stained with antitryptase antibody, antichymase antibody, and Toluidine blue and compared with human mast cells derived from cord blood (CBMCs). Nuclei were stained with 4,6-diamidino-2-phenylindole. Figures were captured by an Olympus BX61 upright fluorescence microscope with 40× (antichymase, antitryptase staining) and 60× (Toluidine blue staining) objectives with a Hamamatsu ORCA RC camera, operated by Velocity software (PerkinElmer Life and Analytical Sciences). Bars in insets represent 10 μm. Controls represent cells stained without antitryptase, antichymase antibody. Figures represent 1 of at least 4 independent experiments. (B) Quantification of tryptase- and chymase-positive cells in culture during mast cell differentiation. (C) Activity of tryptase measured as a change in fluorescence activity of tosyl-Gly-Pro-Arg-pNA in mast cell lysates after 20-minute incubation at 37°C. Unit is defined as the amount of enzymatic activity that induces a 0.001 change in optical density at 405 nm/minute (n = 3). (D) Inhibition of tryptase activity by tryptase antagonist Polybrene, activity measured as in panel C (n = 3).

Figure 3

Expression of FcϵRI and c-Kit on ESMCs. FACS analysis of ESMCs. Staining of cells with anti-IgE antibody after exposure of cells with human IgE for 5 days (black), without IgE pretreatment (empty), and isotype control (empty dotted line). (A) ESMCs differentiated by coculture with OP9. (B) ESMCs differentiated by EB formation. (C) CBMCs. (D) Expression of c-Kit in ESMCs differentiated by EB formation. Isotype control (black), and anti–c-Kit antibody (gray). (E) Double staining of ESMCs by anti-IgE and anti–c-Kit antibody. Results shown are representative of 6 experiments.

Figure 4

Activation of ESMCs by cross-linking of FcϵRI and PGE₂. (A) Phosphorylation of p42/44 ERK assayed by FACS using a phosphospecific antibody that recognizes phosphorylation of T202 and Y204. ESMCs (left panel) or CBMCs (right panel) were activated by FcϵRI cross-linking or by PGE₂ activation (black represents unactivated; green, PGE₂; red, anti-IgE; and blue, PGE₂ and anti-IgE). (B) Quantitative analysis (n = 6) of T202/Y204 phosphorylation in ESMCs stimulated by PGE₂ (white bar), FcϵRI (hatched bar), and FcϵRI and PGE₂ together (dark bar). (C) Intracellular calcium release in ESMCs (left panel) or CBMCs (right panel) and activated by FcϵRI cross-linking 3 μg/mL anti-IgE antibody (blue) or 1μM PGE₂ (red). Point of stimulation is shown by arrow. Calcium mobilization is expressed as the ratio of fluorescence of Fura-2 measured at 340 and 380 nm. Typical results from at least 2 experiments are shown. (D) Histamine release in ESMCs (left panel) or CBMCs (right panel) preincubated with hIgE and cross-linked with various concentrations of anti-IgE antibody alone or together with 10⁻⁶M PGE₂. Histamine release is presented as a percentage of total histamine assayed by lysis of cells by Triton X-100 (n = 3). *Significant difference from 0 μg/mL anti-IgE (P < .05). (E) Release of TNF-α was evaluated in ESMCs (left panel) and CBMCs (right panel) preincubated with hIgE and cross-linked with 3 μg/mL anti-IgE antibody alone or together with 10⁻⁶M PGE₂ (n = 3). (F) PGD₂ release from ESMCs (left panel) or CBMCs (right panel) measured using PGD₂ methoxime enzyme immunoassay kit (n = 3). *Significantly different from untreated cells (P < .05). Results shown in right panels in panels C and E were obtained under identical conditions to those in the left panels but on different days.

Figure 5

Human mast cells differentiated from genetically modified hES cells. (A) A reporter gene expressing GFP under EF-1 α promoter was expressed in hES cell clone H1 by lentiviral transfection. hES cells double-positive for GFP and pluripotent marker TRA-1-81 (region R5) were sorted by MoFlow sorter. Left panel: Nontransfected cells, isotype control. Right panel: Transfected cells stained with TRA-1-81. (B) hES cell lines expressing GFP were established. (C) Differentiating EBs formed from GFP-hES cells. (D) GFP-expressing mast cell progenitors releasing from differentiated EBs to medium. Images were captured using an Olympus IX81 inverted fluorescence microscope with a 4× objective. (E) Mast cell progenitors express GFP and CD43. Left panel: Isotype control. Right panel: CD43 antibody. (F) Mast cell progenitors differentiated to mast cells expressing c-Kit, anti–c-kit-APC (gray), and isotype control (empty). (G) Mast cells derived from GFP-hES express tryptase. Top panel: GFP-hES cells. Bottom panels: Mast cells derived from nontransfected H1 clone. Figures were captured by an Olympus BX61 upright fluorescence microscope with a 40× objective with a Hamamatsu ORCA RC camera, operated by Velocity software (PerkinElmer Life and Analytical Sciences).