Figure 2.

G2V ESC-Derived V⁺ Cells Generate Mucosal and Connective Tissue-Type Mast Cells that Express Proteases and Degranulate

(A) Image showing the clonal expansion capacity of a single V⁺ mast cell harvested from stage 2 after 5 days. Area within the dotted region shows part of such a colony (>1,000 cells). Scale bar, 100 μm.

(B) Image of toluidine blue-stained mast cells generated after stage 3 expansion. Lighter blue indicates mucosal mast cells (mMC) and darker blue connective tissue-like mast cells (ctMC).

(C) Mast cell-specific gene expression in undifferentiated ESCs (number of independent experiments [n] = 3), day-10 V⁺ hematopoietic cells (stage 1, n = 3), expanded mast cells (stage 3, n = 4) and mast cells after clonal replating (rpMC, n = 3). RT, reverse transcriptase; CPA, carboxypeptidase; mMCP, mouse mast cell protease.

(D) Image of toluidine blue-stained mast cells generated after two serial clonal replatings of a single cell harvested from stage 2 co-culture. Scale bar, 100 μm.

(E) Quantitative gene expression analysis of mast cell-specific genes in expanded G2V mast cells. Expression levels were normalized to 18S expression and compared with the normalized levels in control mouse ear tissue. Mean ± SEM, n = 3.

(F) ELISA assay showing the relative concentration of released tryptase in the medium of c48/80-treated (5 μM) G2V mast cells compared with untreated cells. Tryptase levels were calculated based on a tryptase standard curve. Level was set as 1 in untreated sample. ^∗p < 0.05, n = 5.

(G) Immunoglobulin E (IgE) activation of degranulation. Functional assay showing the percentage of β-hexosaminidase release in the supernatant of IgE activation/antigen stimulation-treated ESC-MCs and peritoneal MC (P-MC) compared with controls. β-Hexosaminidase enzymatic activity was measured in supernatants and cell pellets and percent release was calculated as described in Experimental Procedures. Unpaired t test, two-tailed p value; n = 4.

Error bars represent means ± SEM.