Figure 2.

Rapid maturation of MC_TCs generated from a CD34⁺c-kit⁺ cell population. (A) Flow cytometric profiles showing expression of CD34⁺c-kit⁺ cells derived from H1/AGM-S3 coculture. (B) Proliferation of CD34⁺CD45⁺, CD34⁺CD43⁺, CD34⁺c-kit⁺, and CD45⁺ cell fractions on D4‒D16 derived from 1 × 10⁴ H1 cells (independent experiments, n = 3; mean ± SD). (C and D) Flow cytometric analysis showing representative phenotypic expression of mesoderm (KDR), endothelial (CD31 and CD144), hematopoietic (CD43 and CD45), and MC (CD13 and IgE-R) markers in the four cell fractions at D8 (C) and D16 (D) during a single culture. (E) Four cell fractions defined by expression of CD34 and c-kit were sorted by FACS from a D8 H1/AGM-S3 coculture. MGG analysis showing typical morphologies of the sorted CD34⁺c-kit⁺, CD34⁺c-kit⁻, CD34⁻c-kit⁺, and CD34⁻c-kit⁻ cell fractions (a) and qPCR analysis showing mRNA expression of c-kit (b), tryptase (c), and chymase (d) in each cell fraction, total coculture D8 cells, and a pure population of MCs. Independent experiments, n = 3; mean ± SD. ND, not detected. Scale bar, 10 µm. (F) Total cell proliferation of the four cell fractions sorted from D8. (G) Total cell proliferation of CB CD34⁺ cells (a), coculture D14 CD34⁺CD45⁺ cells (b), and coculture D8 CD34⁺c-kit⁺ cells (c) in SF-MC culture. (H) Compared with CB CD34⁺ and coculture D14 CD34⁺CD45⁺ progenitor cells, a high proportion (36.3% ± 3.4%) of coculture D8 CD34⁺c-kit⁺ cell-derived c-kit⁺Tryptase⁺ MCs could be observed early on D7 in SF-MC culture and reached a purity of 90.3% ± 1.4% after 21 days in culture (a) that was associated with a rapid increase in co-expression of chymase (90.2% ± 1.1% at D21, b). NS, non-significant.