| Objective:Human embryonic stem cells (hESCs) proliferate infinitely and pluripotent, They could potentially represent an alternative source for studying the ontogeny of hematopoiesis. More recently, human pluripotent stem cells (hiPSCs) have also been established by reprogramming human somatic cells with defined factors. based on human iPS cells, models for diseases have been established and cures are on their way. Until now, there are many reports describe specific and efficient methods to induce hESCs to differentiate into mature blood cells. It is important to determine whether and how these cells behave similarly with their counterparts in vivo produced. Eosinophil are very rarely blood cells, normally comprising 1-3% of peripheral blood cells. But they implicated in the pathogenesis of numerous inflammatory process. We developed a method to induce hES/iPSCs to differentiate into mature eosinophils. This study may provide an experimental model for exploring the early eosinophilpoiesis, especially in uncovering the mechanism of controlling the development of initial innate immune system of human being in normal and diseased individuals.Methods:In our study, the hES/iPSCs were maintained and passaged weekly on irradiated mouse embryonic fibroblast feeder cells(MEF). Small clumps of undifferentiated hES/iPSCs were transferred onto Murine Aorta-Gonad Mesonephros region-derived stromal cells (AGM) to generate hematopoietic progenitor. On days 14 of culture, embryonic stem cell-derived hematopoietic progenitors were transferred into suspension culture by adding a cocktail cytokines favoring eosinophil development. We performed cells surface markers analysis on co-culture and suspension cells at different times by flow cytometry. TO dectect hES/iPSCs-derived hematopoiesis and eosinophil granule protein expression. Cells in suspension culture were centrifuged onto glass slides and stained with May-Grunwald-Gismsa solution for morphological observation. The specific granule protein of eosinophil were confirmed by transmission electron microscopy(TEM). The expression of eosinophil peroxidase (EPO), major basic protein(MBP),2D7,Pro-major basic protein1(ProMBP1) in suspension culture cells were determinded by immunofluorescent staining. The functions of eosinophil were confirmed by degranuation and chemotaxis.Results:The maturation of hESC/iPSCs-derived eosinophils was shown in a time-dependent manner, first co-expressing eosinophil-and basophil-specific markers [(EPO), and 2D7 respectively], then the portion of eosinophil markers gradually increased while that of basophil markers decreased (EPO+ cells from 56.4% at day 7 to 94.4% at day 21, while 2D7+ cells from 62.8% to 25.7%, respectively), typically mimicking the development of eosinophils from human adult hematopoietic progenitors. By flow cytometric analysis, an eosinophil-specific surface marker Siglec-8, was also expressed on these hESC/iPSC-derived eosinophils in a time-dependent manner (from 10.8% at day 7 to 91.3% at day 21), paralleling to those with EPO. The expression of eosinophil-specific granule cationic proteins (EPO, MBP, ECP, EDN) and IL-5 receptor mRNA was also detected by RT-PCR. Furthermore, transmission electron microscopy (TEM) observation confirmed the eosinophils property. Eosinophils derived from hiPSCs hold similar characteristics as those from hESCs. The function of hESC/iPSCs-derived eosinophils had also been investigated. hESC-derived mature epsinophils migrated upon stimulation of various factors. They also exert function to release EDN upon the stimulation of slgA.Conclusions:We have successfully developed a method for efficient production of eosinophil from hESC/iPSCs. Through the experiments, we found the hESC/hiPSCs derived mature eosinophoils developed through a Eo/Baso bipotent pathway, that has been found similar to eosinophils on adult hematopoiesis. Siglec-8 may be a proper surface marker to trace the early development stage of human eosinophils.
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