Generation Of Functional Basophils From Human Mbryonic And Induced Pluripotent Stem Cell-Derived Efinitive Hematopoiesis And Its Bilological Haracteristics

Human embryonic stem cells (hESCs) are cells with an infinite proliferation andmultiplex differentiation potential. They could potentially represent an alternativesource for the research of ontogeny of hematopoiesis and regenerative medicine. Morerecently, induced human pluripotent stem cells (hiPSCs) have also been established byreprogramming human somatic cells with defined factors. Based on human iPSCs andiPSCs, some disease models have been established and individualizational treatment areon their way.hESC/hiPSC-derived hematopoietic cells (HCs) have been proved to be excellenttools in characterization of molecular and cellular mechanisms controlling the normaland diseased differentiation of hematopoietic progenitors and mature, functional bloodcells, including erythrocytes, neutrophils, platelets, megakaryocytes, monocytes,dendritic cells (DC), nature killer (NK) cells, and B-lineage, T-lineage lymphoid cellswhich without functional assays. But basophils from hESC/hiPSC-derived definitivehematopoiesis cells have not been reported.Basophils develop from hematopoietic cells, differentitate and mature in the bonemarrow where they constitute less than1%of peripheral blood leukocytes. Basophilsare key effector cells in response to immune regulation and allergic inflammation,which circulate in the blood and are recruited to allergic inflammatory site.Basophils and eosinophils, basophils and mast cells (MCs) have the similarphenotype and biological function. It has been controversial that basophils developthrough a pathway initially with eosinophils or mast cells. Several groups claimed thatbasophils developed from the common progenitors for basophil and eosinophil lineages.While others provided a new insights, they thought that basophils developed from thecommon progenitor for basophils and mast cells. In our previous study, We have established an efficient method to producefunctionally mature eosinophils and MCs from hESCs and hiPSCs by a three-stepsinduction. So in this paper, we will figure out a better method to produce the basophilisfrom hESCs and hiPSCs in vitro and to explore its biological function.PART ⅠDifferentiation of Human Embryonic and Induced Pluripotent Stem Cells intohematopoietic cellsObject: To establish a stable culture system for differentiation of hESCs andhiPSCs into hematopoietic cells in vitro and to investigate their biologicalcharacteristics.Method: Mouse embryonic fibroblast (MEF) was isolated from ICR mouse,cultured and proliferated in vitro, which as a kind of feeder cells for maintenance ofboth hESCs(H1) and hiPSCs(G1,G4,B6,B7). hESC and hiPSCs colonies were harvestedand co-cultured with murine aorta-gonad-mesonephros stromal cells-3(mAGMS3) anddifferentiated into hematopoietic cells. The phenotype of these hematopoietic cells wereanalyzed by flowcytometry. early stage genes of hESC-derived hematopoiesis weredetect by RT-PCR.Result: As a kind of feeder layer cells, MEF could support hESCs and hiPSCsgrowth and undifferentiation. Multipotential hematopoietic progenitors could begenerated by co-culturing hESC/iPSCs with mouse AGM-derived stromal cells for2weeks. Moreover, we found that multipotential hematopoietic progenitors are capable ofdifferentiating to erythroid progenitor cells, hematopoietic progenitor cells, myeloidprogenitor cells and endothelial progenitor cells with flowcytometric analysis. Theexpression of early stage genes of hematopoiesis (CD34, GATA-1, c-kit) were alsodetected by RT-PCR.Conclusion: In our culture system, hESC/mAGMS3co-culturing generated a largenumber of human hematopoietic definitive progenitors. Part ⅡGeneration of basophils from human embryonic and induced pluripotent stemcell and its biological characteristicsObject: To establish a stable culture system for differentiation of hESCs andhiPSCs-derived definitive hematopoiesis into basophils in vitro and to investigate theirbiological characteristics and their early development.Method: The basophilic granulocytes could be induced by a three-steps inductionmethod. hESCs-derived basophils’ basic features were observed with May-Giemsa andToluidine bule staining. FACS and immunofluoresecence staining were used to analyzethe expression of surface marker and basophil-specific granule cationic proteins onhESCs-derived basophils. The functions of basophils were confirmed by degranulationand chemotaxis assay.Result: High proportion of the cells with rough basophilic granules could beobserved by May-Giemsa staining, while about41.9%of the small granulocytes, but notlarge mast cells, contained metachromatic granules when checked by Toluidine bulestaining. The immunofluoresecence staining of basophils showed that thehESCs-derived basophils expressed2D7(basophil-specific markers) while withouteosinophil peroxidase (EPO) expression, and Pro-major basic protein reversetranscription (ProMBP) expression is higher while Major basic protein (MBP) is lower.Furthermore, by flowcytometric analysis, a basophil surface marker was found to beexpressed on the basophils from hESCs. Compared with eosinophils, hESCs-derivedbasophils didn’t express eosinophil-specific granule cationic proteins mRNA whendetected by RT-PCR. Basophils derived from hiPSCs hold similar characteristic as thosefrom hESCs. The function of hESCs-derived basophils had also been investigated.hESCs-derived mature basophils migrated upon stimulation of various factors(N-Formyl-Methionyl-Leucyl-Phenylalanine (fMLP)，eotaxin，FcεRI(CRA-1)). Theyalso exerted function of releasing histamine upon the stimulation of FcεRI (CRA-1).Conclusion: We have successfully developed an efficient method for induction ofbasophils from human embryonic and induced pluripotent stem cells (hESC/iPSCs).The biological characteristics and function of hESCs-derived basophils is simliar withbasophils of human peripheral blood. Our study may contribute in uncovering the unknown mechanisms controlling the development of human basophil and relateddiseases.