| Background and Objective:Mast cells(MC)and macrophages(MΦ)are very important innate immunity cells,which play key roles in immunity response and also have great heterogeneity.MΦ exist in the form of mononuclear/MΦ both in the blood and variety of tissues with different names according to the morphology and their locations.The MCTC and MCT types of human MC were initially recognized as the basis of the different protease compositions of their secretory granules,with tryptase,chymase,Carboxypeptidase A and Cathepsin G in the former and only tryptase in the latter.MCT are the predominant type found in the lungs,bronchial airway basement membrane and adjacent sub-mucosal glands parts in free form,similar with mouse mucosal MC.While MCTC are the predominant type found in the skin around the vascular connective tissue in the sub-mucosa,and mouse tissue mast cell related similar.A variety of toll-like receptors(TLRs)are found on the surface and body distribution of MC and MΦ,as the one of important pattern recognition receptors in the body,TLRs can effectively identify the pathogen related exogenous danger signals and release endogenous danger signals,playing an important role in the body’s defenses.In the early development of an individual,the origin of the blood cells is characterized by the chronological order.All the blood cells are derived from mesoderm,and the earliest hematopoietic cells are found in the yolk sac.Later it occurred in the Aorta-Gonad-Mesonephros(AGM)region,where the original hematopoietic stem cells(HSCs)are found.Finally,HSCs seed in the fetal liver,where the HSCs can be transplanted.Based on the chronological order and different characteristic of human hematopoiesis,it is divided into primitive hematopoiesis and definitive hematopoiesis.The study of adult hematopoiesis has confirmed that all functional blood cells are derived from bone marrow hematopoietic stem cells(HSCs).However,the study of hematopoiesis during mouse embryonic development showed that there are three waves.Both the primitive hematopoiesis and erythromyeloid progenitors(EMPs)originated from yolk sac can produce MΦ.In addition,during the development of mouse fetal liver,it was found that precursors of MC were highly concentrated in yolk sac and fetal liver blood.The data above showed that there is a strong early embryonic development stage of MΦ and MC,and the study found that two kinds of cells produced in this stage have tissue bias.However,due to the restrictions of ethics and law,it is difficult to study the development of embryonic hematopoiesis directly in human body.As the lack of a suitable model to investigate early human hematopoiesis,previous studies have been based on animal models,such as mice.However,the data from animal studies do not fully indicate the development of the human body.Successful establishment of human embryonic stem cells(hESCs)and human induced pluripotent stem cells(hiPSCs)provided an accessible model for studying early embryonic development of human.Human pluripotent stem cells(hPSCs),including hESCs and hiPSCs,have the ability of self-renewal,proliferation and differentiation into all types cell.It is widely reported that hPSCs can be differentiated into all mature cells of three germ layers.Based on the research of hPSCs-drevied blood cells.the process of early human hematopoiesis can be largely reproduced and provide a good model to the scientific community for more detailed analyzing human embryonic hematopoiesis.In order to study the early development of human MΦ and MC,we need to establish a highly efficient culture system in vitro,to better mimic the in vivo developmental process.After compared the cells derived from the adult hematopoietic stem cells,we study that the function and reorganization of the innate immune cells derived from the early development.Here,we take the initial development of innate immune cells as the research direction,and study the origin and function of macrophages and MC differentiated from hPSCs in vitro.Methods:Based on the co-culture system of hPSCs/AGM-S3,an innate cells cultured method was established.In the main of MC and MΦ,we detected the morphology,phenotype,function as well as explored the origins of the two types cells.The operation methods are as follow:1.Use mouse aorta gonad mesonephros(Aorta-Gonad-Mesonephros,AGM)cells as stromal cell,co-culture with hPSCs in vitro,induced a large amount of hematopoietic stem/progenitor cells,then using directional suspension culture method for MC and MΦ.The process can be divided into three steps:1)Inoculate hPSCs clones into stromal cells,induce hematopoietic and detect the phenotypes of the clones and the expression of surface markers at different time points.2)Suspension culture for 7 days,amplificated the hematopoietic stem/progenitor cells,3)Changing medium for MC or MΦ directional differentiation.2.Observe morphology and phenotype characters of MC and MΦ1)MC:Use MGG,toluidine blue and alcian blue method to observe the morphology of MC,observe the granules of MC by transmission electron microscope,Immunofluorescence(IF)staining analyze the expressing of Tryptase and Chymase in MC,FACS analysis of surface marker on MC.2)MΦ:The three conditions are(a)cord blood mononuclear cells CD34+ derived macrophages,(b)co-culture D3 cells derived macrophages and(c)co-culture day 14 cells,observed the morphology with MGG,detected the expression of surface markers on the three conditions macrophages with multicolor flow cytometric analysis technology(FACS).3.Detecte the function of MC and MΦ1)Function of MC:Detection of histamine release by Enzyme-linked immune(ELISA)analysis,detected the expression level of TLRs with RT-qPCR.2)Using RT-qPCR detected the expression of inflammatory and TLRs under different polarization conditions,detecting phagocytosis of macrophages with low-density lipoprotein.4.Use FACS technology to sort the target cells,find out the progenitor cells of MC.Results:1.Established a high-efficient hPSCs/AGM-S3 co-culture hematopoietic differentiation system,which can produce a large number of MC with high purity and early MΦ1)Through co-culture 14 days then cultured 10 days,the double positive tryptase and chymase MC are found,the percent is up to 98%on 40 days.The number of C-KIT+Tryptase+ MC is 200 times of undifferentiated H1 cells.2)By co-culture 3 days then cultured 14 days,early MΦ were produced.2.Both MC and MΦ have an organized bias1)Immunological staining,IF and electron microscopy,IgE receptor antibody,substrate P,and complex 48/80 can stimulate MC release histamine,based on the data,and confirmed the MC in our culture system is MCTC type.2)Early MΦ can be polarized into M2 type by IL-4.3.MCTC and early MO are different with the cells generated from adult HSCs1)Compared with the MC derived from peripheral blood and cord blood CD34+ cells,hPSCs/AGM-S3-derived MC have high expression of TLR2,TLR4 and TLR9.2)The expression of surface markers of the macrophages produced from the three different culture conditions are significantly different from the others.The expression levels of TLR1-TLR8 were significantly higher in M2 macrophages derived from co-culture day 3 cells.4.MCTC and MΦ were produced before adult HSCs(CD34+CD45+)1)Sorted D8 cells by FACS analysis,the daughter cells from CD34+C-KIT-and CD34+C-KIT-cells fraction,having a high positive expression of tryptase and chymase for 14 days suspension culture.2)There is no double po.sitive CD34 and CD45 cells in co-culture Day3,so the early MΦ was born before adult HSCs.Conclusion:1.The hPSCs/AGM-S3 co-culture system can induce innate immune cells,such as MC and macrophages,and those cells are produced earlier than the adult hematopoietic stem cells,in our system it means CD34+CD45+ cells.2.These early innate immune cells have unique development pathway,and independent from HSCs.3.These early innate cells tend to tissue characteristics and have high expression of TLRs,showing the MCTC and MΦ in tissue have higher ability of recognition,and may play an important role in innate immunity response. |
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