Human Induced Pluripotent Stem Cell Derived Macrophages And Its Immunological Function In Response To Tuberculosis Infection

Macrophages(M?)are the most plastic cells in the hematopoietic system that exist in all tissues and play many different roles in cell development,in vivo balance,tissue repair,and immune response to pathogens.Tuberculosis(TB)is one of the three infectious diseases that seriously threatens human health,which is zoonotic chronic infectious diseases caused by Mycobacterium tuberculosis(MTB).In the immune response to MTB infection,macrophages are the main host cells,so the interaction of macrophages and MTB plays a very important role in the occurrence and development of tuberculosis.At present,human macrophages for research are mostly derived from tumor cell lines and peripheral blood mononuclear cells.The first source of macrophages is prone to genetic changes,resulting in functional loss,the second method is difficult to obtain and the less quantity.How to obtain a large number of functional macrophages is the key to the study,the emergence of induced pluripotent stem cells(iPS)technology provides a novel way to obtain a large number of macrophages in vitro.iPS technology is reprogramming terminally differentiated somatic cells into embryonic stem cells(ES)-like by introduction of specific transcription factor genes in vitro.iPS has the ability of self-renewing and the potential to differentiate into cells from all three germ layers.iPS cells is favored due to avoid ethical and immune rejection and other limitations.It was confirmed that iPS could differentiate into macrophages under specific induction conditions in vitro.At present,the methods to differentiate iPS into macrophages can be divided into three categories:(1)Embryoid body(EB)form;(2)Co-culture with bone marrow stromal cells OP9,S17;(3)Directly differentiated by the addition of different factors.Currently,the research about iPS-derived macrophages(iPS-M?)is mostly focused on the field of tumor immunity.It is still lack of research on the anti-tuberculosis immune mechanisms of iPS-M?.Depend on this,we induced human iPS differentiation into macrophages in vitro,and explored the immune function of macrophages responded to Bacillus Calmette-Guérin(BCG).First,the pluripotency of hiPS was detected through the experiments of alkaline phosphatase(AKP),reverse transcription polymerase chain reaction(RT-PCR),immunocytochemistry.On the basis of this,two kinds of methods were used to induce human iPS differentiation into macrophages,by feeder free embryoid body formation or co-cultured with mouse bone marrow stromal cells OP9.In the meanwhile,we identify iPS-M? through cell morphology observation,Giemsa staining,non-specific esterase staining(?-NAE),phagocytosis and cell surface specific antigen expression detection.Finally,iPS-M? were infected with BCG and we analyzed the production of nitric oxide(NO),and the expression of tumor necrosis factor-alpha(TNF-?),and the effect on apoptosis-related protein cysteine-3(Caspase-3)and B-cell lymphoma-2(Bcl-2).Meanwhile human monocyte cell line THP-1 derived macrophages(THP-1-M?)were used as control.The results showed that AKP staining was positive,specific marker Oct-4 and Nanog were expressed in hiPS.The expression of endodermal gene AFP,mesenchymal marker gene Gata-1 and exoskeletal gene Nestin in EB,indicating that the hiPS had pluripotency,could be used for subsequent differentiation experiments.iPS-M? induced by the methods of formation of EB and co-culture with OP9 appeared the macrophage morphological characteristics(round,oval,fusiform and irregular).Next we identified the characteristic and function of iPS-M? which were induced by the method of formation of EB without feeder layer.The results showed that Giemsa staining and ?-NAE staining were positive,and had the ability of phagocytosis,in addition,expressed the specific surface antigen cluster of differentiation(CD)14,CD11 b,CD40,CD68 and major histocompatibility complex II(MHC-II).Compared with the untreated control group,after 24 hours BCG treated,the expression of TNF-? and the production of NO were significantly increased(P<0.01)and the apoptotic rate was significantly increased(P<0.01),the expression of Caspase-3 was increased significantly(P<0.01),but the expression of Bcl-2 was inhibited significantly(P<0.01).Our research established the method of obtaining functional macrophages from hiPS and detected the immunological mechanisms between iPS-M? and MTB,which provided a novel way to elucidate the mechanism of macrophages in tuberculosis immunization.