Establishment And Application Of Human Embryonic Stem Cells Derived Lung Organoids

The technology to generate human embryonic stem cells(hESCs)derived lung cells have already changed from tranditional two-dimentional(2D)monolayer cell based differentiation to three-dimentional(3D)organoids differention.In vitro hESCs derived lung organoids contain lung proximal and distal cells types,such as ciliated cells(Foxj1+),goblet cells(MUC5AC+),basal cells(P63+),club cells(CC10+),immature alveolar type I cells(AT1,PDPN+/SOX9+)and alveolar type II like cells(AT2,SPC+/SOX9+).Current lung organoids protocols are money consuming and time consuming,and generation of mature AT1 cells(AQP5+/PDPN+/SOX9-)is still challenging.The present study focus on establishment and application of hESCs derived lung organoids(HLOs),mainly studies organoids based lung development and cell therapy.In vitro generation of hESCs derived lung cells mimic human lung development stages in vivo by adding some cytokines and small molecules in the culture system.In the first part of the present study,we employed 6 cytokines and small molecules to generate lung organoids from hESCs.Our qPCR data demonstrated that lung progenitor makers including NKX2.1,P63,SOX2 and SOX9 were up regulated following differentiation(D21 and D31)and down regulated at the end stage(D41),and lung terminal cell types makers such as MUC5AC,CC10,SPC and SPB were up regulated following differentiation and got summit at the end stage(D41).Immunofluorescence staining results conformed the expression of marker mentioned above,such as NKX2.1,P63,MUC5AC,SOX9,SPC and SPB.Transmission electron microscopy(TEM)analysis of D41HLOs revealed lamellar bodies,which are specific secretory organelles surfactant protein trafficking and secretion in AT2 cells.TEM also showed multiple cilia with a "9+2"structure consisting of nine outer microtubule doublets and a central pair of microtubule singlets,the specific features of motile cilia.These data indicated that we successfully established hESCs derived lung organoids platform by a simple protocol.In the next part of the present study we transplanted D21,D31 and D41 lung organoids into NSG mice to investigate differentiation and mature.Data revealed that D21 lung organoids short-term transplantation(14 days)possessed lung progenitor state(NKX2.1+,P63+,SOX9+,AQP5-,SPC-),D21 lung organoids long-term transplantation(100-120 days)possessed lung alveolar bi-potential progenitor cells(BP cells,PDPN+/SPC+/SOX9+),D41 lung organoids long-term transplantation(100-120 days)possessed mature AT1 like cells(PDPN+,AQP5+/SPB-/SOX9-).Furthermore,transplanted lung organoids contained vascular network and neuronal network resemble human lung.These finding indicate that hESCs derived lung organoids provide a promising model to study human lung development.In the third part of current study,we investigated hESCs derived cells based cell therapy on lung injury mouse model.After donor cells(GFP labeled D25HLOs cells,divided into pool graft and CD47high graft group)intratracheal transplantation for 5 weeks,donor cells survival and formation of sequential pseudostratified epithelium like state at the airways lumen in the lung of model NSG mouse.Further analysis revealed that donor cells differentiated into several proximal lung cell types,such as ciliated cells(acetylated tubulin+/GFP+),club cells(CC10+/GFP+)and basal cells(P63+/GFP+,only in pool group).After donor cells transplantation 6 weeks,mouse lung function results revealed that Penh,EF50 and Rpef got alleviating compared to control group(n=5-6,*P<0.05).After donor cells transplantation 16 weeks,dissection of transplanted mouse lung did not observe tumor formation.Donor cells survival and generation of pseudostratified epithelium state at the airways lumen in the lung of model NSG mouse.Meanwhile,donor cells differentiated into several proximal lung cell types after 16 weeks transplantation.Compared to pool cells group,CD47high cells group generated more ciliated cells and possessed taller cell height of pseudostratified epithelium like state.Transcriptome data revealed that CD4high cells highly expressed epithelial cell,lung progenitor,lung proximal cell and embryonic lung development associated genes.These data indicated that CD47high cells are lung determined cells.In summary,the main achievements of the current study are as follow:we firstly generate hESCs derived lung organoids differentiation platform only used 6 cytokines and small molecules.hESCs lung organoids transplantation study generated human lung BP cells(PDPN+/SPC+/SOX9+)at the first time and also generated mature AT1 like cells(PDPN+,AQP5+/SPB-/SOX9-).Transplanted lung organoids contained vascular network and neuronal network resemble human lung.Furthermore,hESCs derived lung organoids cells intratracheal transplantation into lung injury NSG mouse model generated sequential pseudostratified epithelium like state at the airways lumen,and differentiation into several proximal lung cell types after 5 and 16 weeks transplantation,and without tumor formation.Furthermore,after donor cells transplantation 6 weeks,mouse lung function including Penh,EF50 and Rpef got alleviating.Our data demonstrated hESCs lung organoids possessed promising application prospects in lung development and cell therapy.