Mechanism Of ID2 In Lung Bud Organoids On Promoting Pulmonary Angiogenesis

Objective Lung disease associated pulmonary hypertension is a type of pulmonary artery-related secondary disease,which manifests as irreversible lesions of the lung parenchyma and lung interstitium.Lung transplantation is the main treatment option for terminal patients.However,due to the lack of donors,most patients cannot get healthy lungs that match the type.Human embryonic stem cells(hESCs)are pluripotent,and lung cells derived from hESCs are expected to become transplantable seed cells.Inhibitor Of DNA Binding-2(ID2),as a differentiation inhibitor,has the function of regulating lung differentiation and development,and plays an important role in the early stages of organogenesis and bronchial branching.However,how ID2 affects the formation of lung buds in the early stages of lung development,especially in the lung parenchyma,and how it affects the formation of pulmonary blood vessels by endothelial cells(ECs)during lung interstitial development is not yet clear.This study aims to clarify the mechanism of ID2 in early lung development.Method Firstly,establish a method for directed differentiation of hESCs into lung bud organoids(LBOs)and ECs.Secondly,CRISPR/Cas9 system and Lentiviral infection were used to edit ID2 in human embryonic cells(hESCs)to construct ID2 knockout strains and ID2 overexpression strains.hESCs and ID2 gene-edited cell strains were further differentiated into lung bud organoids(LBOs)or endothelial cells(ECs).Flow cytometry was performed to analyse the differentiation efficiency,quantitative PCR was used to detect genes expression during differentiation,immunofluorescence staining was performed to identify the surface characteristic molecules of differentiated cells,and Western blot was applied to check proteins expression.Finally,3D co-culture of ECs and LBOs was performed to compare the effect of LBOs derived from different hESCs on the tubular formation of ECs.Results There is no difference in pluripotency gene expression between normal hESCs,ID2 specific knockout strains and ID2 overexpression strains.In the differentiation of lung bud organoids,ID2 promotes the formation of endoderm and lung bud organoids.In the differentiation of ECs,ID2 can promote cell differentiation into the mesoderm,the lack of ID2 can relatively promote the production of endothelial cells,and leads to impaired endothelial cell tube-forming function.Organoid cultured lung buds can promote the tube-forming function of pulmonary vascular endothelial cells,and the defect of ID2 weakens this promotion.Conclusions 1.ID2 deletion generate excessive endothelial cells with defective tube function.2.ID2 can promote the early development of the lung and the regulatory effect of lung buds on pulmonary blood vessel formation.