Abstract: Objective To investigate the role of inhibitor of DNA binding-2 (ID2) in pulmonary vasculature formation in early lung development. Methods CRISPR/Cas9 system was used to edit ID2 in human embryonic stem cells (hESCs) to construct ID2 knockout strains, hESCs and ID2 knockout strains were further differentiated into endothelial cells(ECs) or lung bud organoids (LBOs).Quantitative PCR was used to detect ID2, CD31, NKX2.1 and other genes expression during differentiation, flow cytometry was performed to analyze the differentiation efficiency, Western blot was applied to check ID2, CD31 and PAX9 protein expression, 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 ID2 promoted cells to enter the mesoderm stage during the differentiation of hESCs into ECs, but ID2 deletion led to the formation of a large number of ECs with tubular defects. ID2 promoted the differentiation of hESCs into LBOs.In the 3D co-culture system, LBOs could promote ECs tube-forming ability, while the deficiency of ID2 weakened this promotion. Conclusions In early stage of pulmonary tissue development, ID2 affects the differenti- ation and function of ECs and LBOs, loss of ID2 in lung bud reduces the angiogenesis of surrounded ECs.

Key words: inhibitor of DNA binding-2 (ID2), lung bud organoids(LBOs), endothelial cells(ECs)