A novel human fetal lung-derived alveolar organoid model reveals mechanisms of surfactant protein C maturation relevant to interstitial lung disease
During human lung development the distal tip epithelial cells act as multipotent progenitors. From ~15 pcw (post conception weeks) the human tip epithelium retains progenitor marker expression, but also upregulates markers of the AT2 lineage. Immature AT2 cells appear in the tissue from 17 pcw. We have recently shown that 16-22 pcw epithelial tip cells can be expanded as organoids and differentiated to AT2 cells. However, the differentiated AT2 cells were not proliferative, limiting their use for functional studies and genetic manipulation. We have now developed a highly robust, efficient, and scalable culture condition (AT2 medium) that induces the differentiation of 16-22 pcw fetal lung tip cells into mature AT2 cells which grow as expandable 3D organoids. In this study, we characterise the fetal-derived AT2 cells, showing that they are stable over long-term passaging, efficiently process and secrete surfactant, and can differentiate into AT1 cells in vitro and in mouse lung transplantation assays. Forward genetic screen identifies candidate effectors of SFTPC trafficking which we validate using CRISPR interference (CRISPRi) in the AT2 organoids. We show that the trafficking of SFTPC requires ubiquitination by HECT domain E3 ligases, particularly Itch, and that their depletion phenocopies the pathological redistribution seen for the SFTPCI73T variant. Overall design: Four biological AT2 organoid lines cultured in AT2 medium were used for RNA seq analysis; each at early and late passages. Four biological AT2 organoid lines cultured in AT2 medium were used for single cell RNA seq. >> AT2_merged.gene_counts_length_scaled.tsv << A comparison between the RNA sequencing data of AT2 organoids and publicly available data of fetal organoid types and other alveolar cell types was performed: fetal early tip progenitor organoids GSM5393370 and GSM5393371; fetal late tip progenitor organoids6 GSM5393372 and GSM5393373; PSC-iAT2s7 GSM5578511, GSM5578512 and GSM5578513; cultured adult AT2 cells7 GSM5578508, GSM5578509 and GSM5578510; freshly isolated adult AT2 cells8 GSM2537127, GSM2537128 and GSM2537129 Re-analysed Samples in GSE184143/PRJNA763363, GSE96642/PRJNA379280, GSE178528/PRJNA739556: data column header; re-analyzed sample accns and title adult_AT2_1; GSM2537127 SRR5345478 SAMN06603895 Adult AEC2_A adult_AT2_2; GSM2537128 SRR5345479 SAMN06603915 Adult AEC2_B adult_AT2_3; GSM2537129 SRR5345480 SAMN06603914 Adult AEC2_C adult_AT2_CKDCI_1; GSM5578508 SRR15902408 SAMN21438592 adult_AT2_CKDCI_MRC5_1 adult_AT2_CKDCI_2; GSM5578509 SRR15902409 SAMN21438593 adult_AT2_CKDCI_MRC5_2 adult_AT2_CKDCI_3; GSM5578510 SRR15902410 SAMN21438594 adult_AT2_CKDCI_MRC5_3 Early_tip_progenitor_1; GSM5393372 SRR14869977 SAMN19792069 14404_folded_P2_RNA-seq Early_tip_progenitor_2; GSM5393373 SRR14869978 SAMN19792068 14387_folded_P3_RNA-seq Late_tip_progenitor_1; GSM5393370 SRR14869975 SAMN19792071 BRC1915_CS20_P5_RNA-seq Late_tip_progenitor_2; GSM5393371 SRR14869976 SAMN19792070 13393_CS23_P5_RNA-seq PSC_iAT2_3D_CKDCI_1; GSM5578511 SRR15902411 SAMN21438595 iAT2_3D_CKDCI_1 PSC_iAT2_3D_CKDCI_2; GSM5578512 SRR15902412 SAMN21438596 iAT2_3D_CKDCI_2 PSC_iAT2_3D_CKDCI_3; GSM5578513 SRR15902413 SAMN21438597 iAT2_3D_CKDCI_3