{"id":["DOI:10.1016/j.stem.2025.09.007"],"title":["Hypoxia promotes airway differentiation in the human lung epithelium"],"publication_date":["2025-11-06"],"authors":{"ORCID:0000-0003-1216-7740":{"id":["ORCID:0000-0003-1216-7740"],"family_name":["Dong"],"personal_name":["Ziqi"],"affiliations":{"ROR:013meh722":{"organization_name":["University of Cambridge"]},"ROR:00f54p054":{"organization_name":["Stanford University"]},"ROR:00fp3ce15":{"organization_name":["The Gurdon Institute"]}}},"ORCID:0009-0002-4330-5333":{"id":["ORCID:0009-0002-4330-5333"],"family_name":["Niek"],"personal_name":["Wit"],"affiliations":{"ROR:013meh722":{"organization_name":["University of Cambridge"]}}},"AUTHTEMP:00077":{"id":["AUTHTEMP:00077"],"family_name":["Agarwal"],"personal_name":["Aastha"],"affiliations":{"ROR:013meh722":{"organization_name":["University of Cambridge"]},"ROR:052gg0110":{"organization_name":["University of Oxford"]}}},"ORCID:0000-0002-5926-7768":{"id":["ORCID:0000-0002-5926-7768"],"family_name":["Reid"],"personal_name":["Adam James"],"affiliations":{"ROR:00fp3ce15":{"organization_name":["The Gurdon Institute"]}}},"ORCID:0000-0001-9236-8221":{"id":["ORCID:0000-0001-9236-8221"],"family_name":["Dubal"],"personal_name":["Dnyanesh"],"affiliations":{"ROR:01vdt8f48":{"organization_name":["MRC Mitochondrial Biology Unit"]}}},"ORCID:0000-0002-8230-4256":{"id":["ORCID:0000-0002-8230-4256"],"family_name":["Beier"],"personal_name":["Sina"],"affiliations":{"ROR:00fp3ce15":{"organization_name":["The Gurdon Institute"]}}},"ORCID:0000-0002-1327-2334":{"id":["ORCID:0000-0002-1327-2334"],"family_name":["Mahbubani"],"personal_name":["Krishnaa T."],"affiliations":{"ROR:013meh722":{"organization_name":["University of Cambridge"]}}},"ORCID:0000-0002-0633-3696":{"id":["ORCID:0000-0002-0633-3696"],"family_name":["Saeb-Parsy"],"personal_name":["Kourosh"],"affiliations":{"ROR:013meh722":{"organization_name":["University of Cambridge"]}}},"ORCID:0000-0002-2744-0810":{"id":["ORCID:0000-0002-2744-0810"],"family_name":["van den Ameele"],"personal_name":["Jelle"],"affiliations":{"ROR:013meh722":{"organization_name":["University of Cambridge"]}}},"ORCID:0000-0002-0248-1632":{"id":["ORCID:0000-0002-0248-1632"],"family_name":["Nathan"],"personal_name":["James A."],"affiliations":{"ROR:013meh722":{"organization_name":["University of Cambridge"]}}},"ORCID:0000-0001-7426-3792":{"id":["ORCID:0000-0001-7426-3792"],"family_name":["Rawlins"],"personal_name":["Emma L."],"affiliations":{"ROR:00fp3ce15":{"organization_name":["The Gurdon Institute"]}},"HDBItheme":["Cardiopulmonary"]}},"abstract":["Human lungs experience dynamic oxygen tension during development. Here, we show that hypoxia directly regulates human lung epithelial cell identity using tissue-derived organoids. Fetal multipotent lung epithelial progenitors remain undifferentiated in a self-renewing culture condition under normoxia but spontaneously differentiate toward multiple airway cell types and inhibit alveolar differentiation under hypoxia. Using chemical and genetic tools, we demonstrate that hypoxia-induced airway differentiation depends on hypoxia-inducible factor (HIF) activity, with HIF1α and HIF2α differentially regulating progenitor fate decisions. KLF4 and KLF5 are direct HIF targets that promote basal and secretory cell fates. These findings reveal roles for hypoxia and HIF activity in the developing human lung epithelium and have implications for aberrant cell fate changes in pathological lungs."],"HDBItheme":["Cardiopulmonary"],"datasets":{"GEO:GSE273089":{"id":["GEO:GSE273089"],"title":["Hypoxia promotes airway differentiation in the human lung epithelium"],"description":["Expression profiling by high throughput sequencing\nHuman embryos develop under physiological hypoxia, but how hypoxia directly affects human organogenesis remains unknown. We have investigated the effects of hypoxia on human lung epithelia using organoids. First trimester lung epithelial progenitors remain undifferentiated under normoxia, but initiate spontaneous differentiation towards multiple airway cell types, and inhibit alveolar differentiation under hypoxia. Genetic and chemical manipulation experiments showed that these effects were dependent on HIF (Hypoxia-Inducible Factor) activity, with HIF1α and HIF2α differentially regulating progenitor fate decisions. We identified the cell fate-determining transcription factors KLF4/KLF5 and ASCL1 as direct targets of the HIF pathway, promoting progenitor differentiation to basal and neuroendocrine cells respectively. Chronic hypoxia also induces transdifferentiation of human alveolar type 2 cells into airway cells via the HIF pathway, suggesting that the developmental response to hypoxia is conserved into adulthood and potentially contributes to chronic lung disease.\nTime series single cell RNA-seq of wild-type human lung progenitor organoids cultured under hypoxia for 0, 8, 16, 24, 32 days. Each time point has 2 biological replicates. Libraries prepared with Evercode Whole Transcriptome v2 kit. Each sublibrary is a mixture of all 10 barcoded samples (5 time points x 2 replicates). Sample loading information is attached for demultiplexing."],"authors":{"ORCID:0000-0003-1216-7740":{"id":["ORCID:0000-0003-1216-7740"],"family_name":["Dong"],"personal_name":["Ziqi"],"affiliations":{"ROR:013meh722":{"organization_name":["University of Cambridge"]},"ROR:00f54p054":{"organization_name":["Stanford University"]},"ROR:00fp3ce15":{"organization_name":["The Gurdon Institute"]}}},"ORCID:0000-0001-7426-3792":{"id":["ORCID:0000-0001-7426-3792"],"family_name":["Rawlins"],"personal_name":["Emma L."],"affiliations":{"ROR:00fp3ce15":{"organization_name":["The Gurdon Institute"]}},"HDBItheme":["Cardiopulmonary"]}},"tags":["sequencing"]},"GEO:GSE272859":{"id":["GEO:GSE272859"],"title":["Hypoxia promotes airway differentiation in the human lung epithelium [DamID-seq]"],"description":["Human embryos develop under physiological hypoxia, but how hypoxia directly affects human organogenesis remains unknown. We have investigated the effects of hypoxia on human lung epithelia using organoids. First trimester lung epithelial progenitors remain undifferentiated under normoxia, but initiate spontaneous differentiation towards multiple airway cell types, and inhibit alveolar differentiation under hypoxia. Genetic and chemical manipulation experiments showed that these effects were dependent on HIF (Hypoxia-Inducible Factor) activity, with HIF1α and HIF2α differentially regulating progenitor fate decisions. We identified the cell fate-determining transcription factors KLF4/KLF5 and ASCL1 as direct targets of the HIF pathway, promoting progenitor differentiation to basal and neuroendocrine cells respectively. Chronic hypoxia also induces transdifferentiation of human alveolar type 2 cells into airway cells via the HIF pathway, suggesting that the developmental response to hypoxia is conserved into adulthood and potentially contributes to chronic lung disease."],"authors":{"ORCID:0000-0003-1216-7740":{"id":["ORCID:0000-0003-1216-7740"],"family_name":["Dong"],"personal_name":["Ziqi"],"affiliations":{"ROR:013meh722":{"organization_name":["University of Cambridge"]},"ROR:00f54p054":{"organization_name":["Stanford University"]},"ROR:00fp3ce15":{"organization_name":["The Gurdon Institute"]}}},"ORCID:0009-0002-4330-5333":{"id":["ORCID:0009-0002-4330-5333"],"family_name":["Niek"],"personal_name":["Wit"],"affiliations":{"ROR:013meh722":{"organization_name":["University of Cambridge"]}}},"ORCID:0000-0001-9236-8221":{"id":["ORCID:0000-0001-9236-8221"],"family_name":["Dubal"],"personal_name":["Dnyanesh"],"affiliations":{"ROR:01vdt8f48":{"organization_name":["MRC Mitochondrial Biology Unit"]}}},"ORCID:0000-0002-2744-0810":{"id":["ORCID:0000-0002-2744-0810"],"family_name":["van den Ameele"],"personal_name":["Jelle"],"affiliations":{"ROR:013meh722":{"organization_name":["University of Cambridge"]}}},"ORCID:0000-0002-0248-1632":{"id":["ORCID:0000-0002-0248-1632"],"family_name":["Nathan"],"personal_name":["James A."],"affiliations":{"ROR:013meh722":{"organization_name":["University of Cambridge"]}}},"ORCID:0000-0001-7426-3792":{"id":["ORCID:0000-0001-7426-3792"],"family_name":["Rawlins"],"personal_name":["Emma L."],"affiliations":{"ROR:00fp3ce15":{"organization_name":["The Gurdon Institute"]}},"HDBItheme":["Cardiopulmonary"]}},"tags":["sequencing"]},"GEO:GSE272860":{"id":["GEO:GSE272860"],"title":["Hypoxia promotes airway differentiation in the human lung epithelium [Bulk RNA-seq]"],"description":["Expression profiling by high throughput sequencing\nHuman embryos develop under physiological hypoxia, but how hypoxia directly affects human organogenesis remains unknown. We have investigated the effects of hypoxia on human lung epithelia using organoids. First trimester lung epithelial progenitors remain undifferentiated under normoxia, but initiate spontaneous differentiation towards multiple airway cell types, and inhibit alveolar differentiation under hypoxia. Genetic and chemical manipulation experiments showed that these effects were dependent on HIF (Hypoxia-Inducible Factor) activity, with HIF1α and HIF2α differentially regulating progenitor fate decisions. We identified the cell fate-determining transcription factors KLF4/KLF5 and ASCL1 as direct targets of the HIF pathway, promoting progenitor differentiation to basal and neuroendocrine cells respectively. Chronic hypoxia also induces transdifferentiation of human alveolar type 2 cells into airway cells via the HIF pathway, suggesting that the developmental response to hypoxia is conserved into adulthood and potentially contributes to chronic lung disease.\nBulk RNA-seq of human lung progenitor organoids transfected with CRISPRi and gRNA lentiviral constructs of non-targeting control or HIF1a-gRNA. Organoids cultured under normoxia or hypoxia for 9 days. In total 3 conditions: normoxia + NTC; hypoxia + NTC; hypoxia + HIF1a-targeting. Each condition has 2 different gRNA replicates and 3 biological replicates, in total 6 replicates."],"authors":{"ORCID:0000-0003-1216-7740":{"id":["ORCID:0000-0003-1216-7740"],"family_name":["Dong"],"personal_name":["Ziqi"],"affiliations":{"ROR:013meh722":{"organization_name":["University of Cambridge"]},"ROR:00f54p054":{"organization_name":["Stanford University"]},"ROR:00fp3ce15":{"organization_name":["The Gurdon Institute"]}}},"ORCID:0000-0001-7426-3792":{"id":["ORCID:0000-0001-7426-3792"],"family_name":["Rawlins"],"personal_name":["Emma L."],"affiliations":{"ROR:00fp3ce15":{"organization_name":["The Gurdon Institute"]}},"HDBItheme":["Cardiopulmonary"]}},"tags":["sequencing"]},"GEO:GSE296547":{"id":["GEO:GSE296547"],"title":["Hypoxia promotes airway differentiation in the human lung epithelium [scRNAseq_hypoxia_AT2]"],"description":["Expression profiling by high throughput sequencing\nHuman early embryos develop under physiological hypoxia, but how hypoxia regulates human lung organogenesis remains little known. We have investigated oxygen availability effects on the human lung epithelium using organoids. We find first-trimester lung epithelial progenitors remain undifferentiated under normoxia, but spontaneously differentiate towards multiple airway cell types and inhibit alveolar differentiation under hypoxia. Using chemical and genetic tools, we demonstrate that hypoxia-induced airway differentiation is dependent on HIF (Hypoxia-Inducible Factor) pathways, with HIF1α and HIF2α differentially regulating fate decisions. Transcription factors KLF4 and KLF5 are direct targets of the HIF pathway and promote progenitor differentiation to basal and secretory cells. Chronic hypoxia also reprogrammed human alveolar type 2 cells to airway cells via the HIF pathway. Our results reveal roles for hypoxia and HIF signalling during human lung development and have implications for aberrant cell fate decisions in chronic lung diseases.\nTime series single cell RNA-seq of human fetal lung-derived AT2 (fdAT2) cells isolated from one donor cultured under hypoxia for 0, 6, 15, 30 days, and back to normoxia for 6, 15, 30 days. The two sub-libraries were prepared with Evercode Whole Transcriptome v3 mini kit. Each sublibrary is a mixture of all 7 barcoded samples (7 time points). Sample loading information is attached for demultiplexing."],"authors":{"ORCID:0000-0003-1216-7740":{"id":["ORCID:0000-0003-1216-7740"],"family_name":["Dong"],"personal_name":["Ziqi"],"affiliations":{"ROR:013meh722":{"organization_name":["University of Cambridge"]},"ROR:00f54p054":{"organization_name":["Stanford University"]},"ROR:00fp3ce15":{"organization_name":["The Gurdon Institute"]}}},"ORCID:0000-0001-7426-3792":{"id":["ORCID:0000-0001-7426-3792"],"family_name":["Rawlins"],"personal_name":["Emma L."],"affiliations":{"ROR:00fp3ce15":{"organization_name":["The Gurdon Institute"]}},"HDBItheme":["Cardiopulmonary"]}},"tags":["sequencing"]}}}