{"doi":"10.1016/j.xpro.2025.103667","title":"Protocol for the differentiation of human alveolar epithelial type I cells from pluripotent stem cell-derived type II-like cells","abstract":"Pulmonary alveolar epithelial type I (AT1) cells have a flattened morphology to permit the diffusion of oxygen into the capillaries and historically have been difficult to isolate or maintain in culture. Here, we present a protocol for generating human alveolar type I-like cells (induced pluripotent stem cell-derived AT1s [iAT1s]) from induced pluripotent stem cell-derived alveolar epithelial type II cells (iAT2s) in vitro . We describe steps to plate iAT1s in either 3D or air-liquid interface cultures and to analyze or isolate iAT1s via flow cytometry. For complete details on the use and execution of this protocol, please refer to Burgess et al. 1 • Steps for differentiating AT1-like cells from iPSC-derived AT2 cells in 3D culture • Instructions for analyzing AT1-like cells using flow cytometry • Guidance on transitioning AT1-like cells to an air-liquid interface culture Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Pulmonary alveolar epithelial type I (AT1) cells have a flattened morphology to permit the diffusion of oxygen into the capillaries and historically have been difficult to isolate or maintain in culture. Here, we present a protocol for generating human alveolar type I-like cells (induced pluripotent stem cell-derived AT1s [iAT1s]) from induced pluripotent stem cell-derived alveolar epithelial type II cells (iAT2s) in vitro . We describe steps to plate iAT1s in either 3D or air-liquid interface cultures and to analyze or isolate iAT1s via flow cytometry.","journal":"STAR Protocols","year":2025,"id":550260,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":2,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9492,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1203466,"name":"Lauren J. Ayers","orcid":null,"position":1,"is_corresponding":false},{"id":579037,"name":"Kasey Minakin","orcid":null,"position":2,"is_corresponding":false},{"id":275576,"name":"Konstantinos–Dionysios Alysandratos","orcid":"0000-0002-5611-0343","position":3,"is_corresponding":false},{"id":266489,"name":"Xaralabos Varelas","orcid":"0000-0002-2882-4541","position":4,"is_corresponding":false},{"id":228731,"name":"Darrell N. Kotton","orcid":"0000-0002-9604-8476","position":5,"is_corresponding":false},{"id":790268,"name":"Claire L. Burgess","orcid":"0000-0001-9609-8839","position":0,"is_corresponding":true}],"reference_count":10,"raw_metadata":null,"created_at":"2026-07-19T02:54:16.596730Z","pmid":"40048418","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}