Human Pluripotent Stem Cell-Derived Alveolar Organoids for Gene-editing and Lung Adenocarcinomas Modeling

Human pluripotent stem cell (hPSC)-derived alveolar organoids have emerged as valuable tools for studying lung development, modeling pulmonary diseases, and drug discovery, though their application has been hindered by laborious differentiation protocols and technical complexity. Here, we present an hPSC_x0002_derived alveolar organoid (hALO) system with exceptional long-term expandability (>30 passages), efficient cryopreservation resilience, and streamlined production achieved through earlier 3D culture initiation and elimination of cell sorting requirements. Transcriptomic analysis across passages confirmed hALOs contain alveolar progenitors and AT2 lineages, recapitulating pseudoglandular-to-canalicular development while partially maintaining adult AT2 immune-related functions. The system permits alveolar epithelial differentiation via pharmacological modulation of WNT/YAP signaling or through orthotopic transplantation, while multiplex genetic engineering enables programmable disease modeling and adenocarcinoma pathogenesis studies. These versatile capabilities establish hALOs as a robust dual-phase platform for mechanistic investigation of lung epithelial biology and disease modeling across in vitro and in vivo environments. scRNA-seq of pluripotent stem cell-derived alveolar organoids at early (P3), middle (P7) and late (P20) passages

人多能干细胞（human pluripotent stem cell, hPSC）来源的肺泡类器官已成为研究肺发育、构建肺部疾病模型及开展药物发现的重要工具，但其应用曾因分化流程繁琐、技术复杂度较高而受到限制。本研究报道了一种人多能干细胞来源的肺泡类器官（hALO）培养体系，该体系具备优异的长期扩增能力（可传代超过30次）、高效的冷冻复苏能力，并通过提前启动三维培养、省去细胞分选步骤实现了生产流程的简化。转录组测序分析证实，不同传代批次的hALOs均包含肺泡祖细胞与肺泡II型上皮细胞（alveolar type 2, AT2）谱系，可重现肺发育从假腺期到小管期的进程，同时部分保留了成人肺泡II型上皮细胞的免疫相关功能。该体系可通过药物调控WNT/YAP信号通路诱导肺泡上皮细胞分化，或通过原位移植实现体内分化；结合多重基因编辑技术，还可实现定制化疾病模型构建以及腺癌发病机制研究。这些多样的功能使hALOs成为一款稳定的双相平台，可用于肺上皮生物学的机制探究，以及在体外与体内环境中开展疾病模型构建。针对不同传代阶段（早期P3、中期P7、晚期P20）的多能干细胞来源肺泡类器官的单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）