Lung Organoid Models Enable Interrogation of Progenitor Cell Function and Cellular Heterogeneity of the Developing Human Lung

Abstract

The development and functional maturation of the human lung are intricate processes regulated by a complex interplay of cellular and molecular events. Understanding the underlying mechanisms governing lung development is crucial for unraveling the pathogenesis of various respiratory diseases and advancing regenerative medicine approaches. In recent years, the emergence of deep sequencing techniques and the expansion of organoid technology has provided a powerful platform for investigating human tissues and to model organ development in previously unattainable ways. This dissertation presents a comprehensive exploration of lung development through the utilization of both these tools. Chapter 1 presents a thorough review of the current knowledge surrounding lung development including key molecular and cellular processes involved in lung organogenesis, including epithelial-mesenchymal signaling, branching morphogenesis, and alveolar maturation. Additionally, the limitations and advantages of organoid models as tools for recapitulating lung development are critically evaluated. To investigate lung development using organoid models, the dissertation presents a series of projects that encompass various aspects of lung organogenesis. The first project, in Chapter 2, encompasses the characterization of SCGB3A2+ Lower Airway Progenitor (LAP) cells, a novel, previously undefined cell type that we identified during human lung development using single-cell sequencing. The utilization of lung organoid models coupled with novel lineage tracing techniques elucidates the function of LAP cells as a progenitor for pulmonary neuroendocrine cells and a subset of multiciliated cells with regional specificity. Together this work highlights the importance of understanding normal development in a uniquely human context. The second project, presented in Chapter 3, illustrates an optimized protocol for generating lung organoids from pluripotent stem cells that mimic a specialized cell population, the bud tip progenitor, which are the progenitor of all epithelial cell types in the lung. Their functional competence to give rise to both airway and alveolar differentiation is also demonstrated. Finally, the resulting organoids are benchmarked using single-cell sequencing, presenting a clear example of how this data serves as a roadmap to improve hPSC-derived lung organoid models, refine culture methods, and evaluate transcriptional similarity to primary tissue and other organoid models. Overall, this dissertation provides a comprehensive investigation into lung development using organoid models. The findings contribute to our understanding of the cellular and molecular mechanisms governing lung organogenesis and emphasize the need for robust and accurate in vitro models to advance our understanding of human biology and improve clinical translatability. Moreover, this research underscores the significance of organoid technology as a versatile tool for studying organ development, disease modeling, and personalized medicine.