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Bud tip progenitors (BTPs) in the developing lung give rise to all epithelial cell types found in the airways and alveoli. This work aimed to develop an iPSC organoid model enriched with NKX2-1+ BTP-like cells. Building on previous studies, we optimized a directed differentiation paradigm to generate spheroids with more robust NKX2-1 expression. Spheroids were expanded into organoids that possessed NKX2-1+/CPM+ BTP-like cells, which increased in number over time. Single cell RNA-sequencing analysis revealed a high degree of transcriptional similarity between induced BTPs (iBTPs) and in vivo BTPs. Using FACS, iBTPs were purified and expanded as induced bud tip progenitor organoids (iBTOs), which maintained an enriched population of bud tip progenitors. When iBTOs were directed to differentiate into airway or alveolar cell types using well-established methods, they gave rise to organoids composed of organized airway or alveolar epithelium, respectively. Collectively, iBTOs are transcriptionally and functionally similar to in vivo BTPs, providing an important model for studying human lung development and differentiation.

Recent advances using single cell genomic approaches have identified new epithelial cell types and uncovered cellular heterogeneity in the murine and human lung (1). Here, using scRNA-seq and microscopy we identify and describe a secretory-like cell that is enriched in the small airways of the developing human lung and identified by the unique co-expression of SCGB3A2/SFTPB/CFTR. To place these cells in the hierarchy of airway development, we apply a single cell barcode-based lineage tracing method track the fate of SCGB3A2/SFTPB/CFTR cells during airway organoid differentiation in vitro (2). Lineage tracing revealed that these cells have distinct developmental potential from basal cells, giving rise predominantly to pulmonary neuroendocrine cells (PNECs) and a subset of multiciliated cells distinguished by high C6 and low MUC16 expression. We conclude that SCGB3A2/SFTPB/CFTR cells act as a progenitor cell contributing to the cellular diversity and heterogeneity in the developing human airway.SIGNIFICANCE STATEMENTThe current study identifies a novel secretory cell type that is present predominantly in the small airway of the developing human lung. These secretory cells are defined by co-expression of SCGB3A2/SFTPB/CFTR, and functional studies show that this cell gives rise to pulmonary neuroendocrine cells and a sub-population of multiciliated cells, thereby leading to cellular heterogeneity.

Bud tip progenitors (BTPs) in the developing lung give rise to all epithelial cell types found in the airways and alveoli. The current work aimed to develop an iPSC organoid model enriched with stable NKX2-1+ BTP-like cells. Building on prior work, we optimized a directed differentiation paradigm to generate spheroids with robust NKX2-1 expression. Spheroids were expanded into organoids that possessed NKX2-1+/CPM+ BTP-like cells, which increased in number over time. Single cell RNA-sequencing analysis revealed a high degree of transcriptional similarity between induced BTPs (iBTPs) and in vivo BTPs. Using FACS, iBTPs can be purified and expanded as induced bud tip organoids (iBTO), which maintain an enriched population of bud tip progenitors. When iBTOs are directed to differentiate into airway or alveolar cell types using well-established methods, they give rise to organoids composed of organized airway or alveolar epithelium, respectively. Collectively, iBTOs are transcriptionally and functionally similar to in vivo BTPs, providing an important model to study human lung development and differentiation.SUMMARY STATEMENTiPSC-derived lung bud tip progenitors emerge in organoid culture, can be isolated and expanded, are transcriptionally similar to primary bud tip progenitors, and can differentiate into airway or alveolar organoids.

Mapping the spatial distribution and molecular identity of constituent cells is essential for understanding tissue dynamics in health and disease. We lack a comprehensive map of human distal airways, including the terminal and respiratory bronchioles (TRBs), which are implicated in respiratory diseases1-4. Here, using spatial transcriptomics and single-cell profiling of microdissected distal airways, we identify molecularly distinct TRB cell types that have not-to our knowledge-been previously characterized. These include airway-associated LGR5+ fibroblasts and TRB-specific alveolar type-0 (AT0) cells and TRB secretory cells (TRB-SCs). Connectome maps and organoid-based co-cultures reveal that LGR5+ fibroblasts form a signalling hub in the airway niche. AT0 cells and TRB-SCs are conserved in primates and emerge dynamically during human lung development. Using a non-human primate model of lung injury, together with human organoids and tissue specimens, we show that alveolar type-2 cells in regenerating lungs transiently acquire an AT0 state from which they can differentiate into either alveolar type-1 cells or TRB-SCs. This differentiation programme is distinct from that identified in the mouse lung5-7. Our study also reveals mechanisms that drive the differentiation of the bipotent AT0 cell state into normal or pathological states. In sum, our findings revise human lung cell maps and lineage trajectories, and implicate an epithelial transitional state in primate lung regeneration and disease.

The human respiratory epithelium is derived from a progenitor cell in the distal buds of the developing lung. These ""bud tip progenitors'' are regulated by reciprocal signaling with surrounding mesenchyme; however, mesenchymal heterogeneity and function in the developing human lung are poorly understood. We interrogated single-cell RNA sequencing data from multiple human lung specimens and identified a mesenchymal cell population present during development that is highly enriched for expression of theWNT agonist RSPO2, and we found that the adjacent bud tip progenitors are enriched for the RSPO2 receptor LGR5. Functional experiments using organoid models, explant cultures, and FACS-isolated RSPO2(+) mesenchyme show that RSPO2 is a critical niche cue that potentiates WNT signaling in bud tip progenitors to support their maintenance and multipotency., This project has been made possible in part by grant number CZF2019-002440 from the Chan Zuckerberg Initiative DAF, an advised fund from the Silicon Valley Community Foundation, and in part by the NIH-NHLBI (R01HL119215) funding to J.R.S.; R.F.C.H. was supported by a NIH Tissue En- gineering and Regenerative Medicine Training Grant (NIH-NIDCR T32DE 007057) and by a Ruth L. Kirschstein Predoctoral Individual National Research Service Award (NIH-NHLBI F31HL152531); A.J.M. was supported by a Ruth L. Kirschstein Predoctoral Individual National Research Service Award (NIH- NHLBI F31HL142197); E.M.H. was supported by a Ruth L. Kirschstein Predoc- toral Individual National Research Service Award (NIH-NHBLI F31HL146162); T.F. was supported by a NIH Tissue Engineering and Regenerative Medicine Training Grant (NIH-NIDCR T32DE007057); A.S.C. was supported by the T32 Michigan Medical Scientist Training Program (5T32GM007863-40); I.G. and the University of Washington Laboratory of Developmental Biology was supported by NIH award number 5R24HD000836 from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD). B.R. is a fellow of the Branco Weiss Foundation (Switzerland) and the National Research Foundation (Singapore), and an A*STAR and EMBO Young Investi- gator. This work was supported by an inaugural Use-Inspired Basic Research (UIBR) central fund from the Agency for Science, Technology and Research (A*STAR) and National Medical Research Council Open Fund– Individual Research Grants (OF-YIRG,#OFYIRG18May-0053; and OF-IRG, #OFIRG20- Nov-0057), to E.S.-R and B.R., respectively. We would like to thank Judy Opp and the University of Michigan Advanced Genomics core for the operation of the 103 chromium single-cell capture plat- form, the University of Michigan Microscopy core for providing access to confocal microscopes, the Flow Cytometry core for providing access to flow cytometers, and the Vector core for providing lentivirus production and adeno- virus purification and expansion services. We would also like to thank the Uni- versity of Washington Laboratory of Developmental Biology. Lastly, we would like to thank Michael Dame and the Translational Tissue Modeling Laboratory (TTML) for providing helpful suggestions regarding the use of WNT3a afamin conditioned media as well as Lindy K. Brastrom for her careful technical editing of the manuscript.

SUMMARYMammalian respiratory system development is regulated by complex reciprocal signaling events that take place between epithelial cells and the surrounding mesenchymal cells; however, mesenchymal heterogeneity and function in the developing human lung is poorly understood. We interrogated single cell RNA sequencing data from multiple human lung specimens and identified a mesenchymal cell population present during development that is highly enriched for expression of the WNT agonistR-SPONDIN2(RSPO2), and we found that adjacent epithelial bud tip progenitors are enriched for the RSPO2 receptorLGR5. By carrying out functional experiments using organoid models, lung explant cultures, and FACS-isolatedRSPO2+mesenchyme, we show that RSPO2 is a critical niche cue that potentiates WNT signaling in human lung progenitors to maintain their multipotency.

The human respiratory epithelium is derived from a progenitor cell in the distal buds of the developing lung. These "bud tip progenitors" are regulated by reciprocal signaling with surrounding mesenchyme; however, mesenchymal heterogeneity and function in the developing human lung are poorly understood. We interrogated single-cell RNA sequencing data from multiple human lung specimens and identified a mesenchymal cell population present during development that is highly enriched for expression of the WNT agonist RSPO2, and we found that the adjacent bud tip progenitors are enriched for the RSPO2 receptor LGR5. Functional experiments using organoid models, explant cultures, and FACS-isolated RSPO2

An abundance of information about lung development in animal models exists; however, comparatively little is known about lung development in humans. Recent advances using primary human lung tissue combined with the use of human in vitro model systems, such as human pluripotent stem cell-derived tissue, have led to a growing understanding of the mechanisms governing human lung development. They have illuminated key differences between animal models and humans, underscoring the need for continued advancements in modeling human lung development and utilizing human tissue. This review discusses the use of human tissue and the use of human in vitro model systems that have been leveraged to better understand key regulators of human lung development and that have identified uniquely human features of development. This review also examines the implementation and challenges of human model systems and discusses how they can be applied to address knowledge gaps.