The cell type specific sequences of transcriptional programs during lung regeneration have remained elusive. Using time-series single cell RNA-seq of the bleomycin lung injury model, we resolved transcriptional dynamics for 28 cell types. Trajectory modeling together with lineage tracing revealed that airway and alveolar stem cells converge on a unique Krt8 + transitional stem cell state during alveolar regeneration. These cells have squamous morphology, feature p53 and NFkB activation and display transcriptional features of cellular senescence. The Krt8+ state appears in several independent models of lung injury and persists in human lung fibrosis, creating a distinct cell-cell communication network with mesenchyme and macrophages during repair. We generated a model of gene regulatory programs leading to Krt8+ transitional cells and their terminal differentiation to alveolar type-1 cells. We propose that in lung fibrosis, perturbed molecular checkpoints on the way to terminal differentiation can cause aberrant persistence of regenerative intermediate stem cell states.

The respiratory system, which includes the trachea, airways, and distal alveoli, is a complex multi-cellular organ that intimately links with the cardiovascular system to accomplish gas exchange. In this review and as members of the NIH/NHLBI-supported Progenitor Cell Translational Consortium, we discuss key aspects of lung repair and regeneration. We focus on the cellular compositions within functional niches, cell-cell signaling in homeostatic health, the responses to injury, and new methods to study lung repair and regeneration. We also provide future directions for an improved understanding of the cell biology of the respiratory system, as well as new therapeutic avenues.

Highlights d Supervised scRNA-seq uncovers unexpected heterogeneity within club-like cells d Specialized epithelial progenitors with unique features hide among mature cells d Different stem/progenitors are activated in region and injurydependent manner d Transplantation of expanded progenitors rescues lung function of injured mice

No abstract

revious studies in mice have shown that mouse alveolar type 2 cells (mAEC2s) are the resident stem cell population in the alveoli that constitute the entire gas exchange surface of the lung 1,2 . In idiopathic pulmonary fibrosis (IPF), the most deadly and prevalent form of diffuse parenchymal lung disease, human alveolar type 2 cells (hAEC2s) are lost from the alveoli, concurrent with the appearance of metaplastic alveolar KRT5 + basal cells, which normally appear in the conducting airways [3][4][5][6][7][8][9] . Rigorous genetic lineage tracing has shown that metaplastic KRT5 + cells in the murine alveoli are not derived from mAEC2s, but rather from KRT5 − /SOX2 + progenitors in the mouse airway after severe alveolar injury from fibrosis or viral infections 5,6,[10][11][12] . However, it is not clear whether a similar population in the human airway exists that contributes to metaplastic basal cells, as the airways contain key anatomic differences across the two species 13 . This is a clinically relevant question, because the extent of alveolar KRT5 + basal cells directly correlates with mortality in IPF 14 . In this study, we made a surprising finding that hAEC2s, but not mAEC2s, can readily transdifferentiate into KRT5 + basal cells in organoid culture and xenotransplant. Moreover, we define pro-fibrotic mesenchymal niche-derived factors that promote hAEC2-to-basal cell transdifferentiation. Finally, quantitative spatial analysis of IPF lung tissue reveals that basal cells and advanced alveolar-basal intermediates are surrounded by aberrant, CTHRC1 hi pro-fibrotic mesenchyme. These results identify hAEC2s as a source of metaplastic KRT5 + basal cells in severe alveolar injuries and provide a potential explanation for the reported appearance of aberrant hAEC2s with basaloid features in the transcriptomes of IPF and other severe lung injures such as COVID pneumonia 8,9 .

Choroid plexus epithelial cells (CPECs) have essential developmental and homeostatic roles related to the cerebrospinal fluid (CSF) and blood-CSF barrier they produce. Accordingly, CPEC dysfunction has been implicated in many neurological disorders, such as Alzheimer’s disease, and transplant studies have provided proof-of-concept for CPEC-based therapies. However, such therapies have been hindered by the inability to expand or generate CPECs in culture. During development, CPECs differentiate from preneurogenic neuroepithelial cells and require Bone Morphogenetic Protein (BMP) signaling, but whether BMPs suffice for CPEC induction is unknown. Here we provide evidence for BMP4 sufficiency to induce CPEC fate from neural progenitors derived from mouse embryonic stem cells (ESCs). CPEC specification by BMP4 was restricted to an early time period after neural induction in culture, with peak CPEC competency correlating to neuroepithelial cells rather than radial glia. In addition to molecular, cellular, and ultrastructural criteria, derived CPECs (dCPECs) had functions that were indistinguishable from primary CPECs, including self-assembly into secretory vesicles and integration into endogenous choroid plexus epithelium following intraventricular injection. We then used BMP4 to generate dCPECs from human ESC-derived neuroepithelial cells. These findings demonstrate BMP4 sufficiency to instruct CPEC fate, expand the repertoire of stem cell-derived neural derivatives in culture, and herald dCPEC-based therapeutic applications aimed at the unique interface between blood, CSF, and brain governed by CPECs.

Aberrant epithelial reprogramming can induce metaplastic differentiation at sites of tissue injury, culminating in transformed barriers composed of scar and metaplastic epithelium. While the plasticity of epithelial stem cells is well-characterized, the identity and role of the niche has not been delineated in metaplasia. Here we show that Gli1 + mesenchymal stromal cells (MSCs), previously shown to contribute to myofibroblasts during scarring, promote metaplastic differentiation of airway progenitors into KRT5+ basal cells. During fibrotic repair, Gli1 + MSCs integrate hedgehog activation to upregulate BMP antagonism in the progenitor niche that promotes metaplasia. Restoring the balance towards BMP activation attenuated metaplastic KRT5+ differentiation while promoting adaptive alveolar differentiation into SFTPC+ epithelium. Finally, fibrotic human lungs demonstrate altered BMP activation in the metaplastic epithelium. These findings show that Gli1 + MSCs integrate hedgehog signaling as a rheostat to control BMP activation in the progenitor niche to determine regenerative outcome in fibrosis.

Lung disease is a major health burden accounting for one in six deaths globally 1 . The lung's large surface area is exposed to a great variety of environmental and microbial insults causing injuries to its epithelium that require a regenerative response mediated by tissue-resident stem and progenitor Lung injury activates quiescent stem and progenitor cells to regenerate alveolar structures. The sequence and coordination of transcriptional programs during this process has largely remained elusive. Using single cell RNA-seq, we first generated a whole-organ bird's-eye view on cellular dynamics and cell-cell communication networks during mouse lung regeneration from ~30,000 cells at six timepoints. We discovered an injury-specific progenitor cell state characterized by Krt8 in flat epithelial cells covering alveolar surfaces. The number of these cells peaked during fibrogenesis in independent mouse models, as well as in human acute lung injury and fibrosis. Krt8+ progenitors featured a highly distinct connectome of receptor-ligand pairs with endothelial cells, fibroblasts, and macrophages. To 'sky dive' into epithelial differentiation dynamics, we sequenced >30,000 sorted epithelial cells at 18 timepoints and computationally derived cell state trajectories that were validated by lineage tracing genetic reporter mice. Airway stem cells within the club cell lineage and alveolar type-2 cells underwent transcriptional convergence onto the same Krt8+ progenitor cell state, which later resolved by terminal differentiation into alveolar type-1 cells. We derived distinct transcriptional regulators as key switch points in this process and show that induction of TNF-alpha/NFkappaB, p53, and hypoxia driven gene expression programs precede a Sox4, Ctnnb1, and Wwtr1 driven switch towards alveolar type-1 cell fate. We show that epithelial cell plasticity can induce non-gradual transdifferentiation, involving intermediate progenitor cell states that may persist and promote disease if checkpoint signals for terminal differentiation are perturbed.