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 .

GWAS have repeatedly mapped susceptibility loci for emphysema to genes that modify hedgehog signaling, but the functional relevance of hedgehog signaling to this morbid disease remains unclear. In the current study, we identified a broad population of mesenchymal cells in the adult murine lung receptive to hedgehog signaling, characterized by higher activation of hedgehog surrounding the proximal airway relative to the distal alveoli. Single-cell RNA-sequencing showed that the hedgehog-receptive mesenchyme is composed of mostly fibroblasts with distinct proximal and distal subsets with discrete identities. Ectopic hedgehog activation in the distal fibroblasts promoted expression of proximal fibroblast markers and loss of distal alveoli and airspace enlargement of over 20% compared with controls. We found that hedgehog suppressed mesenchymal-derived mitogens enriched in distal fibroblasts that regulate alveolar stem cell regeneration and airspace size. Finally, single-cell analysis of the human lung mesenchyme showed that segregated proximal-distal identity with preferential hedgehog activation in the proximal fibroblasts was conserved between mice and humans. In conclusion, we showed that differential hedgehog activation segregates mesenchymal identities of distinct fibroblast subsets and that disruption of fibroblast identity can alter the alveolar stem cell niche, leading to emphysematous changes in the murine lung.

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.

We engineered an ultrasensitive reporter of p16 INK4a , a biomarker of cellular senescence. Our reporter detected p16 INK4a -expressing fibroblasts with certain senescent characteristics that appeared shortly after birth in the basement membrane adjacent to epithelial stem cells in the lung. Furthermore, these p16 INK4a+ fibroblasts had enhanced capacity to sense tissue inflammation and respond through their increased secretory capacity to promote epithelial regeneration. In addition, p16 INK4a expression was required in fibroblasts to enhance epithelial regeneration. This study highlights a role for p16 INK4a+ fibroblasts as tissue-resident sentinels in the stem cell niche that monitor barrier integrity and rapidly respond to inflammation to promote tissue regeneration.