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.

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.

As a secreted morphogen, Sonic Hedgehog (SHH) determines differential cell fates, behaviors, and functions by forming a gradient of Hedgehog (Hh) activation along an axis of Hh-receptive cells during development. Despite clearly delineated roles for Hh during organ morphogenesis, whether Hh continues to regulate cell fate and behavior in the same fashion in adult organs is less understood. Adult organs, particularly barrier organs interfacing with the ambient environment, are exposed to insults that require renewal of cellular populations to maintain structural integrity. Understanding key aspects of Hh’s ability to generate an organ could translate into conceptual understanding of Hh’s ability to maintain organ homeostasis and stimulate regeneration. In this review, we will summarize the current knowledge about Hh signaling in regulating adult lung regeneration and maintenance, and discuss how alteration of Hh signaling contributes to adult lung diseases.

Understanding differential lineage potential of orthologous stem cells across species can shed light on human disease. Here, utilizing 3D organoids, single cell RNAseq, and xenotransplants, we demonstrate that human alveolar type 2 cells (hAEC2s), unlike murine AEC2s, are multipotent and able to transdifferentiate into KRT5+ basal cells when co-cultured with primary fibroblasts in 3D organoids. Trajectory analyses and immunophenotyping of epithelial progenitors in idiopathic pulmonary fibrosis (IPF) indicate that hAEC2s transdifferentiate into metaplastic basal cells through alveolar-basal intermediate (ABI) cells that we also identify in hAEC2-derived organoids. Modulating hAEC2-intrinsic and niche factors dysregulated in IPF can attenuate metaplastic basal cell transdifferentiation and preserve hAEC2 identity. Finally, hAEC2s transplanted into fibrotic immune-deficient murine lungs engraft as either hAEC2s or differentiated KRT5+ basal cells. Our study indicates that hAEC2s-loss and expansion of alveolar metaplastic basal cells in IPF are causally connected, which would not have been revealed utilizing murine AEC2s as a model. Highlights Human AEC2s transdifferentiate into KRT5+ basal cells when accompanied by primary adult human lung mesenchyme in 3D organoid culture. Alterations of hAEC2-intrinsic and niche factors dysregulated in IPF can modify metaplastic hAEC2 transdifferentiation. hAEC2s engraft into fibrotic lungs of immune-deficient mice and transdifferentiate into metaplastic basal cells. Transcriptional trajectory analysis suggests that hAEC2s in IPF gives rise to metaplastic basal cells via alveolar-basal intermediate cells.

Most of the lung’s gas-exchange surface forms during alveologenesis and its disruption causes bronchopulmonary dysplasia (BPD) in infants, characterized by alveolar simplification and myofibroblast accumulation. BPD also increases the risk of adult emphysema, marked by alveolar loss. Despite this connection, mechanisms linking these conditions and effective treatments are still lacking. We identify hedgehog-interacting protein ( HHIP ), associated with both BPD and emphysema, as a critical regulator of alveologenesis. During this process, Hhip -expressing cells expanded, accompanied by hedgehog (Hh) signaling inhibition and myofibroblast transition. Stromal-specific Hhip deletion led to hyperactivation of Hh-IGF1 signaling axis, causing persistent SMA ⁺ myofibroblasts and epithelial stem/progenitor cell senescence. Hyperactivation of this pathway was also observed in human BPD and hyperoxia-induced BPD models. Early Hhip deficiency resulted in adult emphysema with myofibroblast accumulation. We developed a therapeutic Fc-fused HHIP protein that mitigated BPD in neonatal mice and prevented adult emphysema. These findings establish HHIP as a critical regulator of alveologenesis and a therapeutic target for BPD and emphysema.