Single-cell RNA-sequencing has transformed our understanding of alveolar epithelial type 2 (AT2) cells and alveolar lipofibroblasts (LIFs) during lung injury and repair. Both cell types undergo dynamic transitions through intermediate states that determine whether the lung proceeds toward regeneration or fibrosis. Emerging evidence highlights reciprocal paracrine signaling between AT2/AT1 transitional cells and LIF-derived myofibroblasts (aMYFs) as a key regulatory axis. Among these, amphiregulin (AREG)–EGFR signaling functions as a central profibrotic pathway whose inhibition can restore alveolar differentiation and repair. The human WI-38 fibroblast model provides a practical platform to study the reversible LIF–MYF switch and screen antifibrotic and pro-regenerative compounds. Candidate therapeutics including metformin, haloperidol and FGF10 show promise in reprogramming fibroblast and epithelial states through metabolic and signaling modulation. Integrating WI-38-based assays, alveolosphere co-cultures, and multi-omics profiling offers a translational framework for identifying interventions that halt fibrosis and actively induce lung regeneration. This review highlights a unifying framework in which epithelial and mesenchymal plasticity converge to define repair outcomes and identifies actionable targets for promoting alveolar regeneration in chronic lung disease.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease with limited treatment options. Depression and anxiety are common comorbidities in patients with IPF, and emerging evidence suggests that neuroactive pathways may also influence fibrotic remodeling. On this basis, we investigated desipramine, a tricyclic antidepressant, as a potential modulator of fibroblast state in lung fibrosis. Desipramine was identified in an FDA-approved compound screen as a pro-lipogenic hit in TGF-β-stimulated fibroblasts and was subsequently evaluated across a stepwise validation pipeline of increasing biological complexity. In WI-38 fibroblasts, desipramine was well tolerated at 10 μM and reduced myofibroblast-associated features while increasing lipid-associated staining. In a fibroblast-supported alveolosphere assay, desipramine altered qualitative organoid clustering and changed the transcript levels of specific mesenchymal markers under profibrotic stimulation, whereas direct treatment of MLE-12 epithelial cells did not elicit a consistent response. While desipramine demonstrated pro-lipogenic and anti-myofibroblastic phenotypic shifts in reductionist 2D cultures, these effects failed to translate robustly into complex 3D human lung tissue slices or in vivo disease models. Ultimately, our findings highlight the critical necessity of utilizing complex translational pipelines to rigorously validate early screening hits before therapeutic efficacy is assumed.

The heparan sulfate mimetic OTR4120, previously described for its antifibrotic activity, acts as a regenerative agent in the lung by reprogramming fibroblasts toward a regenerative state that sustains AT2 identity and activates embryonic epithelial programs. To define its primary cellular target and mode of action, we performed single-cell RNA sequencing on human fibroblasts exposed to TGF-β1 or vehicle, followed by treatment with OTR4120 or vehicle. This analysis revealed that OTR4120 reprograms TGF-β1–induced myofibroblasts toward a regenerative, lipofibroblast-like transcriptional state. To assess the functional consequences of this fibroblast reprogramming on epithelial behavior, we established a mixed-species lung organoid model combining human fibroblasts and primary mouse AT2 cells, enabling compartment-specific transcriptional profiling. In the presence of OTR4120, organoid number and size were increased irrespective of fibroblast activation state, while AT2 cells retained lineage identity and exhibited activation of embryonic and regenerative gene programs. Crosstalk analysis identified ligand–receptor interactions associated with regenerative signaling between fibroblasts and epithelial cells, consistent with a fibroblast-driven regenerative switch. In conclusion, our results establish OTR4120 as a regenerative agent in the lung that extends beyond antifibrotic activity by reprogramming fibroblasts and promoting epithelial regeneration. AT2 cells emerge as active contributors to this process, highlighting a fibroblast–epithelial regenerative axis with potential relevance for lung diseases characterized by epithelial injury, including chronic obstructive pulmonary disease and bronchopulmonary dysplasia.

Idiopathic Pulmonary Fibrosis (IPF) is a fatal, progressive lung disease characterized by excessive extracellular matrix (ECM) deposition and impaired lung function. Current treatments are limited and not curative. RGTA® compounds, heparan sulfate mimetics like OTR4120, have shown the ability to modulate ECM and support tissue repair, making them strong candidates for IPF therapy. To evaluate OTR4120’s antifibrotic potential, we used ex vivo human precision-cut lung slices (hPCLS), WI-38 cells, an in vitro model for myofibroblast differentiation, and an in vivo bleomycin-induced lung fibrosis mouse model. Collagen deposition in hPCLS was quantified via second harmonic generation (SHG) microscopy. Transcriptomic changes in WI-38 cells undergoing myofibroblast differentiation and treated with OTR4120 were analyzed via qPCR, bulk RNA-Seq, and pathway analyses. SHG microscopy revealed a significant reduction in collagen deposition in hPCLS treated with the fibrotic cocktail and OTR4120. In WI-38 cells, OTR4120 treatment downregulated myofibroblast markers in both preventive and therapeutic approaches. Bulk RNA-Seq highlighted significant impacts on fibrosis-related processes, with marked downregulation of TGF-β1 signaling while the Reactome analysis revealed a reduction in immune-related pathways, suggesting other immunomodulatory effects. Co-treatment with the TGF-β1 receptor inhibitor SB431542 further decreased MYF markers, indicating other mechanisms at play. In the in vivo bleomycin-induced lung fibrosis mouse model, OTR4120 promoted fibrosis resolution and restored alveolar architecture. In conclusion, OTR4120 demonstrates potent antifibrotic effects, positioning it as a promising treatment for fibrosis.