Aug 2026· Frontiers in Cell and Developmental Biology· 0 citations· 76 references
TL;DR
A humanized multi-lineage lung model that captures epithelial-mesenchymal co-development and responsiveness to TGF-β1 stimulation and sensitivity to pirfenidone highlight its potential for antifibrotic drug screening.
Abstract
Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible interstitial lung disease with limited therapeutic options. Existing hiPSC-derived lung organoid models are largely restricted to single epithelial lineages and cannot endogenously integrate multiple pulmonary cell types, limiting mechanistic studies and drug screening.
We established a staged directed differentiation protocol to sequentially differentiate hiPSCs into definitive endoderm, anterior foregut endoderm, and lung progenitor cells, ultimately generating a multi-lineage lung model on Transwell inserts. The model was characterized by bright-field microscopy, hematoxylin-eosin (H&E) staining, scanning and transmission electron microscopy, immunofluorescence, quantitative real-time PCR (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), and single-cell RNA sequencing. Fibrosis-like phenotypes were induced by transforming growth factor-beta 1 (TGF-β1) stimulation. Transcriptomic similarity to human IPF was assessed by RNA sequencing (RNA-seq) combined with bidirectional gene set enrichment analysis (GSEA), and drug responsiveness was validated with pirfenidone.
Without genetic editing or exogenous cell supplementation, the model endogenously generated proximal duct-like epithelium, distal alveolar epithelium, fibroblasts, endothelial cells, and CD68
+
macrophage-like cells confirmed by immunofluorescence. Single-cell sequencing identified 13 transcriptional subclusters and revealed epithelial-mesenchymal co-development. TGF-β1 stimulation induced epithelial barrier disruption, ferroptosis-like mitochondrial ultrastructural alterations, extracellular matrix (ECM) remodeling, and aberrant secretion of multiple IPF-associated biomarkers. Transcriptomic analysis showed that upregulated genes in the fibrosis model group were significantly enriched in ECM organization, cell adhesion, and the PI3K-Akt pathway. Protein-protein interaction (PPI) network analysis identified
COL1A1
,
FN1
, and integrin family members as central hubs. Bidirectional GSEA validation confirmed that the transcriptomic signature of the model was highly similar to human IPF, with differentially expressed genes significantly enriched in three independent IPF cohorts. Pirfenidone reversed TGF-β1-induced ECM deposition and myofibroblast activation, and partially restored type II alveolar epithelial (AT2) cell marker expression.
We successfully established a humanized multi-lineage lung model that captures epithelial-mesenchymal co-development
in vitro
. Its responsiveness to TGF-β1 stimulation and sensitivity to pirfenidone highlight its potential for antifibrotic drug screening. This platform offers a humanized tool with the potential to be standardized for investigating cell fate determination during early lung development and the pathogenesis of IPF.
Idiopathic pulmonary fibrosis (IPF) is a progressive, fatal interstitial lung disease driven by aberrant repair after repetitive alveolar micro-injury. The classical paradigm - that fibrosis is a passive consequence of epithelial injury followed by fibroblast activation - has been substantially reshaped by single-cell RNA sequencing (scRNA-seq) and, more recently, spatial transcriptomics. These technologies have resolved the lung into a constellation of molecularly distinct cell states and exposed a highly structured fibrotic niche in which epithelial, mesenchymal, immune, and endothelial compartments are engaged in active, ligand-receptor-mediated crosstalk. This review synthesizes the emerging single-cell and spatial atlas of human PF/ILD, with a focus on four themes: (i) the ectopic KRT5⁻/KRT17⁺ aberrant basaloid epithelium and the persistence of a KRT8⁺ alveolar differentiation intermediate; (ii) the lipofibroblast-to-myofibroblast trajectory and the collagen-producing fibroblast atlas that explains heterogeneous drug response; (iii) immune remodeling, particularly the metabolic reprogramming of SPP1⁺/MERTK⁺ macrophages and the exhaustion of CD8⁺ T cells; and (iv) the spatial organization of disease-associated niches at the fibroblast-focus edge. We then translate these findings into a framework for molecular subtyping and precision medicine, arguing that the future of IPF therapy lies in disrupting specific niche communication axes rather than broadly antagonizing collagen synthesis. Beyond cataloguing cell types, we emphasize the systems-level principle that emerges from the single-cell literature: IPF is not a disease of one cell type gone wrong, but a disease of the communication network that binds multiple compartments into a self-sustaining, maladaptive ecosystem.
Ruicheng Deng, Shumin Jiang, Xianfeng Han et al.· Cytokine & growth factor rev...· 0 citations
Dysregulated alveolar epithelial repair is a central aspect of Idiopathic Pulmonary Fibrosis but it has proven challenging to reliably model alveolar epithelial cell biology in-vitro. We previously reported persistent activation of Hypoxia-inducible Factor 2 (HIF2) is a hallmark of aberrant epithelial cell phenotypes in IPF. We hypothesized that HIF2 activation primes AECs for aberrant differentiation. In these studies, we investigated how primary human alveolar epithelial organoids respond to a commonly-used commercial alveolar differentiation media intended to facilitate AT2->AT1 differentiation (ADM; which includes human serum and withdrawal of multiple growth factors/inhibitors) in isolation and following biased HIF activation. Alveolar organoids from donor lungs were expanded and transitioned to ADM with pharmacologic modulators to establish HIF-biased signaling. Multimodal analysis integrating scRNA-seq, quantitative label-free proteomics, and Cell Painting revealed that ADM induces expression of several AT1 markers, yet these conditions are insufficient to generate mature AT1-like cells but drives the emergence of KRT17+/KRT5- "aberrant basaloid" -like cells which demonstrate high transcriptional similarity to populations in end-stage IPF lungs (p = 3x10-286). HIF2-biased activation significantly exacerbated these aberrant transitions. Gene module co-expression analysis (hdWGCNA) linked ADM to cytoskeletal modulation and HIF2-biased signaling to metabolic shifts and cytoskeletal rearrangement. Proteomics reinforced protein-level induction of aberrant markers (e.g., GDF15) and revealed novel signatures, including increased complement production. Orthogonal transmission electron microscopy confirmed ultrastructural remodeling, including lysosomal inclusions and mitochondrial modulation. Collectively, these data establish that ADM models disease-emergent transitional states rather than homeostatic AT1 differentiation. These data further position HIF2 as a critical potentiator of aberrant alveolar epithelial cell states in PF.
H. David, A. Dietrich, C. Calvi et al.· American Journal of Respirat...· 0 citations
INTRODUCTION
Acute kidney injury (AKI) is a severe clinical syndrome associated with high morbidity and mortality, and it possesses high risk of progression to chronic kidney disease (CKD). Renal infiltration of inflammatory cells, particularly macrophages, plays a key role in driving the progression from AKI to CKD.
METHODS
We performed single-cell RNA sequencing and spatial transcriptomics analyses on mouse kidney tissues after unilateral ischemia-reperfusion injury (UIRI) at multiple time points (sham, 12 h, 1 day, 4 days, and 10 days post-UIRI). Immunofluorescence, flow cytometry, Western blotting, co-immunoprecipitation, surface plasmon resonance and immunohistochemical staining were applied to investigate protein expression and function.
RESULTS
Six distinct mononuclear phagocyte (MNP) subpopulations were identified and each of them exhibited a unique set of gene expression pattern. In particular, we uncovered an injury-induced, spatially distinct macrophage subset with specific profibrotic properties, named as pro-fibrotic macrophages (PFMs). During the progression of AKI to CKD, PFMs secreted thrombospondin-1 (THBS1), which interacted with and activated LDL receptor-related protein 1 (LRP1) on adjacent interstitial fibroblasts, thereby promoting fibroblast activation and proliferation. In cultured kidney fibroblasts, THBS1 induced fibroblast activation and matrix production, and blockade or knockdown of LRP1 abolished this effect. Furthermore, THBS1 activated a cascade of LRP1 downstream signaling mediators including focal adhesion kinase, protein kinase B, and extracellular signal-regulated kinase 1 and 2. THBS1 also promoted TGF-β1 activation in a LRP1-dependent manner in fibroblasts. In humans, urinary THBS1 levels predicted the rate of CKD progression.
CONCLUSION
These studies identify a unique subset of pro-fibrotic macrophages that drive fibroblast activation through THBS1/LRP1 signaling in a spatially confined setting. Our findings suggest that PFMs-derived THBS1 contributes to the progression from AKI to CKD.
Liuyan Xiao, Yinyi Long, Xue Hong et al.· Journal of Advanced Research· 0 citations
Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease characterized by excessive extracellular matrix deposition and irreversible architectural distortion of the lung. Fibrotic remodeling is driven by dynamic interactions among endothelial, fibroblast, epithelial and immune cells. Although pulmonary endothelial cells (ECs) are increasingly recognized as important contributors to IPF pathogenesis, the molecular and cellular events underlying endothelial dysfunction remains poorly understood. Using integrative multi-omics analyses of human IPF lungs combined with functional in vitro assays, we identify ACKR1-expressing venous endothelial cells (ACKR1+ VECs) as critical regulators of a pathogenic niche that promotes lung fibrosis. Single-cell RNA sequencing and spatial transcriptomics analyses reveal that ACKR1+ VECs exhibit a distinct pro-fibrotic and pro-inflammatory transcriptional program enriched for hypoxia responses, extracellular matrix remodeling, and immune cell recruitment. In both mouse and human fibrotic lungs, ACKR1+ VECs localize adjacent to fibroblastic foci and are surrounded by pro-fibrotic CD68+/CCR5+/SPP1+ macrophages-monocytes, suggesting a spatial organized cellular crosstalk supporting fibrotic remodeling. Consistent with these findings, in vitro co-culture assays using ACKR1+ VECs isolated from IPF lungs demonstrate that these cells drive myeloid recruitment and fibroblast activation through ACKR1 dependent mechanisms. Silencing of ACKR1 in IPF-derived VECs suppressed inflammatory and fibrotic transcriptional programs, and pharmacological inhibition of ACKR1 attenuated stromal and immune remodeling and reduced bleomycin-induced lung fibrosis in vivo. Together, these findings identify ACKR1+ VECs as key orchestrators of fibrosis progression and establish ACKR1 and the pathogenic vasculature as promising therapeutic targets for IPF. Clinical Relevance Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease with limited treatment options. We identify ACKR1-expressing venous endothelial cells as key drivers of inflammatory and fibrotic remodeling and show that pharmacologic inhibition of ACKR1 attenuates experimental lung fibrosis. These findings establish endothelial ACKR1 as a promising therapeutic target and highlight the pulmonary vasculature as a novel avenue for disease-modifying therapies in IPF.
K. Kontodimas, A. Raslan, Benjamin Spira et al.· bioRxiv· 0 citations
RATIONALE
Restrictive allograft syndrome (RAS) is a major cause of mortality following lung transplantation due to progressive fibrosis of the lung allograft with no therapeutic options. Knowledge of the cellular and molecular mechanisms driving fibrosis in RAS remains limited.
OBJECTIVE
To characterize the cellular and molecular changes in human RAS lungs through single-cell transcriptomic profiling.
METHODS
Single-nucleus RNA-sequencing (snRNA-seq) was performed in peripheral lung tissues from 15 RAS patients undergoing lung re-transplantation, and from 9 healthy control lungs. Findings were validated and extended using histologic techniques including immunofluorescence, RNA in situ hybridization, Elastica-van-Gieson immunohistochemistry, quantitative histological analyses, and micro-CT scans.
MEASUREMENTS AND MAIN RESULTS
snRNA-seq analysis of RAS lungs revealed previously undescribed aberrant basaloid cells, ectopic COL15A1+ peribronchial vascular endothelial cells (pVECs), and CTHRC1+ fibrotic fibroblasts. Histologic stains disclosed distinctive distribution patterns: aberrant basaloid cells, primarily localized at the fibrotic edge, together with juxtaposed CTHRC1+ fibrotic fibroblasts and ectopic COL15A1+ pVECs form the fibrotic niche of alveolar fibroelastosis (AFE). PRX+ alveolar microvasculature is partially lost in AFE areas. Micro-CT scans revealed changes from pulmonary to systemic perfusion, facilitated by COL15A1+ pVECs. Last, our data reveals potential therapeutic targets in RAS, including integrin αvβ6, activator of TGFβ.
CONCLUSION
Considering the multifaceted differences of RAS and idiopathic pulmonary fibrosis, we revealed a surprising general principle of an entity-spanning composition of the fibrotic niche by aberrant basaloid cells localized at the fibrotic edge, ectopic COL15A1+ pVECs and CTHRC1+ fibrotic fibroblasts. This suggests a flexible but cellular pathogenesis-guided transferability of potential therapeutic approaches between progressive fibrotic lung diseases.
L. Leiber, Leonard Christian, L. Neubert et al.· European Respiratory Journal· 0 citations
INTRODUCTION
Pulmonary fibrosis is an interstitial lung disease characterized by diverse etiologies and unknown mechanisms. Phase separation has been implicated in the development of numerous diseases, but its role in the pathogenesis of pulmonary fibrogenesis remains unclear. In this study, we explored whether heterogeneous nuclear ribonucleoprotein L (hnRNP L) can form phase-separation condensates that contribute to the progression of pulmonary fibrosis.
METHODS
Pulmonary fibrosis was modeled in vitro using TGF-β1-treated fibroblasts and in vivo by intratracheal bleomycin administration in mice. Phase-separation formation was evaluated by droplet formation and fluorescence recovery after photobleaching experiments. Adeno-associated virus vectors overexpressing full-length hnRNP L or hnRNP L-Δ26-111 & 274-374 (truncation of amino acid fragments 26-111 and 274-374) were constructed and delivered into murine lungs using a Penn-Century MicroSprayer device. Micro-computed tomography imaging, forced vital capacity tests, hematoxylin and eosin staining, and Masson's trichrome staining were performed to assess the anti-fibrotic effect of disrupting hnRNP L phase separation. RNA-seq was employed to explore the underlying molecular targets of hnRNP L.
RESULTS
In vivo and in vitro results revealed that the role of hnRNP L in promoting fibroblast differentiation depended on its phase separation, and the amino acid fragments 26-111 and 274-374 were essential to this process. Phenotypic, physiological, and histological analyses demonstrated that hnRNP L overexpression accelerated the development of pulmonary fibrogenesis. In contrast, truncated hnRNP L-Δ26-111 and 274-374 improved lung tissue structure and function and reduced collagen deposition by blocking hnRNP L phase separation. RNA-seq results showed that a total of 1,155 dysregulated genes (FDR < 0.05, |logFC| > 1) were detected after silencing hnRNP L using small interfering RNA (siRNA) in TGF-β1-treated MRC-5 cells. Most differentially expressed genes were related to lung fibrosis, including TGFBR1, CXCL8, WWTR1, FAP, VIM, α-SMA, and collagen. The RNA levels of these genes were evidently regulated by silencing or overexpressing hnRNP L. Notably, we observed that hnRNP L phase separation promoted histone modifications such as H3K27ac, H4K8la, H4K12la, and H4K16la, which are associated with active chromatin.
DISCUSSION
Our findings reveal that TGF-β1-enhanced phase separation of the chromatin-associated RBP hnRNP L promotes gene transcription and facilitates profibrotic phenotypes in lung myofibroblasts, suggesting a phase-separation-based therapeutic strategy.
CONCLUSION
Blocking hnRNP L phase separation inhibited fibroblast-to-myofibroblast differentiation, attenuating pulmonary fibrogenesis. This study reveals a novel regulatory mechanism by which phase separation contributes to the development of pulmonary fibrosis.
Mingze Wang, HuiLing Yang, Yujie Wang et al.· Current Gene Therapy· 0 citations
