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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session C & D

I18 - The role of decellularized extracellular matrix and laminins in lung epithelial cell differentiation

Friday, October 10, 2025
10:00 AM - 11:00 AM PDT
Location: Exhibit Hall F, G & H

Introduction: : The lung performs gas exchange through a specialized tissue architecture that depends critically on interactions between the extracellular matrix (ECM) and epithelial cells. The extracellular matrix (ECM) functions as a dynamic network of proteins, providing mechanical support and bioactive signals to cells. Proteomic data have shown that ECM composition varies between airway and alveolar regions of the lung [1]. We previously found that ECM drive pulmonary airway epithelial basal cells towards region-specific differentiation when cultured ex vivo in engineered trachea and lungs [2]. Here, we focus specifically on how ECM composition affects airway epithelial basal cell differentiation and function. Epithelial basement membrane contains heterotrimer glycoproteins called laminins, which are the first ECM proteins laid down in embryogenesis during lineage specification [3]. Recent evidence indicates that distinct laminin subtypes can affect mammary epithelial cell differentiation, and that aged lung ECM can influence laminin production in epithelial cells, though their role in pulmonary epithelial cell differentiation remains unknown [4]. To investigate how variation in regional ECM composition, including differences in laminin content, influences lung epithelial cell differentiation, we cultured airway basal cells in 3D lung hydrogels derived from distinct regions of the lung and on individual laminin isoforms in 2D culture. This work provides new insights into epithelial-ECM interactions, which will help us better understand lung development and diseases like pulmonary fibrosis.

Materials and

Methods: : Airway basal cells isolated from male WT Sprague-Dawley rats were cultured in region-specific decellularized human lung ECM (dECM) hydrogels provided by Dr. Daniel Weiss [1]. These hydrogels were generated by decellularizing whole human lungs (a process in which cells are removed via chemical and enzymatic treatments while ECM proteins are preserved), micro-dissecting the tissue into alveolar, vascular, airway, and bulk (whole lung) regions, then pepsin-digesting and lyophilizing the tissue. Rat airway basal cells were seeded at 500,000 cells/mL in a hydrogel comprised of dECM and MatrigelTM (50:50) and cultured in differentiation media. Cells were cultured in pure Matrigel as a control (Figure 1A). Resulting organoids underwent qRT-PCR to assess differentiation through expression of lung cell type markers compared to starting cells: Krt5 (basal cells), Scgb1a1 (airway secretory cells), Aqp5 (alveolar type I cells, ATI), and Sfpc (alveolar type II cells, ATII). Immunofluorescent (IF) staining was used to confirm protein-level expression and localization of these markers. Additionally, full-length laminin isoforms present in the lung (LN-3B32, LN-411, LN-511, LN-512, Biolamina) were coated on Transwell inserts or tissue culture plastic at 1 μg/cm² and cultured with human or rat airway basal cells for 21 days (Figure 1B). These experiments are ongoing but will be characterized by RT-qPCR and IF for expression of the same lung cell type markers as above.

Results, Conclusions, and Discussions:: Organoids grown in airway dECM appear empty, whereas organoids grown in alveolar dECM appear to be filled with secretions (Figure 1C). This could indicate dead cells or potentially surfactant production by cells in the alveolar condition, a physiologic function that facilitates gas exchange in the alveoli [5]. The bulk and Matrigel conditions produced diverse organoid types, likely due to non-specific biochemical cues present in bulk dECM from the amalgamation of various lung regions. The vascular condition showed little to no organoid formation. This was expected as vascular ECM interfaces with endothelium rather than epithelium. At the gene expression level, we see a significant reduction in Krt5 expression in the vascular condition over the Matrigel control, possibly due to the non-epithelial nature of this dECM (Figure 1D). Alternatively, we see a significant increase in Aqp5 and Sftpc expression in the alveolar condition over Matrigel, suggesting enhanced alveolar epithelial character in cells cultured in alveolar dECM. Scgb1a1 expression increased in all organoid conditions over starting cells, but was not significantly different between conditions. Airway dECM did not exhibit any significant differences in the genes tested. Here, we have demonstrated that region-specific ECM composition can influence lung epithelial cell fate in a 3D hydrogel platform. Preliminary studies of human epithelial basal cells cultured in wells coated with laminin isoforms show differences in cell phenotype (Figure 1E). Ongoing work is focused on the specific role of laminins in this process. Future work involves investigating integrin-mediated mechanisms by which ECM proteins and laminins control epithelial cell fate. These findings will provide greater context for how ECM composition influences cell behavior in homeostasis, development, and disease. This work could impact how we understand lung diseases like pulmonary fibrosis, where changes to the ECM may drive cell-based pathologies.

Acknowledgements and/or References (Optional): : [1] Hoffman ET, Uhl FE, Asarian L, Deng B, Becker C, Uriarte JJ, Downs I, Young B, Weiss DJ. Regional and disease specific human lung extracellular matrix composition. Biomaterials. 2023 Feb;293:121960. doi: 10.1016/j.biomaterials.2022.121960. Epub 2022 Dec 24. PMID: 36580718; PMCID: PMC986808.
[2] Greaney AM, Adams TS, Brickman Raredon MS, Gubbins E, Schupp JC, Engler AJ, Ghaedi M, Yuan Y, Kaminski N, Niklason LE. Platform Effects on Regeneration by Pulmonary Basal Cells as Evaluated by Single-Cell RNA Sequencing. Cell Rep. 2020 Mar 24;30(12):4250-4265.e6. doi: 10.1016/j.celrep.2020.03.004. PMID:  32209482; PMCID: PMC7175071. 
[3] Developmental regulation of laminin accumulation in the extracellular matrix of a mouse muscle cell line. Olwin, Developmental Biology 1989. 
[4] Godin LM, Sandri BJ, Wagner DE, Meyer CM, Price AP, Akinnola I, Weiss DJ, Panoskaltsis-Mortari A. Decreased Laminin Expression by Human Lung Epithelial Cells and Fibroblasts Cultured in Acellular Lung Scaffolds from Aged Mice. PLoS One. 2016 Mar 8;11(3):e0150966. doi: 10.1371/journal.pone.0150966. PMID: 26954258; PMCID: PMC4783067. 
[5] Liu MY, Chen B, Borji M, Garcia de Alba Rivas C, Dost AFM, Moye AL, Movval Abdulla N, Paschini M, Rollins SD, Wang R, Schnapp LM, Khalil HA, Wu CJ, Sharma NS, Kim CF. Human Airway and Alveolar Organoids from BAL Fluid. Am J Respir Crit Care Med. 2024 Jun 15;209(12):1501-1504. doi: 10.1164/rccm.202310-1831LE. PMID: 38652140; PMCID: PMC11208964.