Poster C4 - Engineered Hydrogels Towards Modeling Alveolar Epithelial Differentiation during Fibrosis

Thursday, October 24, 2024

2:45 PM – 3:45 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Esther Gao

Undergraduate student
University of Michigan Ann Arbor, United States

Co-Author(s)

DA

Donia Ahmed, M.S.

PhD Candidate
University of Michigan, United States

Primary Investigator(s)

Claudia Loebel, MD PhD (she/her/hers)

Assistant Professor
Department of Biomedical Engineering, University of Michigan/Department of Materials Science & Engineering, University of Michigan, United States

Introduction: Idiopathic pulmonary fibrosis (IPF) is characterized by excessive deposition of aberrant extracellular matrix (ECM) within the lungs [1]. As the disease progresses, the ECM becomes more dense and rigid, leading to dynamic changes in mechanical properties within the cellular microenvironment. Within the distal lung, type 2 alveolar epithelial cells (AT2) are the stem/progenitor cells that proliferate and differentiate into type 1 alveolar epithelial cells (AT1) to facilitate alveolar regeneration and repair [2]. However, during fibrosis, incomplete AT2-to-AT1 differentiation has recently been described, which causes accumulation of pre-AT1 transitional state cells (PATS) [2] (Figure 1A). Despite recent evidence showing that increased mechanical forces (i.e. breathing) promote AT2 differentiation, there are limited reports investigating whether changes in ECM mechanical properties (i.e., elastic modulus) promote or impair alveolar epithelial cell differentiation during fibrosis [3]. In this study, we develop a hydrogel platform of varying elastic moduli to probe the role of ECM mechanical properties on epithelial cell function and differentiation.

References: [1] Burgstaller F., et al., Euro Respir J 20, 1 (2017), [2] Peng J., et al., Am Respir Crit Care Med 201, 11 (2020), [3] Shiraishi K., et al., Cell 186, 7 (2023)

Materials and

Methods: Norbornene modified hyaluronic acid (NorHA) hydrogels were prepared in PBS with 2 wt% NorHA, 10% LAP photo-initiator, 2 mM thiolated RGD, and varying concentrations of dithiothreitol (DTT) to modulate hydrogel elastic moduli. 2D hydrogels were photo-crosslinked (UV light, 5 minutes, 3 mW/cm2) on methacrylate-coated glass coverslips then seeded with mouse lung epithelial cells (mLE-12). Hydrogel mechanical properties were measured using photo-rheology (0.5% strain, 0.5 Hz). Cells were then cultured for 3 days in DMEM/F12 with 10% fetal bovine serum (FBS) prior fixation for analysis. Cell morphology was assessed by fluorescent labeling of cell membrane (Cell Mask) and F-actin cytoskeleton (Phalloidin), and cell area was quantified using ImageJ-based cell segmentation.

Results, Conclusions, and Discussions:

Results: By varying the amount of DTT crosslinker concentration, we fabricated NorHA hydrogels with either 2.2 kPa ± 0.24 kPa, 6.6 kPa ± 0.81 kPa, and 10.3 kPa ± 2.63 kPa (Figure 1B). Within 3 days of culture, mLE-12 cells showed high adhesion to all NorHA gels, with increased spread morphologies on the stiffer ~10 kPa hydrogels (Figure 1C). Quantification of average cell areas at day 3 showed an average of 229.5 ± 72 μm^2 for ~2.0 kPa hydrogels and 308.8 ± 160 μm^2 for ~10 kPa hydrogels (Figure 1D, indicating that higher elastic moduli increase cell spreading of lung epithelial cells).

Discussion and

Conclusion: Using NorHA hydrogels we were able to fabricate an engineered platform to recapitulate the mechanical properties of healthy (2-6 kPa) and fibrotic (10-50 kPa) lung ECM. Given that alveolar epithelial cells typically experience a 2D surface within the alveoli, our 2D hydrogel system will enable studies of mechanical forces that direct alveolar epithelial cell phenotype and differentiation in health and disease. Ongoing work is to expand moduli range to target different phases of disease and to characterize epithelial cell phenotype (AT2, PATS, AT1) by immunofluorescence and quantitative polymerase chain reaction (qPCR).

Acknowledgements (Optional):