ABS 039: Retinoic Acid Receptor Inhibition Promotes Hillock Stem Cell Expansion

Juliette Gaytan ¹ ² ⁶, Viral S. Shah ¹ ³ ⁵, Jayraj Rajagopal ¹ ³ ⁴ ⁵ ⁶

¹ Simches 4, Northeast Corridor, Massachusetts General Hospital
² University of Notre Dame, Notre Dame
³ Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, Massachusetts General Hospital
⁴ Department of Pathology, Massachusetts General Hospital, Harvard Medical School
⁵ Broad Institute of MIT and Harvard
⁶ Harvard Stem Cell Institute

The Van Wickle Journal (2026) Volume 2, ABS039

Introduction: The airway is constantly exposed to environmental insults that damage the airway surface. After injury, the airway must regenerate the epithelial surface to maintain its critical functions. Recently, the Rajagopal lab identified a new stem cell reservoir, called the hillock, which serves as the primary cellular source of airway regeneration after injury. Hillocks are stratified epithelial structures lacking ciliated cells and containing a distinct population of highly proliferative, injury-resistant basal stem cells found in mouse and human large airways. However, there is no established in vitro model to study hillocks. When hillock basal cells are cultured they no longer demonstrate the aforementioned properties of hillocks. Thus, the primary objective of this study was to develop an approach to culture hillocks in vitro while maintaining their defining characteristics. Unlike their pseudostratified counterparts, hillock stem cells exhibit a distinct transcriptional signature characterized by enhanced retinoic acid (RA) anabolism. I hypothesize that modulating retinoic acid metabolism could be critical for the successful in vitro culture hillock epithelia. To test this hypothesis, a six-point dose gradient (0-10µM) of Agn193109, a pan-retinoic acid receptor antagonist, was applied to murine in vitro airway basal cell culture isolated from hillocks and non-hillock regions. Cultures were categorized as hillock-enriched, ciliated, or mixed populations, and epithelial morphology and lineage distribution were monitored over time. High-dose Agn treatment promoted widespread loss of defined architecture, suggesting a cell-instinsic response to RA inhibition. However, hillock expansion occurred at the expense of ciliated cell development. Lower concentrations allowed partial reemergence of ciliated cell differentiation. These findings identify RA signaling as a key regulator of hillock expansion and epithelial regeneration, with potential implications for therapies aimed at improving airway resistance after injury. Future studies must evaluate potential physiologic consequences of hillock expansion.

Methods: Tracheas were dissected from KRT13-tdTomato; FoxJ1-GFP; CreER lineage-labeled mice, and airway basal stem cells were isolated for in vitro culture. The KRT13-tdTomato and FoxJ1-GFP labeling system was used to distinguish hillock basal cells from non-hillock and ciliated cell populations. Cells were cultured on collagen-coated plates in basal media supplemented with growth factors and differentiated using standard air-liquid interface (ALI) culture to recapitulate airway epithelium. Resulting cultures produced KRT13+ basal cells, FoxJ1+ ciliated cells, and mixed populations co-expressing both markers. To investigate the role of RA signaling in maintaining hillock characteristics, cultures were treated with a six-point dose gradient of Agn193109 (0 nM–10 µM), a pan-retinoic acid receptor inhibitor, to determine conditions that best recapitulate in vivo stratified, KRT13⁺ structure. Media was replaced every 2-3 days, and cultures were maintained for one month while epithelial morphology and lineage distribution were monitored.

Results: Low concentrations of RA receptor inhibition (0,1,10nM Agn193109) produced limited KRT13+ differentiation, with KRT13+ cells remaining primarily as isolated single cells while FoxJ1+ ciliated cells remained abundant. Significant overlap between KRT13+ and FoxJ1+ populations persisted across low-dose conditions, indicating minimal effects on differentiation. In contrast, higher concentrations (100 nM, 1 µM, and 10 µM) drove dose-dependent hillock differentiation. At 100nM, KRT13+ clusters formed as ciliogenesis decreased. At 1µM, stratified KRT13+ structures resembling in vivo hillocks replaced most ciliated cells. At 10 µM, cultures were dominated by KRT13⁺ cells with minimal ciliogenesis, indicating excessive RA inhibition drives hillock over-differentiation in airway cultures.

Discussion: These findings identify RA signaling as a key regulator of hillock basal cell differentiation, demonstrating that hillock cells respond functionally to RA inhibition. Specifically, 1 µM Agn193109 most effectively recapitulated the stratified KRT13⁺ hillock structures observed in vivo while limiting excessive over-differentiation. This work establishes a foundation for developing an in vitro model to study hillock biology and airway regeneration. Future studies should quantify hillock expansion under varying levels of RA inhibition and determine whether cultured hillocks retain the injury-resistant properties observed in vivo. Additionally, future work will investigate whether human hillock structures can be successfully recapitulated in vitro.