3D Culture Reverses Limbal Niche Cell Replicative Aging via FOSL1 Upregulation

Wang X, Li S, Liu Z, Guo X, Shen J, Zhou T, Liao S, Huang X, Wang W, Xu L, Zang X, Li G (2026)

Publication Type: Journal article

Publication year: 2026

Journal

Aging Cell Wiley

Book Volume: 25

Article Number: e70705

Journal Issue: 9

DOI: 10.1111/acel.70705

Abstract

Limbal niche cells (LNCs) serve as essential regulators of limbal microenvironmental homeostasis and corneal epithelial wound repair, representing a promising therapeutic resource for limbal stem cell deficiency (LSCD). However, their clinical application is constrained by replicative aging during in vitro expansion. In this study, we investigated whether a three-dimensional (3D) Matrigel-based culture system could modulate replicative aging in LNCs. Compared with conventional two-dimensional (2D) culture, 3D-cultured LNCs restored stemness marker expression and enhanced proliferative capacity. Concurrently, these cells displayed reduced senescence-associated β-galactosidase (SA-β-gal) activity and decreased expression of senescence-associated proteins, including p16, p21, p53, and γ-H2AX. Single-cell RNA sequencing (scRNA-seq) analysis revealed prominent upregulation of FOS-like antigen 1 (FOSL1). FOSL1 is a component of the AP-1 transcription factor family and participates in cell proliferation and stress adaptation. Functional assays using an in vitro replicative aging model showed that FOSL1 knockdown in early-passage (P4) LNCs accelerated senescence, whereas FOSL1 overexpression in late-passage (P11) LNCs attenuated senescence. Mechanistically, FOSL1 knockdown induced mitochondrial dysfunction characterized by elevated levels of mitochondrial superoxide and cellular reactive oxygen species (ROS), as well as a decrease in mitochondrial membrane potential, while FOSL1 overexpression preserved mitochondrial integrity and function. Collectively, our findings demonstrate that 3D culture reverses LNC replicative aging through FOSL1-mediated enhancement of mitochondrial function, providing a microenvironment-based strategy to counteract replicative aging in adult stem cells for corneal regenerative therapy.

Authors with CRIS profile

Shen Li Department of Ophthalmology

Involved external institutions

Huazhong University of Science & Technology CN China (CN) Qilu Hospital of Shandong University CN China (CN)

How to cite

APA:

Wang, X., Li, S., Liu, Z., Guo, X., Shen, J., Zhou, T.,... Li, G. (2026). 3D Culture Reverses Limbal Niche Cell Replicative Aging via FOSL1 Upregulation. Aging Cell, 25(9). https://doi.org/10.1111/acel.70705

MLA:

Wang, Xuying, et al. "3D Culture Reverses Limbal Niche Cell Replicative Aging via FOSL1 Upregulation." Aging Cell 25.9 (2026).

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