Growing Eye Cells in 3D Reverses Aging and Restores Their Healing Power

Deep in the corner of the eye, in a ring of tissue called the limbus, live stem cells that keep the cornea clear and the vision sharp. These limbal epithelial stem cells depend on an entourage of supporting cells, known as limbal niche cells, to stay healthy and to repair the corneal surface after injury. When this support system fails, patients can develop limbal stem cell deficiency, a devastating condition in which the opaque conjunctiva invades the cornea, wounds refuse to heal, and sight deteriorates. Limbal niche cells, which arise from the stroma just beneath the limbal epithelium and display mesenchymal stem cell properties, have shown striking therapeutic promise in animal models of corneal injury, reducing epithelial defects, curbing new blood vessel growth, and promoting scarless healing. But like any cell grown in a laboratory dish, they face a formidable obstacle: replicative aging.

Replicative aging is the progressive decline that cells undergo as they divide again and again during in vitro expansion. It is distinct from the abrupt, irreversible state of full senescence; instead, it is a gradual erosion of function that unfolds long before cell division stops completely. The hallmarks are familiar to anyone who works with cultured cells: rising levels of the cell cycle inhibitors p16, p21, and p53, increased activity of the senescence-associated beta-galactosidase enzyme, and a slow decay of mitochondrial performance. Mitochondria sit at the center of this decline. As cells keep dividing, their respiratory machinery falters, ATP output drops, and reactive oxygen species spilling from the electron transport chain damage mitochondrial DNA and oxidize mitochondrial proteins. Quality control systems such as mitophagy struggle to keep pace, lesions accumulate, gene expression profiles shift, and eventually proliferation halts altogether. For researchers hoping to manufacture enough limbal niche cells for clinical use, this aging bottleneck has been a persistent barrier.

A new study published in Aging Cell suggests a way around it. A research team based at Tongji Hospital in Wuhan, China, reports that growing limbal niche cells in a three-dimensional Matrigel-based culture system, rather than on the conventional flat, two-dimensional plastic surface, effectively reverses their replicative aging. The work, which combines classic cell biology with single-cell RNA sequencing and genetic gain- and loss-of-function experiments, identifies the transcription factor FOSL1 as a pivotal mediator of this rejuvenation. The findings not only illuminate how three-dimensional architecture preserves cellular youth, but also point toward FOSL1 as a potential target for revitalizing aged cells destined for regenerative therapies.

The rationale for the three-dimensional approach grew out of earlier work. Flat culture surfaces fail to reproduce the rich microenvironment of living tissue, and stem cells grown on them for extended periods tend to drift away from their native identity, losing function as they go. Mesenchymal stem cells grown in three dimensions, by contrast, spontaneously form spheroids and display stronger secretory profiles, better adaptive capacity, and enhanced immunomodulatory behavior, and several studies have suggested that such conditions can ameliorate aging phenotypes. The Tongji group had previously observed that limbal niche cells reseeded onto a Matrigel scaffold in the fourth generation showed a striking rebound in embryonic stem cell marker expression. The new study set out to test whether three-dimensional culture could do more than preserve youthfulness, namely whether it could actually turn back the aging clock.

To build the three-dimensional system, the researchers mixed limbal niche cells with Matrigel at a fifty percent volume ratio and deposited the suspension as small hemispherical droplets in culture plates, which were inverted briefly to encourage gelation and prevent cells from settling. Within this soft, matrix-rich environment, the cells did something remarkable: they self-assembled into spheroids whose diameters grew steadily over six days of culture, a sign of active proliferation and matrix remodeling. The cells used in the experiments were isolated from corneal tissue donated by individuals aged sixty to eighty, digested from limbal tissue with collagenase, and purified through serial passaging until they expressed mesenchymal markers such as vimentin, CD73, and CD90 while losing epithelial markers.

When the team compared aged limbal niche cells maintained in standard two-dimensional culture with counterparts of the same passage grown as three-dimensional spheroids, the differences were unmistakable. The three-dimensionally cultured cells showed markedly higher expression of the proliferation marker Ki67 and stronger growth in CCK-8 proliferation assays across seventy-two hours. Their expression of the stemness markers SOX2, OCT4, and NANOG, measured both by immunofluorescence and quantitative PCR, rebounded significantly. Meanwhile, the aging indicators moved in the opposite direction: far fewer cells stained positive for senescence-associated beta-galactosidase, and the levels of p16, p21, p53, and the DNA damage marker gamma-H2AX all fell. In short, the cells grown in three dimensions looked and behaved younger than their flat-grown siblings.

To understand how this rejuvenation operated at single-cell resolution, the researchers performed single-cell RNA sequencing on early-passage and late-passage cells grown in both formats. Unsupervised clustering revealed ten distinct transcriptional populations. Two of these, clusters C2 and C3, were enriched for cell cycle genes but also carried strong signatures of cellular senescence and p53 signaling, suggesting they were in a precarious transitional state between proliferation and stress-induced aging. These senescence-prone clusters were abundant in two-dimensional cultures but shrank dramatically in the three-dimensional environment. Conversely, a cluster designated C5, defined by enrichment in DNA replication, cell cycle progression, and mitotic nuclear division, expanded significantly under three-dimensional conditions. The rejuvenation, in other words, was not a uniform shift across all cells but a subpopulation remodeling: three-dimensional culture pruned the aging-prone cells and selectively amplified a proliferative, DNA-replication-active fraction.

The search for the molecular driver of this remodeling led to FOSL1, an AP-1 family transcription factor previously known for roles in proliferation, differentiation, and stress adaptation, and for maintaining the stemness of limbal epithelial stem cells in the cornea. By intersecting genes that decline as cells age in two dimensions with genes that rise in three-dimensional culture, the team identified twenty-one candidate rejuvenation regulators, with FOSL1 at the center of a protein interaction network that included fellow AP-1 members FOS and JUN as well as stress-responsive factors MAF and EGR1. FOSL1 expression fell sharply between early and late passages, and functional experiments confirmed its importance: silencing FOSL1 in young cells accelerated aging, increasing senescence staining and aging markers while suppressing proliferation, whereas overexpressing FOSL1 in aged cells did the reverse, restoring proliferative capacity and reducing senescence markers.

Mitochondria emerged as a key venue of FOSL1’s anti-aging activity. When FOSL1 was knocked down in early-passage cells, intracellular reactive oxygen species and mitochondrial superoxide rose, transmission electron microscopy revealed swollen mitochondria with disrupted cristae and vacuolization, and JC-1 staining showed a collapse of mitochondrial membrane potential. Overexpressing FOSL1 in aged cells improved mitochondrial ultrastructure, restored more ordered cristae, and partially recovered membrane potential. The authors suggest that FOSL1 may coordinate a dual defense, enhancing antioxidant capacity through pathways such as NRF2 while preserving mitochondrial health to curb oxidant generation at its source, thereby breaking the vicious cycle in which reactive oxygen species and mitochondrial damage feed each other.

The clinical implications are considerable. Limbal niche cell therapies for limbal stem cell deficiency have been constrained by the limited expansion capacity of conventional two-dimensional culture, which cannot yield sufficient cell numbers before aging sets in. A scalable three-dimensional system that keeps cells youthful, combined with genetic or pharmacological activation of FOSL1, could provide abundant, standardized cell products for bioengineered corneal constructs or direct subconjunctival injection. The authors caution, however, that the findings rest on in vitro experiments and still require validation in animal models of corneal injury, that the downstream targets of FOSL1 remain to be fully mapped, and that FOSL1’s well-documented oncogenic role in epithelial tumors demands careful, locally confined delivery strategies. Within the limbal niche, where the factor is physiologically expressed and even reduced in diseases such as keratoconus, it appears to act as a homeostatic guardian rather than a tumor driver. If future work confirms its safety, FOSL1 could become a molecular lever for turning aged eye-support cells young again.

Subject of Research: Reversal of replicative aging in limbal niche cells through three-dimensional culture and FOSL1 upregulation

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

Article References: 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). 3D Culture Reverses Limbal Niche Cell Replicative Aging via FOSL1 Upregulation. Aging Cell, 25(9), Article e70705. https://doi.org/10.1111/acel.70705

Image Credits: AI Generated

DOI: 10.1111/acel.70705

Keywords: limbal niche cells, cornea, replicative aging, 3D cell culture, FOSL1, mitochondria, senescence, limbal stem cell deficiency, single-cell RNA sequencing, AP-1 transcription factor, regenerative medicine, reactive oxygen species