A three-dimensional culture reduced senescence markers in corneal niche cells and pointed researchers to FOSL1
A three-dimensional culture reduced senescence markers in corneal niche cells and pointed researchers to FOSL1
On September 8, in a paper published in Aging Cell, researchers described experiments with limbal cells from human donor tissues. The cells grew for six days in Matrigel droplets, a laboratory gel in which they assembled into spheroid clusters. Compared with conventional flat culture, these cells divided more actively and showed fewer signs of replicative senescence, the changes that accumulate when cells are passaged repeatedly in the laboratory.
The limbus is the border between the transparent cornea and the sclera. The connective tissue of this zone harbors limbal niche cells: they maintain the microenvironment for epithelial stem cells that renew the corneal surface. With repeated passaging, limbal niche cells divide more slowly and acquire markers of replicative senescence. This narrows the supply of cellular material available for subsequent work.
This line of research grew out of a 2012 study by the same group. In that earlier work, transferring already expanded cells into three-dimensional Matrigel restored some of the protein markers associated with a stem cell-like state. The new study poses the question more precisely: how does a three-dimensional environment alter senescence markers, and which protein participates in this shift.
In the new paper, cells recovered from three-dimensional culture contained a higher fraction of dividing cells and a lower fraction of cells with senescence markers. When the researchers compared gene activity in individual cells, two clusters with senescence signatures shrank in the three-dimensional condition, while a cluster of cells actively replicating DNA before division expanded.
To search for a regulator, the authors cross-referenced genes whose activity declined with successive passages in flat culture and rose after transfer to the three-dimensional environment. Among the 21 candidates was FOSL1, a transcription factor that regulates the activity of other genes. FOSL1 levels were lower in late-passage cells than in early-passage cells. When the authors knocked down FOSL1 in early-passage cells, the cells divided less frequently and displayed more senescence markers; when they overexpressed FOSL1 in late-passage cells, division increased and senescence markers diminished.
The same manipulations affected mitochondria, the organelles that supply cells with energy. When FOSL1 was reduced, reactive oxygen species increased, mitochondria swelled, their internal cristae became disorganized, and membrane potential dropped. When FOSL1 was overexpressed in late-passage cells, reactive oxygen species declined, and mitochondrial structure and membrane potential recovered.
In this laboratory model, the culture environment set the cellular state that the cells carried into the next stage of work.