Rapid Senescence Onset in Skin Cells Aids Wound Healing

Cellular senescence, a state in which cells stop proliferating, is often associated with aging and pathologies like cancer.¹ Recently, though, researchers found senescent cells at the site of injuries.^2,3

“But what hasn't been really discovered is when the senescent cells show up, what do they do,” said Mikolaj Ogrodnik, a regenerative biologist at the Ludwig Boltzmann Institute for Traumatology. Ogrodnik was interested in studying the transition from homeostasis to wound healing in the skin, especially the role of senescent cells in this process.

In a new study published in Nature Cell Biology, Ogrodnik and his team studied senescent cells in vivo during tissue repair in mouse and pig skin and showed that these cells appear rapidly in response to an injury.⁴ The team also demonstrated that the presence of senescent cells early in the repair process is important for proper healing. The findings reveal a new regulatory mechanism of inducing senescence in cells, traditionally thought of as a slow process, that could guide treatments targeting these cells in wound healing and other diseases.

To study in vivo injury responses, the researchers excised a small piece of tissue, including the dermal tissue, from mouse skin and quantified the number of cells that expressed the protein p21, a cell cycle arrest protein and marker for senescence. They saw high concentrations of p21-expressing (p21⁺) cells including keratinocytes and fibroblasts throughout the duration of wound repair, approximately 28 days, with the largest proportion of p21⁺ cells present on day three. Unwounded mouse skin did not contain any p21⁺ cells.

Ogrodnik and his team confirmed that these p21-expressing cells were senescent by assessing other key traits of senescence. Using a proliferation marker, they showed that p21⁺ cells in wounded skin did not divide. These cells were also larger on average than their p21^- counterparts and contained more lipid droplets, two other features of senescent cells.

After confirming that the team was in fact studying senescent cells in skin wounds, the researchers turned to single-cell RNA sequencing to explore the functions of these cells. They saw that cells that expressed high levels of cyclin-dependent kinase inhibitor 1A (Cdkn1a), the gene for p21, also had increased expression of genes related to inflammation and migration, while genes involved in adhesion were decreased. This corresponded with p21⁺ keratinocytes and fibroblasts present at the leading edge of cells migrating to repair the wound. The findings demonstrated functional roles for senescent cells in wound healing.

Since the researchers already saw high quantities of senescent cells at wound sites three days after injury, they explored earlier time points to determine when these cells initiate senescence. They used a pig model, since pig skin more closely resembles human skin and they could take multiple samples from the same animal. The researchers started seeing p21 expression in cells within minutes after creating an injury, and they saw a significantly greater frequency of p21⁺ cells in the tissue compared to unwounded skin at 90 minutes. They observed a similar pattern in mice. These findings showed that senescence can be induced rapidly in some cells, “which, to my knowledge, is almost unprecedented,” Ogrodnik said.

One of the challenges in studying cellular senescence in vivo is the difficulty of confirming that the senescent phenotype is stable. To address this, Ogrodnik and his team used a lineage tracing assay that coupled p21 expression with that of a reporter protein. Using this approach, they showed that cells that begin to express p21 at the time of injury continued to express this protein throughout the duration of the injury response. “This [technique] is something which is also very, very exciting, and I am hoping to share that with the scientific community. So, maybe we can start doing it much more frequently from now on,” Ogrodnik said.

After confirming that senescent cells maintain this phenotype, the researchers explored how these cells disappeared after the wound response finished. They used their same lineage tracing approach to confirm that between 16 and 20 days after the injury onset, the mice shed senescent keratinocytes through the epidermal layer while dermal senescent cells underwent apoptosis.

Continue reading below...

Webinar

Best Practices for qPCR Assay Design and Optimization

Explore practical strategies for designing, optimizing, and validating qPCR assays to improve specificity, reproducibility, and confidence across routine and high-throughput gene expression studies.

Read More

“That's a very important concept because in the field we always think that actually senescent cells that are not eliminated efficiently, those are the ones that become a problem. But in this case, being cells that spontaneously die, essentially the problem doesn't come in,” said Marco Demaria, a biologist at the University of Groningen who also studies the mechanisms of cellular senescence but was not involved in the study.

Next, the researchers investigated the mechanism of rapid senescence onset in wound healing. From their single-cell RNA sequencing data, Ogrodnik and his team knew that although cells in unwounded mouse skin did not express p21, they did express the Cdkn1a transcripts. Since the expression level for this mRNA did not increase immediately after injury, it suggested to the researchers that the switch to senescence in these skin cells was transcription independent. They confirmed this hypothesis by administering inhibitors of either transcription or translation to ex vivo wounded pig skin. Only the translation inhibitors prevented the appearance of p21⁺ senescent cells.

To explore the mechanism controlling this senescence switch in skin cells, the team isolated Cdkn1a transcripts along with any binding proteins from unwounded and wounded pig skin and identified the proteins by mass spectrometry. While the p21 mRNA in unwounded skin was bound to splicing factors and ribonucleoproteins that prevent its export from the nucleus, wounded skin was associated with translation factors.

“That's really something that mechanistically addresses something very new,” said Demaria, adding that the findings point to a new regulatory method in senescence and that could indicate the potential for subtypes of senescent cells.

Finally, the researchers demonstrated that p21⁺ senescent cells are critical to the early stages of proper wound healing. Treating mice with an inhibitor of its mRNA and protein prior to wounding delayed tissue repair. However, when the researchers administered this inhibitor three days after the initial wound, healing occurred at a comparable rate to un-inhibited wounded skin.

Ogrodnik and his team concluded that rapid onset senescence in the skin is important for proper wound healing. These findings could have critical implications for treatments looking to eliminate these cells. “Not only the quantity but also the timing when senescent cells are inhibited or eliminated might have core meaning for the whole therapy,” Ogrodnik said.

Demaria agreed, adding that the findings also support developing new ways to study senescence in vitro. “The point here is that, in vivo, we might have cells that enter senescence very rapidly which we have essentially ignored in the past because of the fact that our cell culture systems do not represent that,” he said.

Ogrodnik said that the findings could also help answer questions in senescence research about the origins of its physiological role. “Maybe it is actually the response to injury as one of the main evolutionary drivers for having cellular senescence,” he said.