Senolytic Peptide FOXO4-DRI Selectively Removes Over Half of Senescent Cells in Expanded Human Chondrocytes

Autologous chondrocyte implantation (ACI) is a cell-based therapy for treating articular cartilage injuries, a condition that can lead to post-traumatic osteoarthritis. A critical step in ACI is expanding a patient's own cartilage cells (chondrocytes) in the lab to get enough cells for implantation. However, this extensive in vitro expansion process inevitably causes a significant portion of the chondrocytes to enter a state of cellular senescence. These senescent cells are problematic because they impair the quality and quantity of the new cartilage formed after implantation. A new class of drugs called senolytics aims to selectively destroy these harmful senescent cells. One such agent, the peptide FOXO4-DRI, has shown promise in killing senescent fibroblasts by targeting the p53 pathway. This study investigates if FOXO4-DRI could be used to 'clean up' expanded chondrocyte populations before they are implanted, thereby improving the outcome of ACI.

Researchers isolated human chondrocytes from healthy donors and expanded them in vitro. They created two groups of cells based on population doubling level (PDL): an early-passage PDL3 group serving as a minimally expanded control, and a late-passage PDL9 group to simulate chondrocytes ready for implantation in ACI. The PDL9 cells, which had a high burden of senescence, were treated with the senolytic peptide FOXO4-DRI. The primary endpoints were the change in cell number (viability) and the reduction in senescence markers in the PDL9 group compared to untreated PDL9 controls and the PDL3 group. The chondrogenic (cartilage-forming) potential of the treated cells was then assessed using a standard pellet culture assay, and the expression of senescence-associated secretory factors in the resulting cartilage tissue was measured.

Treatment with FOXO4-DRI demonstrated high selectivity for senescent cells. The peptide did not significantly affect the cell number of the minimally expanded PDL3 chondrocytes. In contrast, it induced significant cell death in the highly expanded PDL9 chondrocytes, which are laden with senescent cells.

The treatment of FOXO4-DRI removed more than half of the cells in the PDL9 culture, indicating successful clearance of the target senescent population.

Following this clearance, the remaining population of FOXO4-DRI treated PDL9 chondrocytes showed a significantly reduced level of senescence compared to the untreated PDL9 control group. However, this purification step did not translate into better cartilage formation in the subsequent assay. The pellet culture experiment revealed that FOXO4-DRI pre-treatment did not enhance the chondrogenic potential of the PDL9 cells. Despite this, the cartilage tissue that did form from the treated cells showed lower expression of senescence-relevant secretory factors compared to cartilage from the untreated PDL9 group, suggesting a less inflammatory phenotype.