Purpose

Combination of chondrocytes with autologous or allogeneic mesenchymal stem cells (MSCs) has enabled the development of single-stage cell-based cartilage regeneration procedures, with proven clinical success. With repeat long-term sampling, the MSCs population dies and disappears. Characterization of the mechanism and role of MSC death in the therapeutic effect of MSCs and chondrocytes is currently unknown and hence the aim of the current study.

Methods and Materials

One human chondrocyte donor was cultured with 3 different human adipose derived MSCs at distinct ratios (100% chondrocytes, 80% aMSCs + 20% chondrocytes, 100% aMSCs) for 7 days in pellets (figure 1a). The cells were cultured under hypoxia (2% oxygen) in chondrocyte proliferation medium. RNA sequencing and bioinformatic analysis was performed followed by western blot and immunofluorescence.

Results

Up and down regulation of specific genes was detected using MORPEUS heat map analysis (figure 1b). Among the 362 significantly up regulated genes, 92 were present in all the 3 co-cultures (figure 1c). These genes were analysed using ClueGo software to determine their GO-pathway. To further asses the interaction between the up regulated genes STRING software was used. Of the 92 genes, 51 had interconnections. Of this subgroup, a literature research showed that 73% of these genes play a role in cell death pathways like autophagy and/or apoptosis (figure 2).

To determine which of the two pathways was involved in cell death, the protein levels of cleaved caspase 3 as marker for apoptosis and P62, LC3I & LC3II as markers for autophagy were analysed. The results showed no support for activation of caspase-dependent cell death at the protein level. Instead markers for autophagy were clearly and substantially upregulated.

Conclusion

In single-stage cell-based cartilage regeneration procedures, MSCs disappear by a caspase independent autophagy pathway. We postulate that the self-sacrifice of the MSCs by autophagy generates a chondrocyte stimulatory environment boosting cartilage formation.