Sacrificial cell death of adipose mesenchymal stem cells by autophagy facilitates cartilage formation in co-cultures with chondrocytes

Objective: Cartilage formation is stimulated in mixtures of chondrocytes and human adipose-derived mesenchymal stem cells (MSCs) both in vitro and in vivo. During co-culture, human MSCs perish through an unknown process. The goal of this study is to elucidate the mechanism by which adipose tissue-derived MSC cell death occurs in the presence of chondrocytes. Methods: Human primary chondrocytes were co-cultured with human MSCs derived from three donors. The cells were cultured in mono-culture or co-culture (20% chondrocytes and 80% MSCs) in pellets (200.000 cell/pellet) for 7 days in chondrocyte proliferation media in hypoxia (2% O2). RNA sequencing was performed to assess for differences in gene expression between monocultures or co-culture. Western blot and immune fluorescence assays were performed to quantify the level of Caspase-3, LC3B and P62. Results: RNA sequencing revealed significant up-regulation of >90 genes in the three co-cultures when compared to monocultures. STRING analysis showed interconnections between >50 of these genes. Remarkably, 75% of these genes play a role in cell death pathways such as apoptosis and autophagy. Western blotting shows a clear up-regulation of the autophagic machinery with no substantial activation of the apoptotic pathway. Conclusion: In co-cultures of human MSCs with primary chondrocytes, MSCs perish through autophagy. We propose that this sacrificial cell death may perhaps contribute to the trophic effects of MSCs on cartilage formation. Overall design: Cell pellets were generated by seeding 200,000 cells per well in a 96 well plate. Cells were cultured as monocultures or as co-cultures with a ratio of 80% MSCs/20% human chondrocytes. Both MSCs and chondrocytes were cultured in chondrogenic proliferation media (Gilco's advance MEM, 10% FBS, 1% pen/strep, 1% GLUTAmax) under hypoxia (2% oxygen). Medium was changed every three days. Pellets were harvested on day 7.