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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE142831 | GEO | 2020/06/30

REPOSITORIES: GEO

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Project description:Avascular soft tissues of the skeletal system, including articular cartilage, have an extremely limited healing capacity, in part due to their low metabolic activity. No drugs are available that can prevent or slow the development of osteoarthritis (OA) after joint injury. Therefore, mesenchymal stromal cell (MSC)-based regenerative therapies are increasingly common in the treatment of OA, but questions regarding their clinical efficacy and mechanisms of action remain unanswered. Our group recently reported that mitochondrial dysfunction is one of the earliest responses of cartilage to injury, resulting in chondrocyte death, extracellular matrix degeneration, and ultimately OA. MSCs have been found to rescue injured cells and improve healing by donating healthy mitochondria in highly metabolic tissues, but mitochondrial transfer has not been investigated in cartilage. To investigate how gene expression changes in stressed chondrocytes, and therefore how they might elicit mitochondrial donation from MSCs, we developed a custom quantitative PCR panel of relevant genes involved in chondrocyte metabolism and OA. We cultured chondrocytes under conditions known induce chondrocyte mitochondrial dysfunction, including stimulation with rotenone/antimycin and non-physiologic culture conditions (hyperoxia and hyperglycemia). We found that expression of gap junction alpha 1 (GJA1), the gene that encodes the Cx43 protein, was increased in hyperoxia. The senescence associated markers, cyclin-dependent kinase inhibitor 2A (CDKN2A) and tissue inhibitor of metalloproteinases 1 (TIMP1), were also increased in hyperoxia. In addition, hyperoxia caused chondrocytes to increase the expression of the antioxidant enzyme, SOD2. This work provides insights into the chemical and environmental conditions that can alter gene expression in chondrocytes. Further, this provides supporting evidence to our hypothesis that stressed chondrocytes may trigger mitochondrial donation from MSCs may via direct or indirect signaling involving the inflammation- and senescence-related genes identified in the present study.

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

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