Objective

Cartilage formation is stimulated in mixtures of chondrocytes and human adipose–derived mesenchymal stromal cells (MSCs) both in vitro and in vivo. During coculture, human MSCs perish. 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 cocultured with human MSCs derived from 3 donors. The cells were cultured in monoculture or coculture (20% chondrocytes and 80% MSCs) in pellets (200,000 cells/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 coculture. Immune fluorescence assays were performed to determine the presence of caspase-3, LC3B, and P62.

Results

RNA sequencing revealed significant upregulation of >90 genes in the 3 cocultures when compared with 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. Immunofluorescence shows a clear upregulation of the autophagic machinery with no substantial activation of the apoptotic pathway.

Conclusion

In cocultures of human MSCs with primary chondrocytes, autophagy is involved in the disappearance of MSCs. We propose that this sacrificial cell death may contribute to the trophic effects of MSCs on cartilage formation.

中文翻译：

---

自噬与软骨细胞共培养中的间充质干细胞死亡有关。

客观的

在体外和体内，软骨细胞和人脂肪来源的间充质基质细胞 (MSCs) 的混合物刺激软骨形成。在共培养过程中，人类 MSCs 会死亡。本研究的目的是阐明在软骨细胞存在的情况下脂肪组织来源的 MSC 细胞死亡发生的机制。

方法

人原代软骨细胞与来自 3 个供体的人 MSCs 共培养。_{细胞在缺氧（2% O 2} ）的软骨细胞增殖培养基中以单培养或共培养（20% 软骨细胞和 80% MSCs）在颗粒（200,000 个细胞/颗粒）中培养 7 天。进行 RNA 测序以评估单一培养物或共培养物之间基因表达的差异。进行免疫荧光测定以确定 caspase-3、LC3B 和 P62 的存在。

结果

与单一培养相比，RNA 测序显示 3 个共培养中 > 90 个基因显着上调。STRING 分析显示 > 50 个这些基因之间存在相互联系。值得注意的是，这些基因中有 75% 在细胞死亡途径中发挥作用，例如细胞凋亡和自噬。免疫荧光显示自噬机制明显上调，而凋亡途径没有实质性激活。

结论

在人 MSCs 与原代软骨细胞的共培养中，自噬参与了 MSCs 的消失。我们提出这种牺牲性细胞死亡可能有助于 MSCs 对软骨形成的营养作用。