Senescence-associated metabolomic phenotype in primary and iPSC-derived mesenchymal stromal cells. Senescence-associated metabolomic phenotype in primary and iPSC-derived mesenchymal stromal cells

Long-term culture of primary cells is reflected by functional and secretory changes, which ultimately result in replicative senescence. In contrast, induced pluripotent stem cells (iPSCs) do not show any signs of cellular aging while in the pluripotency state, whereas little is known how they senesce upon differentiation. Furthermore, it is largely unclear how the metabolome of cells changes during replicative senescence and if such changes are consistent across different cell types. In this study, we have directly compared culture expansion of primary mesenchymal stromal cells (MSCs) and iPSC-derived MSCs (iMSCs) until they reached growth arrest after a mean of 21 and 17 cumulative population doublings, respectively. Both cell types acquired similar changes in morphology, in vitro differentiation potential, up-regulation of senescence-associated beta-galactosidase, and senescence-associated DNA methylation changes. Furthermore, MSCs and iMSCs revealed overlapping gene expression changes during culture expansion, particularly in functional categories related to metabolic processes. We subsequently compared the metabolome of MSCs and iMSCs at early and senescent passages and observed various significant and overlapping senescence-associated changes in both cell types, including down-regulation of nicotinamide ribonucleotide and up-regulation of orotic acid. Replicative senescence of both cell types was consistently reflected by the metabolic switch from oxidative to glycolytic pathways. Taken together, long-term culture of iPSC-derived MSCs evokes very similar molecular and functional changes as observed in primary MSCs. Replicative senescence is associated with a highly reproducible senescence-associated metabolomics phenotype, which may be used to monitor the state of cellular aging. Overall design: Bone marrow mesenchymal stromal cells were cultured until reach senescence, reprogrammed to induced pluripotent stem cells and finally differentiated back to induced mesenchymal stromal cells.

原代细胞（primary cells）的长期培养可通过功能与分泌变化体现，此类变化最终会导致复制性衰老（replicative senescence）。与之相反，诱导多能干细胞（induced pluripotent stem cells, iPSCs）在多能状态下不会表现出任何细胞衰老迹象，但目前对于其分化后的衰老机制尚不清楚。
此外，目前尚不清楚细胞代谢组在复制性衰老过程中会发生何种变化，以及此类变化是否在不同细胞类型中保持一致。
本研究直接比较了原代间充质基质细胞（primary mesenchymal stromal cells, MSCs）与诱导多能干细胞来源的间充质基质细胞（iPSC-derived MSCs, iMSCs）的培养扩增过程，直至二者分别在平均累积群体倍增数为21次和17次时达到生长停滞。
两种细胞类型均出现了相似的形态学改变、体外分化潜能变化、衰老相关β-半乳糖苷酶（senescence-associated beta-galactosidase）表达上调，以及衰老相关DNA甲基化修饰变化。
此外，在培养扩增过程中，MSCs与iMSCs呈现出重叠的基因表达谱改变，尤其富集于与代谢过程相关的功能类别。
随后，我们比较了早期传代与衰老传代的MSCs和iMSCs的代谢组，发现两种细胞类型均存在多种显著且重叠的衰老相关代谢变化，包括烟酰胺核糖核苷酸（nicotinamide ribonucleotide）表达下调与乳清酸（orotic acid）表达上调。
两种细胞类型的复制性衰老均一致地表现为从氧化代谢途径向糖酵解途径的代谢转换。
综上，诱导多能干细胞来源的间充质基质细胞的长期培养所引发的分子与功能变化，与原代MSCs中观察到的变化高度相似。
复制性衰老与一种高度可重复的衰老相关代谢组学表型相关，该表型可用于监测细胞衰老状态。
总体实验设计：将骨髓间充质基质细胞培养至衰老状态，随后将其重编程为诱导多能干细胞，最终再分化为诱导性间充质基质细胞。