Atmospheric oxygen inhibits growth and differentiation of marrow-derived mouse mesenchymal stem cells via a p53-dependent mechanism: implications for long-term culture expansion.
Atmospheric oxygen inhibits growth and differentiation of marrow-derived mouse mesenchymal stem cells via a p53-dependent mechanism: implications for long-term culture expansion.
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DOI：
10.1002/stem.1069
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发表时间：
2012-05
期刊：
影响因子：
5.2
通讯作者：
Phinney, Donald G.
中科院分区：
文献类型：
--
作者：
Boregowda, Siddaraju V.;Krishnappa, Veena;Chambers, Jeremy W.;Lograsso, Philip V.;Lai, Wen-Tzu;Ortiz, Luis A.;Phinney, Donald G.
大规模扩增人类间充质干细胞（MSC）通常用于临床治疗。相比之下，由于啮齿动物生物学的独特性，开发大规模扩增原代小鼠间充质干细胞的方案变得更加困难。目前，分离小鼠间充质干细胞的既定方法选择在大气氧中长期（数月）扩张后从骨髓培养物中出现的快速分裂的亚群。在此，我们证明，暴露于大气中的氧气会快速诱导原代小鼠 MSC 中 p53、TOP2A 和 BAX 的表达以及线粒体 ROS 的产生，从而导致氧化应激、细胞活力降低和细胞增殖抑制。或者，在 5% 氧气中进行采集和培养可支持骨髓中 CD45−ve/CD44+ve 细胞部分的更多增殖，在免疫耗竭后产生增加的 MSC 产量，并支持持续的 MSC 生长，导致第四代时累积细胞产量增加 2300 倍。在 5% 氧气中培养的 MSC 也表现出增强的三系分化。氧诱导的应激反应依赖于p53，因为siRNA介导的野生型细胞中p53的敲低或p53−/− MSC暴露于大气氧气未能诱导ROS产生、降低活力或阻止细胞生长。这些数据表明，小鼠 MSC 在大气氧气中的长期培养扩增会选择 p53 功能缺失或受损的克隆，从而使细胞能够逃避氧诱导的生长抑制。相比之下，在 5% 氧气中扩增会产生大量原代小鼠 MSC，即使在体外长期扩增后，它们仍保留对大气氧的敏感性，因此保留了功能性 p53 蛋白。
Large scale expansion of human mesenchymal stem cells (MSCs) is routinely performed for clinical therapy. In contrast, developing protocols for large scale expansion of primary mouse MSCs has been more difficult due to unique aspects of rodent biology. Currently, established methods to isolate mouse MSCs select for rapidly dividing subpopulations that emerge from bone marrow cultures following long-term (months) expansion in atmospheric oxygen. Herein, we demonstrate that exposure to atmospheric oxygen rapidly induced p53, TOP2A and BAX expression and mitochondrial ROS generation in primary mouse MSCs resulting in oxidative stress, reduced cell viability and inhibition of cell proliferation. Alternatively, procurement and culture in 5% oxygen supported more prolific expansion of the CD45−ve/CD44+ve cell fraction in marrow, produced increased MSC yields following immuno-depletion, and supported sustained MSC growth resulting in a 2300-fold increase in cumulative cell yield by 4th passage. MSCs cultured in 5% oxygen also exhibited enhanced tri-lineage differentiation. The oxygen-induced stress response was dependent upon p53 since siRNA mediated knockdown of p53 in wild type cells or exposure of p53−/− MSCs to atmospheric oxygen failed to induce ROS generation, reduce viability, or arrest cell growth. These data indicate that long-term culture expansion of mouse MSCs in atmospheric oxygen selects for clones with absent or impaired p53 function, which allows cells to escape oxygen-induced growth inhibition. In contrast, expansion in 5% oxygen generates large numbers of primary mouse MSCs that retain sensitivity to atmospheric oxygen, and therefore a functional p53 protein, even after long-term expansion in vitro.
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影响因子：
4
作者：
Chambers, Jeremy W.;Cherry, Lisa;Laughlin, John D.;Figuera-Losada, Mariana;LoGrasso, Philip V.
通讯作者：
LoGrasso, Philip V.
影响因子：
7.8
作者：
Busuttil, RA;Rubio, M;Vijg, J
通讯作者：
Vijg, J
影响因子：
4
作者：
Baddoo, M;Hill, K;Phinney, DG
通讯作者：
Phinney, DG
影响因子：
6.5
作者：
Meirelles, LD;Nardi, NB
通讯作者：
Nardi, NB
影响因子：
4.3
作者：
Lebedeva, Maria A.;Eaton, Jana S.;Shadel, Gerald S.
通讯作者：
Shadel, Gerald S.