Mitochondrial Function Determines the Viability and Osteogenic Potency of Human Mesenchymal Stem Cells
Mitochondrial Function Determines the Viability and Osteogenic Potency of Human Mesenchymal Stem Cells
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DOI：
10.1089/ten.tec.2009.0247
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发表时间：
2010-06-01
影响因子：
3
通讯作者：
Lehenkari, Petri
中科院分区：
文献类型：
--
作者：
Pietila, Mika;Lehtonen, Siri;Lehenkari, Petri
先进疗法医药产品（ATMPs）推出了创新的细胞产品。然而，对atmp表征的监管要求目前无法充分解决此类产品的安全性问题。由于最近的研究强调了线粒体在人间充质干细胞（hMSCs）成骨分化中的作用，我们基于线粒体内膜电位（Delta psi(m)）的分析，详细研究了作为atmp使用的hMSCs的活力和成骨分化能力。采用流式细胞术测定5,5',6,6'-四氯-1,1',3,3'-四乙基苯并咪唑碘化碳菁（JC-1），碘化丙啶荧光和AnnexinV分别测定培养的hMSCs的δ psi(m)，质膜完整性和磷脂酰丝氨酸在质膜中的组织。在甲萘醌临界浓度（20 μ M）下培养细胞，诱导细胞凋亡。以3-（4,5-二甲基噻唑-2-基）-2,5-二苯基溴化四唑（MTT）作为细胞增殖指标，以碱性磷酸酶活性和钙沉积作为成骨分化指标。根据JC-1荧光、细胞形态、磷脂酰丝氨酸的组织和质膜完整性，我们可以将细胞分为四类，代表不同的细胞质量。首次证实了JC-1与成骨分化之间的强相关性，因此该分析工具不仅适用于确定细胞活力，而且适用于预测hMSC的成骨分化。
Advanced therapies medicinal products (ATMPs) have introduced innovative cell-based products. However, the regulatory demands for characterization of ATMPs are currently unable to adequately address the safety of such products. As recent studies have emphasized the role of mitochondria in the osteogenic differentiation of human mesenchymal stem cells (hMSCs), we have studied in detail the viability and osteogenic differentiation potency of the hMSCs intended for use as ATMPs based on analyses of the mitochondrial inner membrane potential (Delta psi(m)). Flow cytometric measurement of 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1), propidium iodide fluorescence, and AnnexinV was employed to determine Delta psi(m), plasmamembrane integrity, and organization of phosphatidylserine in plasma membrane, respectively, in cultured hMSCs. Apoptosis was induced by incubating cells at critical concentration (20 mu M) of menadione. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used as an indicator for cell proliferation and alkaline phosphatase activity and calcium deposition as indicators of osteogenic differentiation. Based on JC-1 fluorescence, cell morphology, organization of phosphatidylserine, and plasma membrane integrity, we could sort cells into four categories that represented different cell quality. A strong correlation between JC-1 and osteogenic differentiation was demonstrated for the first time and thus this analytical tool is suitable not only to determine cell viability but also to predict osteogenic differentiation of hMSC.