An in vitro expansion score for tissue-engineering applications with human bone marrow-derived mesenchymal stem cells
An in vitro expansion score for tissue-engineering applications with human bone marrow-derived mesenchymal stem cells
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DOI：
10.1002/term.1734
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发表时间：
2016-02-01
影响因子：
3.3
通讯作者：
Stoyanov, Jivko V.
中科院分区：
文献类型：
--
作者：
Bertolo, Alessandro;Mehr, Marco;Stoyanov, Jivko V.
人骨髓间充质干细胞（MSCs）在体外的生长潜力有限，并且由于复制衰老而停止分裂，从组织工程的角度来看，这具有实际意义，例如定义正确的分化和移植起点。在失去所需分化潜能之前的培养时间是不同的，这反映了患者的可变性，这是细胞扩增的一个问题。本研究旨在建立一个评分集，用于量化MSCs的衰老状态，并预测细胞是否保留向成骨、脂肪和软骨表型分化的能力，该评分集基于集落形成单位（CFU）测定、群体倍增时间（PDT）、衰老相关-半乳糖苷酶（SA- Gal）活性、细胞大小、端粒长度和体外培养MSCs的基因表达超过11代。这组形态学、生理和遗传衰老标记与MSCs的分化能力相关。通过标记基因和蛋白表达评价分化效率。cfu随传代次数的增加而降低，SA—Gal活性和PDT增加；然而，与MSCs分化潜能的相关性有时是出乎意料的。衰老相关基因在晚传代细胞中的表达高于早传代细胞。早期传代细胞具有高效的成骨分化，中期传代细胞在软骨分化中表现最好。晚期传代细胞仅保留成脂分化潜能。基于这个标记集，我们提出了一个衰老评分，其中组合标记提供了可靠的msc质量控制，而不仅仅依赖于机械传代数。版权所有：John Wiley & Sons， Ltd。
Human bone marrow-derived mesenchymal stem cells (MSCs) have limited growth potential in vitro and cease to divide due to replicative senescence, which from a tissue-engineering perspective has practical implications, such as defining the correct starting points for differentiation and transplantation. Time spent in culture before the loss of required differentiation potential is different and reflects patient variability, which is a problem for cell expansion. This study aimed to develop a score set which can be used to quantify the senescent state of MSCs and predict whether cells preserve their ability to differentiate to osteogenic, adipogenic and chondrogenic phenotypes, based on colony-forming unit (CFU) assay, population doubling time (PDT), senescence-associated -galactosidase (SA--Gal) activity, cell size, telomere length and gene expression of MSCs cultured in vitro over 11 passages. This set of morphological, physiological and genetic senescence markers was correlated to the ability of MSCs to differentiate. Differentiation efficiency was assessed by marker genes and protein expression. CFUs decreased with increasing passage number, whereas SA--Gal activity and PDT increased; however, the correlation with MSCs' differentiation potential was sometimes unexpected. The expression of genes related to senescence was higher in late-passage cells than in early-passage cells. Early-passage cells underwent efficient osteogenic differentiation, with mid-passage cells performing best in chondrogenic differentiation. Late-passage cells preserve only adipogenic differentiation potential. Based on this marker set, we propose a senescence score in which combined markers give a reliable quality control of MSCs, not depending only on mechanistic passage number. Copyright (c) 2013 John Wiley & Sons, Ltd.