Role of the Apoptosis Pathway in Cryopreservation-Induced Cell Death in Mesenchymal Stem Cells Derived from Umbilical Cord Blood
Role of the Apoptosis Pathway in Cryopreservation-Induced Cell Death in Mesenchymal Stem Cells Derived from Umbilical Cord Blood
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DOI：
10.1089/bio.2014.0005
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发表时间：
2014-08-01
影响因子：
1.6
通讯作者：
Pramanik, Krishna
中科院分区：
文献类型：
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作者：
Bissoyi, Akalabya;Pramanik, Krishna
骨髓间充质干细胞(MSCs)的冷冻保存非常重要，因为它们在临床领域具有商业应用价值。骨髓间充质干细胞在解冻过程中，由于凋亡相关蛋白的激活，很容易受到冷冻诱导的细胞凋亡的影响。但冷冻保存与细胞凋亡之间的关系还不是很清楚。脐血MSCs以Me2SO为冻存保护剂，分别加或不加caspase抑制剂z-VAD-FMK或选择性较强的caspase抑制剂z-IETD-FMK、z-Lehd-FMK和z-DEVD-FMK。为了评估钙介导的途径的效果，对冷冻保存的MSCs进行了含有和不含有钙蛋白酶抑制剂的测试。用流式细胞仪检测细胞存活率、线粒体膜电位和细胞周期分析。Western blotting检测原半胱氨酸天冬氨酸蛋白酶-3、-8、-9、钙蛋白水解酶和BID的表达。冷冻保存的MSCs在解冻后24 h内出现特征性的细胞凋亡。结果表明，冷冻保存的MSCs解冻后，内源性、外源性和钙蛋白途径均被激活。与选择性caspase抑制剂相比，一般的caspase抑制剂能更有效地抑制DNA降解，还能抑制caspase-3和-8的加工以及Bid的切割，从而显示出减少冷冻诱导的细胞凋亡的有益效果。类似地，抑制钙蛋白酶可以减少冷冻诱导的细胞凋亡。这些数据表明，使用标准的冷冻保存方案冷冻保存后，caspase介导的外在和内在通路以及蛋白水解级联钙蛋白被激活。这种激活可能在冷冻保存诱导的细胞死亡过程中发挥重要作用。此外，抑制钙蛋白酶活性和半胱氨酸天冬氨酸氨基转移酶途径可能提高保存效果。
Cryopreservation of mesenchymal stem cells (MSCs) is important because of their commercial applications in the clinical sector. MSCs are vulnerable to cryopreservation-induced apoptosis due to activation of apoptosis-related proteins during thawing. But the relationship between cryopreservation and apoptosis is not well understood. MSCs derived from umbilical cord blood were cryopreserved using Me2SO as the cryoprotective agent, with or without pre-treatment with the general caspase inhibitor z-VAD-FMK, or with the more selective caspase inhibitors z-IETD-FMK, z-LEHD-FMK and z-DEVD-FMK. To evaluate the effect of the calcium-mediated pathway, cryopreserved MSCs were tested with and without a calpain inhibitor. FACS was used to measure cell viability, mitochondrial membrane potential, and cell cycle analysis. Processing of the pro-caspases-3, -8, -9, calpain and Bid were determined by Western blotting. Cryopreservation of MSCs resulted in characteristic apoptosis within 24 h after thawing. Results show that intrinsic, extrinsic, and calpain pathways are activated after cryopreserved MSCs are thawed. Compared to selective caspase inhibitors, a general caspase inhibitor blocked DNA degradation more effectively and also inhibited caspases-3 and -8 processing as well as Bid cleavage, showing the beneficial effect of reducing cryopreservation-induced apoptosis. Similarly, calpain inhibition reduced cryopreservation-induced apoptosis. These data indicate that caspase-mediated extrinsic and intrinsic pathways and the proteolytic calpain cascade were activated after cryopreservation using a standard cryopreservation protocol. This activation might play an important role in the process of cryopreservation-induced cell death. Furthermore, the inhibition of calpain activity and caspase-mediated pathways might improve preservation efficacy.