Decrease of Global Methylation Improves Significantly Hepatic Differentiation of Ad-MSCs: Possible Future Application for Urea Detoxification
Decrease of Global Methylation Improves Significantly Hepatic Differentiation of Ad-MSCs: Possible Future Application for Urea Detoxification
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DOI：
10.3727/096368912x638946
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发表时间：
2013-01-01
影响因子：
3.3
通讯作者：
Nuessler, A. K.
中科院分区：
文献类型：
--
作者：
Seeliger, C.;Culmes, M.;Nuessler, A. K.
肝细胞移植被认为是原位肝移植的一种替代方法。细胞可以用来为等待供体器官的患者架起桥梁，降低急性肝衰竭的死亡率，并支持代谢性肝病。有限的原代人肝细胞可用于此类应用，导致从各种成体干细胞或前体细胞中产生替代的肝细胞样细胞。这项研究的目的是从脂肪来源的间充质干细胞（Ad-MSCs）中产生肝细胞样细胞，用于临床应用，这种细胞是现成的。5-氮杂胞苷诱导肝细胞样细胞的表观遗传变化，与其他补充剂联合使用，与未处理的细胞相比，显著改善了代谢和酶活性。采用四步方案生成具有足够肝脏特征的细胞：5-氮杂胞苷（步骤1）；表皮生长因子（步骤2）；成纤维细胞生长因子-4、地塞米松、胰岛素-转铁蛋白-亚硒酸钠和烟酰胺（步骤3）；还有肝细胞生长因子，地塞米松，胰岛素-转铁蛋白-亚硒酸钠和烟酰胺（第四步）。与未分化细胞相比，生成的分化细胞具有更高的I期（CYP1A1/2、CYP2E1、CYP2B6、CYP3A4）和II期活性。在mRNA水平上还可以检测到CYP3A7的强表达和3A4的弱表达，以及重要的解毒标志物甲胎蛋白和白蛋白。重要的是，尿素代谢（基础、NH4刺激、NH4-和鸟氨酸刺激）与新鲜分离的人肝细胞相当，与冷冻保存的人肝细胞不同，这种活性在冷冻保存6个月后仍能保持。这些发现提示这些细胞可能适合临床应用，特别是用于治疗尿素循环障碍。
Hepatocyte transplantation is considered to be an alternative to orthotopic liver transplantation. Cells can be used to bridge patients waiting for a donor organ, decrease mortality in acute liver failure, and support metabolic liver diseases. The limited availability of primary human hepatocytes for such applications has led to the generation of alternative hepatocyte-like cells from various adult stem or precursor cells. The aim of this study was to generate hepatocyte-like cells from adipose-derived mesenchymal stem cells (Ad-MSCs) for clinical applications, which are available "off the shelf." Epigenetic changes in hepatocyte-like cells were induced by 5-azacytidine, which, in combination with other supplements, leads to significantly improved metabolic and enzymatic activities compared to nontreated cells. Cells with sufficient hepatic features were generated with a four-step protocol: 5-azacytidine (step 1); epidermal growth factor (step 2); fibroblast growth factor-4, dexamethasone, insulin-transferrin-sodium-selenite, and nicotinamide (step 3); and hepatocyte growth factor, dexamethasone, insulin-transferrin-sodium-selenite, and nicotinamide (step 4). Generated differentiated cells had higher phase I (CYP1A1/2, CYP2E1, CYP2B6, CYP3A4) and phase II activities compared to the undifferentiated cells. A strong expression of CYP3A7 and a weak expression of 3A4, as well as the important detoxification markers alpha-fetoprotein and albumin, could also be detected at the mRNA level. Importantly, urea metabolism (basal, NH4-stimulated, NH4- and ornithine-stimulated) was comparable to freshly isolated human hepatocytes, and unlike cryopreserved human hepatocytes, this activity was maintained after 6 months of cryopreservation. These findings suggest that these cells may be suitable for clinical application, especially for treatment of urea cycle disorders.