Alizadeh Effat, Zarghami Nosratollah, Eslaminejad Mohamadreza Baghaban, Akbarzadeh Abolfazl, Barzegar Abolfazl, Mohammadi Seyed Abolghasem
a Department of Medical Biotechnology , Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences , Tabriz , Iran.
b The Umbilical Cord Stem Cell Research Center (UCSRC), Tabriz University of Medical Sciences , Tabriz , Iran.
Artif Cells Nanomed Biotechnol. 2016;44(1):157-64. doi: 10.3109/21691401.2014.928778. Epub 2014 Jun 30.
Adipose tissue-derived mesenchymal stem cells (AT-MSCs) are suitable choices in autologous stem cell treatment of liver-associated diseases due to their hepatic differentiation potential. Dimethyl sulfoxide (DMSO) is an amphipathic molecule with potential of delivering both lipophilic and hydrophilic agents into cells, also a common cryoprotectant for freezing of the cells. DMSO was used in some protocols for induction of AT-MSCs towards hepatocyte like cells. However, the effect of DMSO on hepatogenic differentiation of AT-MSCs were not surveyed, previously. In the present study, we aimed at evaluation of the effect of DMSO on differentiation of AT-MSCs into hepatic lineage.
We isolated mesenchymal stem cells (MSCs) from adipose tissue, and then verifies multi-potency and surface markers of AT-MSCs . Isolated AT-MSCs randomly dispensed in four groups including Group 1: HGF treated, 2: HGF+ DMSO treated, 3: HGF+ DMSO+ OSM treated, and group control for a period of 3 weeks in the expansion medium without serum; EGF and bFGF were also included in the first days of inductions. The morphologic changes during induction period was observed with microscopy. The secretion of albumin (ALB) of the differentiating MSCs was investigated using ELISA, and urea production was evaluated using colorimetric assay. The qRT-PCR was performed for quantitation of hepatocyte marker genes including AFP, ALB, CK18, HNF4a, and HNF6. The glycogen storage of differentiated cells was visualized by periodic-acid Schiff‘s staining.
The results demonstrate that DMSO speeds up hepatic differentiation of AT-MSCs characterized by rapid changes in morphology; higher expression of hepatic marker gene (ALB) in both mRNA and protein level (P < 0.05); also increased transcriptional levels of other liver genes including CK18, HNF4a, and HNF6 (P < 0.01); and moreover, greater percentage of glycogen storage(p < 0.05) in DMSO-treated groups.
DMSO catalyzes hepatic differentiation; therefore, using DMSO for acceleration of the hepatogenic protocols of AT-MSCs appears advantageous.
脂肪组织来源的间充质干细胞（AT-MSCs）因其具有向肝细胞分化的潜能，是肝脏相关疾病自体干细胞治疗的合适选择。二甲基亚砜（DMSO）是一种两亲性分子，有将亲脂性和亲水性药物输送到细胞内的潜能，也是细胞冻存常用的冷冻保护剂。在一些诱导AT-MSCs向类肝细胞分化的方案中使用了DMSO。然而，此前尚未研究DMSO对AT-MSCs肝细胞分化的影响。在本研究中，我们旨在评估DMSO对AT-MSCs向肝系分化的影响。
我们从脂肪组织中分离间充质干细胞（MSCs），然后验证AT-MSCs的多能性和表面标志物。将分离的AT-MSCs随机分为四组，包括第1组：用HGF处理；第2组：用HGF和DMSO处理；第3组：用HGF、DMSO和OSM处理；以及对照组，在无血清的扩增培养基中培养3周；诱导开始的头几天还添加了EGF和bFGF。用显微镜观察诱导期的形态变化。用ELISA法检测分化的MSCs中白蛋白（ALB）的分泌，用比色法评估尿素生成。进行qRT-PCR定量检测包括AFP、ALB、CK18、HNF4a和HNF6在内的肝细胞标志物基因。用高碘酸希夫氏染色观察分化细胞中糖原的储存情况。
结果表明，DMSO加速了AT-MSCs的肝细胞分化，其特征为形态迅速改变；肝标志物基因（ALB）在mRNA和蛋白质水平上均有更高表达（P<0.05）；其他肝脏基因包括CK18、HNF4a和HNF6的转录水平也升高（P<0.01）；此外，DMSO处理组中糖原储存的百分比更高（P<0.05）。
DMSO催化肝细胞分化；因此，使用DMSO加速AT-MSCs的肝细胞生成方案似乎具有优势。
