Du Yingdong, Li Dawei, Han Conghui, Wu Haoyu, Xu Longmei, Zhang Ming, Zhang Jianjun, Chen Xiaosong
Department of Transplantation and Hepatic Surgery, PLA No. 107 Hospital, Yantaii, China.
Department of Urology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
Cell Physiol Biochem. 2017;43(2):611-625. doi: 10.1159/000480533. Epub 2017 Sep 21.
BACKGROUND/AIMS: This study aimed to evaluate the effects of exosomes produced by human-induced pluripotent stem cell-derived mesenchymal stromal cells (hiPSC-MSCs-Exo) on hepatic ischemia-reperfusion (I/R) injury, as well as the underlying mechanisms.
Exosomes derived from hiPSC-MSCs were isolated and characterized both biochemically and biophysically. hiPSC-MSCs-Exo were injected systemically into a murine ischemia/reperfusion injury model via the inferior vena cava, and then the therapeutic effects were evaluated. The serum levels of transaminases (aspartate aminotransferase (AST) and alanine aminotransferase (ALT), as well as histological changes were examined. Primary hepatocytes and human hepatocyte cell line HL7702 were used to test whether exosomes could induce hepatocytes proliferation in vitro. In addition, the expression levels of proliferation markers (proliferation cell nuclear antigen, PCNA; Phosphohistone-H3, PHH3) were measured by immunohistochemistry and Western blot. Moreover, SK inhibitor (SKI-II) and S1P1 receptor antagonist (VPC23019) were used to investigate the role of sphingosine kinase and sphingosine-1-phosphate-dependent pathway in the effects of hiPSC-MSCs-Exo on hepatocytes.
hiPSCs were efficiently induced into hiPSC-MSCs that had typical MSC characteristics. hiPSC-MSCs-Exo had diameters ranging from 100 to 200 nm and expressed exosome markers (Alix, CD63 and CD81). After hiPSC-MSCs-Exo administration, hepatocyte necrosis and sinusoidal congestion were markedly suppressed in the ischemia/reperfusion injury model, with lower histopathological scores. The levels of hepatocyte injury markers AST and ALT were significantly lower in the treatment group compared to control, and the expression levels of proliferation markers (PCNA and PHH3) were greatly induced after hiPSC-MSCs-Exo administration. Moreover, hiPSC-MSCs-Exo also induced primary hepatocytes and HL7702 cells proliferation in vitro in a dose-dependent manner. We found that hiPSC-MSCs-Exo could directly fuse with target hepatocytes or HL7702 cells and increase the activity of sphingosine kinase and synthesis of sphingosine-1-phosphate (S1P). Furthermore, the inhibition of SK1 or S1P1 receptor completely abolished the protective and proliferative effects of hiPSC-MSCs-Exo on hepatocytes, both in vitro and in vivo.
Our results demonstrated that hiPSC-MSCs-Exo could alleviate hepatic I/R injury via activating sphingosine kinase and sphingosine-1-phosphate pathway in hepatocytes and promote cell proliferation. These findings represent a novel mechanism that potentially contributes to liver regeneration and have important implications for new therapeutic approaches to acute liver disease.
背景/目的：本研究旨在评估人诱导多能干细胞衍生的间充质基质细胞（hiPSC-MSCs）产生的外泌体（hiPSC-MSCs-Exo）对肝脏缺血再灌注（I/R）损伤的影响及其潜在机制。
分离源自hiPSC-MSCs的外泌体，并进行生化和生物物理特性鉴定。通过下腔静脉将hiPSC-MSCs-Exo全身注射到小鼠缺血/再灌注损伤模型中，然后评估其治疗效果。检测血清转氨酶（天冬氨酸转氨酶（AST）和丙氨酸转氨酶（ALT））水平以及组织学变化。使用原代肝细胞和人肝细胞系HL7702来测试外泌体是否能在体外诱导肝细胞增殖。此外，通过免疫组织化学和蛋白质印迹法检测增殖标志物（增殖细胞核抗原，PCNA；磷酸化组蛋白-H3，PHH3）的表达水平。此外，使用鞘氨醇激酶抑制剂（SKI-II）和鞘氨醇-1-磷酸受体拮抗剂（VPC23019）来研究鞘氨醇激酶和鞘氨醇-1-磷酸依赖性途径在hiPSC-MSCs-Exo对肝细胞作用中的作用。
hiPSC被有效地诱导为具有典型间充质干细胞特征的hiPSC-MSCs。hiPSC-MSCs-Exo的直径范围为100至200nm，并表达外泌体标志物（Alix、CD63和CD81）。给予hiPSC-MSCs-Exo后，缺血/再灌注损伤模型中的肝细胞坏死和窦状充血明显受到抑制，组织病理学评分较低。与对照组相比，治疗组中肝细胞损伤标志物AST和ALT水平显著降低，给予hiPSC-MSCs-Exo后增殖标志物（PCNA和PHH3）的表达水平显著诱导。此外，hiPSC-MSCs-Exo还以剂量依赖性方式在体外诱导原代肝细胞和HL7702细胞增殖。我们发现hiPSC-MSCs-Exo可以直接与靶肝细胞或HL7702细胞融合，并增加鞘氨醇激酶的活性和鞘氨醇-1-磷酸（S1P）的合成。此外，抑制SK1或S1P1受体完全消除了hiPSC-MSCs-Exo在体外和体内对肝细胞的保护和增殖作用。
我们的结果表明，hiPSC-MSCs-Exo可通过激活肝细胞中的鞘氨醇激酶和鞘氨醇-1-磷酸途径减轻肝脏I/R损伤并促进细胞增殖。这些发现代表了一种可能有助于肝脏再生的新机制，对急性肝病的新治疗方法具有重要意义。
