Department of Laboratory Sciences, School of Paramedical Sciences, Mashhad University of Medical Sciences, Mashhad, Iran.
Persian Cohort Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.
Oxid Med Cell Longev. 2021 Nov 22;2021:4229721. doi: 10.1155/2021/4229721. eCollection 2021.
An alternative treatment to liver transplantation includes the use of differentiated stem cells. Hypoxia has been shown to endow human-induced pluripotent stem cells (hiPSCs) with enhanced hepatic differentiation. We have investigated a new strategy for hepatocyte differentiation from hiPSCs using a three-step differentiation protocol with lentiviral overexpression of hypoxia-microRNA-210 of cells grown on a hybrid scaffold. We analyzed the transduction of the miR-210 lentiviral and definitive endoderm and pluripotency gene markers, including SRY-box 17 (SOX17), forkhead box A2 (FOXA2), and octamer-binding transcription factor 4 (OCT-4) by Real-Time PCR and fluorescent microscope. The scanning electron microscopy (SEM) examined the 3D cell morphological changes. Immunocytochemistry staining was used together with assays for aspartate aminotransferase, alanine aminotransferase, and urea secretion to analyze hepatocyte biomarkers and functional markers consisting of -fetoprotein (AFP), low-density lipoprotein (LDL) uptake, fat accumulation, and glycogen. The flow cytometry analyzed the generation of reactive oxygen species (ROS). Compared to cells transfected with the blank lentiviral vectors as a control, overexpressing miR-210 was at higher levels in hiPSCs. The expression of endodermal genes and glycogen synthesis significantly increased in the differentiated lentiviral miR-210 cells without any differences regarding lipid storage level. Additionally, cells containing miR-210 showed a greater expression of ALB, LDL, AST, ALT, urea, and insignificant lower AFP and ROS levels after 18 days. However, SEM showed no significant differences between cells under the differentiation process and controls. In conclusion, the differentiation of hiPSCs to hepatocyte-like cells under hypoxia miR-210 may be a suitable method for cell therapy and regenerative medicine.
肝移植的一种替代疗法包括使用分化的干细胞。缺氧已被证明能使人类诱导多能干细胞（hiPSCs）增强肝分化。我们使用三步分化方案，通过慢病毒过表达细胞在混合支架上生长的缺氧 microRNA-210，从 hiPSCs 中研究了一种新的肝细胞分化策略。我们通过实时 PCR 和荧光显微镜分析了 miR-210 慢病毒和确定的内胚层和多能性基因标记（包括 SRY 盒 17（SOX17）、叉头框 A2（FOXA2）和八聚体结合转录因子 4（OCT-4）的转导。扫描电子显微镜（SEM）检查了 3D 细胞形态变化。免疫细胞化学染色与天门冬氨酸氨基转移酶、丙氨酸氨基转移酶和尿素分泌分析一起用于分析肝细胞生物标志物和功能性标志物，包括 - 胎蛋白（AFP）、低密度脂蛋白（LDL）摄取、脂肪积累和糖原。流式细胞术分析了活性氧物种（ROS）的产生。与作为对照的空白慢病毒载体转染的细胞相比，hiPSCs 中过表达的 miR-210 水平更高。分化的慢病毒 miR-210 细胞中的内胚层基因表达和糖原合成显著增加，而脂质储存水平没有差异。此外，含有 miR-210 的细胞在 18 天后显示出更高的 ALB、LDL、AST、ALT、尿素表达和微不足道的 AFP 和 ROS 水平降低。然而，SEM 显示分化过程中的细胞与对照之间没有明显差异。总之，缺氧 miR-210 下 hiPSCs 向肝细胞样细胞的分化可能是细胞治疗和再生医学的一种合适方法。
