Jafarpour Z, Soleimani M, Hosseinkhani S, M H M H, Yaghmaei P, Mobarra N, Geramizadeh B
Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
Department of Hematology, Faculty of Medical Science, Tarbiat Modares University, Tehran, Iran.
Int J Organ Transplant Med. 2018;9(2):77-87. Epub 2018 May 1.
Generating hepatocytes with complete liver functions is still a challenge and developing more functional hepatocytes is needed.
To compare various differentiation factors and protocols and introducing a preferable protocol to differentiate human-induced pluripotent stem cells (hiPSCs) into hepatocyte-like cells (HLCs).
After 3 days of the endoderm differentiation of hiPSCs, the cells were incubated with 5 hepatocyte differentiation culture media, protocols (P), for 14 days-P1: hepatocyte growth factor and fibroblast growth factor-4 (FGF-4) for the first week and oncostatin-M and dexamethasone for the second week; P2: similar to P1 but FGF4 was used in both the first and second weeks; P3: similar to P1 but FGF-4 was not used; P4: similar to P1 but FGF-4 and dexamethasone were not used; and P5: similar to P1 but FGF-4 and oncostatin-M were not used. After 17 days, characterization was done by qRT-PCR, immunofluorescence and ELISA.
The mRNA expression levels of hepatocyte markers (albumin, cytokeratin-18, tyrosine aminotransferase, hepatocyte nuclear factor-4α, cytochrome-P450 7A1) increased significantly (p<0.05) in the differentiated cells by 5 different protocols. Furthermore, significant protein expression and secretion of albumin were detected in the differentiated cells by 5 different protocols. In P3, the differentiated cells had the highest exhibit of hepatocyte characteristics and in P4 they had the lowest. Moreover, in P1 and P2 similar results were observed.
Since P3 gave us the best results among all protocols, we recommend it as an efficient protocol to differentiate the functional HLCs from hiPSCs, which can improve cell therapies.
生成具有完整肝功能的肝细胞仍然是一项挑战，需要开发更多功能性肝细胞。
比较各种分化因子和方案，并引入一种更优方案将人诱导多能干细胞（hiPSCs）分化为肝样细胞（HLCs）。
hiPSCs向内胚层分化3天后，将细胞与5种肝细胞分化培养基、方案（P）孵育14天——P1：第一周使用肝细胞生长因子和成纤维细胞生长因子-4（FGF-4），第二周使用制瘤素-M和地塞米松；P2：与P1相似，但第一周和第二周均使用FGF4；P3：与P1相似，但不使用FGF-4；P4：与P1相似，但不使用FGF-4和地塞米松；P5：与P1相似，但不使用FGF-4和制瘤素-M。17天后，通过qRT-PCR、免疫荧光和ELISA进行表征。
通过5种不同方案分化的细胞中，肝细胞标志物（白蛋白、细胞角蛋白-18、酪氨酸转氨酶、肝细胞核因子-4α、细胞色素P450 7A1）的mRNA表达水平显著升高（p<0.05）。此外，通过5种不同方案在分化细胞中检测到白蛋白有显著的蛋白表达和分泌。在P3中，分化细胞表现出最高的肝细胞特征，在P4中则最低。此外，在P1和P2中观察到相似的结果。
由于P3在所有方案中给出了最佳结果，我们推荐它作为从hiPSCs分化功能性HLCs的有效方案，这可以改善细胞治疗。
