Tokiwa Takayoshi, Yamazaki Taisuke, Enosawa Shin
Department of Liver Cell Biology, Kohno Clinical Medicine Research Institute , Tokyo Japan.
† Department of Regeneration Surgery, Research Institute, National Center for Child Health and Development , Tokyo Japan .
Cell Med. 2012 Jun 15;3(1-3):127-135. doi: 10.3727/215517912X639450. eCollection 2012 Jan.
The existence of hepatic stem cells in human livers is controversial. We investigated whether the side population (SP) cells derived from an immortalized human liver epithelial cell line THLE-5b possess the properties of hepatic stem-like cells. SP cells derived from THLE-5b were isolated using flow cytometry and were assayed for the expression of phenotypic markers by reverse transcription polymerase chain reaction and immunostaining. THLE-5b SP cells retained the capacity to generate both SP and non-SP cells, showed a capacity for self-renewal, and were more efficient in colony formation than non-SP cells. Neither the SP nor the non-SP cells formed tumors when transplanted into athymic nude mice or severe combined immunodeficient mice. The expression level of stem cell-associated markers such as an ATP-binding cassette membrane transporter, epithelial cell adhesion molecule, c-kit, Thy-1, and octomer binding transcription factor 4 was higher in SP cells than in non-SP cells. When cultivated as rotation-mediated aggregates, the expression of liver-specific genes including tryptophan oxygenase and CYP3A4 was up-regulated in SP cells, suggesting that THLE-5b SP cells have the ability to differentiate into a hepatocyte phenotype. One of the clonal cell lines derived from the SP cells expressed stem cell-associated markers. These results indicate that SP cells derived from THLE-5b possess hepatic stem-like cell properties and suggest that THLE-5b can be used as a model of normal human liver progenitor or stem cell line.
人类肝脏中肝干细胞的存在存在争议。我们研究了源自永生化人肝上皮细胞系THLE - 5b的侧群（SP）细胞是否具有肝干细胞样细胞的特性。使用流式细胞术分离源自THLE - 5b的SP细胞，并通过逆转录聚合酶链反应和免疫染色检测表型标志物的表达。THLE - 5b SP细胞保留了产生SP细胞和非SP细胞的能力，表现出自我更新能力，并且在集落形成方面比非SP细胞更有效。当将SP细胞和非SP细胞移植到无胸腺裸鼠或严重联合免疫缺陷小鼠中时，两者均未形成肿瘤。SP细胞中干细胞相关标志物如ATP结合盒膜转运蛋白、上皮细胞粘附分子、c - kit、Thy - 1和八聚体结合转录因子4的表达水平高于非SP细胞。当作为旋转介导的聚集体培养时，SP细胞中包括色氨酸加氧酶和CYP3A4在内的肝脏特异性基因的表达上调，表明THLE - 5b SP细胞具有分化为肝细胞表型的能力。源自SP细胞的一个克隆细胞系表达干细胞相关标志物。这些结果表明，源自THLE - 5b的SP细胞具有肝干细胞样细胞特性，并表明THLE - 5b可作为正常人肝祖细胞或干细胞系的模型。
