Survival of primary human hepatocytes and death of induced pluripotent stem cells in media lacking glucose and arginine.
Survival of primary human hepatocytes and death of induced pluripotent stem cells in media lacking glucose and arginine.
复制标题
DOI：
10.1371/journal.pone.0071897
复制
发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Yoshida T
中科院分区：
文献类型：
--
作者：
Tomizawa M;Shinozaki F;Sugiyama T;Yamamoto S;Sueishi M;Yoshida T
致瘤性是从人诱导多能干细胞（hiPS）分化的肝细胞移植的相关风险。肝细胞表达半乳糖激酶和鸟氨酸转氨甲酰酶（OTC）以帮助其自身存活。然而，hiPS细胞不表达这些酶，因此，预期不能在含有半乳糖和鸟氨酸且缺乏葡萄糖和精氨酸的培养基中存活。实时定量聚合酶链反应（PCR）分析半乳糖激酶1（GALK 1）1和GALK 2，鸟氨酸氨甲酰转移酶和苯丙氨酸羟化酶（PAH）的表达。hiPS细胞系201 B7在肝细胞选择培养基（HSM）中培养，该培养基缺乏葡萄糖和精氨酸，但含有半乳糖和鸟氨酸。此外，在苏木精和伊红（H&E）染色、末端脱氧核苷酸转移酶（TdT）介导的dUTP缺口末端标记（TUNEL）后，对培养的细胞进行显微镜分析。对hiPS细胞进行免疫染色以评估其在HSM中的多能性。此外，原代人肝细胞在有或没有hiPS细胞的情况下在HSM中培养。与成人肝脏相比，201 B7中GALK 1、GALK 2、OTC和PAH的表达水平分别为22.2±5.0（平均值±标准差）、14.2% ± 1.1%、1.2% ± 0.2%和8.4% ±0.7%。当在HSM中培养时，hiPS细胞群减少，并且在3天后完全消失。培养的细胞显示其细胞核的浓缩或碎裂，从而提示凋亡。TUNEL染色证实细胞已发生凋亡。201 B7细胞对Nanog、SSEA-4和TRA-1-60呈阳性。当在HSM中单独培养时以及当与hiPS细胞共培养时，原代人肝细胞存活。因此，HSM是一种理想的用于去除hiPS细胞和纯化肝细胞的培养基，而不诱导任何损伤。
Tumorigenicity is an associated risk for transplantation of hepatocytes differentiated from human induced pluripotent stem (hiPS) cells. Hepatocytes express the enzymes galactokinase and ornithine transcarbamylase (OTC) to aid in their own survival. However, hiPS cells do not express these enzymes, and therefore, are not be expected to survive in a medium containing galactose and ornithine and lacking glucose and arginine. Real-time quantitative polymerase chain reaction (PCR) was performed to analyze the expression of galactokinase 1 (GALK1)1 and GALK2, ornithine carbamyltransferase, and phenylalanine hydroxylase (PAH). The hiPS cell line 201B7 was cultured in hepatocyte selection medium (HSM), which lacks glucose and arginine but contains galactose and ornithine. Furthermore, microscopic analysis of the cultured cells was performed after hematoxylin and eosin (H&E) staining, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL). The hiPS cells were immunostained to assess their pluripotency in HSM. In addition, the primary human hepatocytes were cultured with or without hiPS cells in HSM. The expression levels of GALK1, GALK2, OTC, and PAH in 201B7 were 22.2±5.0 (average ± standard deviation), 14.2% ±1.1%, 1.2% ±0.2%, and 8.4% ±0.7% respectively, compared with those in the adult liver. The hiPS cell population diminished when cultured in HSM and completely disappeared after 3 days. The cultured cells showed condensation or fragmentation of their nuclei, thereby suggesting apoptosis. TUNEL staining confirmed that the cells had undergone apoptosis. The 201B7 cells were positive for Nanog, SSEA-4, and TRA-1-60. The primary human hepatocytes survived when cultured alone in HSM and when co-cultured with hiPS cells. Therefore, HSM is and ideal medium for eliminating hiPS cells and purifying hepatocytes without inducing any damage.
登录
查看更多内容
DOI：
10.1083/jcb.52.3.559
发表时间：
1972-03
期刊：
The Journal of cell biology
影响因子：
--
作者：
Leffert HL;Paul D
通讯作者：
Paul D
DOI：
10.1016/0006-291x(84)91173-2
发表时间：
1984-01-01
影响因子：
3.1
作者：
NAKAMURA, T;TERAMOTO, H;ICHIHARA, A
通讯作者：
ICHIHARA, A
影响因子：
29
作者：
Folmes CD;Nelson TJ;Martinez-Fernandez A;Arrell DK;Lindor JZ;Dzeja PP;Ikeda Y;Perez-Terzic C;Terzic A
通讯作者：
Terzic A
影响因子：
64.5
作者：
Takahashi, Kazutoshi;Tanabe, Koji;Yamanaka, Shinya
通讯作者：
Yamanaka, Shinya
影响因子：
46.9
作者：
Nakagawa, Masato;Koyanagi, Michiyo;Yamanaka, Shinya
通讯作者：
Yamanaka, Shinya