在缺乏葡萄糖和精氨酸的培养基中，原代人肝细胞的存活和诱导多能干细胞的死亡。
Survival of primary human hepatocytes and death of induced pluripotent stem cells in media lacking glucose and arginine.
机构信息
Department of Gastroenterology, National Hospital Organization, Shimoshizu Hospital, Yotsukaido City, Chiba, Japan.
出版信息
PLoS One. 2013 Aug 14;8(8):e71897. doi: 10.1371/journal.pone.0071897. eCollection 2013.
BACKGROUND
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.
MATERIALS AND METHODS
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.
RESULTS
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.
CONCLUSION
Therefore, HSM is and ideal medium for eliminating hiPS cells and purifying hepatocytes without inducing any damage.
背景
从人诱导多能干细胞（hiPS）分化而来的肝细胞移植存在致瘤性风险。肝细胞表达半乳糖激酶和鸟氨酸转氨甲酰酶（OTC）等酶，以帮助自身存活。然而，hiPS 细胞不表达这些酶，因此，预计它们无法在含有半乳糖和鸟氨酸、缺乏葡萄糖和精氨酸的培养基中存活。
材料和方法
通过实时定量聚合酶链反应（PCR）分析半乳糖激酶 1（GALK1）1 和 GALK2、鸟氨酸氨甲酰转移酶和苯丙氨酸羟化酶（PAH）的表达。将 hiPS 细胞系 201B7 在缺乏葡萄糖和精氨酸但含有半乳糖和鸟氨酸的肝细胞选择培养基（HSM）中培养。此外，对经苏木精和伊红（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）处理后的培养细胞进行显微镜分析。对 HSM 中的 hiPS 细胞进行免疫染色以评估其多能性。此外，在 HSM 中培养原代人肝细胞并加入或不加入 hiPS 细胞。
结果
与成人肝脏相比，201B7 中的 GALK1、GALK2、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 细胞群体减少，3 天后完全消失。培养的细胞显示核的浓缩或碎裂，提示发生了细胞凋亡。TUNEL 染色证实细胞发生了凋亡。201B7 细胞对 Nanog、SSEA-4 和 TRA-1-60 呈阳性。原代人肝细胞在 HSM 中单独培养时存活，与 hiPS 细胞共培养时也存活。
结论
因此，HSM 是一种理想的培养基，可在不造成任何损伤的情况下消除 hiPS 细胞并纯化肝细胞。
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