Qi Zijuan, Cui Yazhou, Shi Liang, Luan Jing, Zhou Xiaoyan, Han Jinxiang
School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Sciences, Ji'nan, China.
Key Laboratory for Rare Disease Research of Shandong Province, Key Laboratory for Biotech Drugs of the Ministry of Health, Shandong Medical Biotechnological Center, Shandong Academy of Medical Sciences, Ji'nan, China.
Intractable Rare Dis Res. 2018 May;7(2):87-93. doi: 10.5582/irdr.2018.01032.
The aim of the study was to establish an induced pluripotent stem cell line from urine-derived cells (UiPSCs) from a patient with phenylketonuria (PKU) in order to provide a useful research tool with which to examine the pathology of this rare genetic metabolic disease. Urine-derived epithelial cells (UCs) from a 15-year-old male patient with PKU were isolated and reprogrammed with integration-free episomal vectors carrying an OCT4, SOX2, KLF4, and miR-302-367 cluster. PKU-UiPSCs were verified as correct using alkaline phosphatase staining. Pluripotency markers were detected with real-time PCR and flow cytometry. Promoter methylation in two pluripotent genes, and , was analyzed using bisulphite sequencing. An embryoid body (EB) formation assay was also performed. An induced pluripotent stem cell line (iPSC) was generated from epithelial cells in urine from a patient with PKU. This cell line had increased expression of stem cell biomarkers, it efficiently formed EBs, it stained positive for alkaline phosphatase (ALP), and it had a marked decrease in promoter methylation in the and genes. The PKU-UiPSCs created here had typical characteristics and are suitable for further differentiation.
本研究的目的是从一名苯丙酮尿症（PKU）患者的尿液来源细胞（UiPSCs）中建立诱导多能干细胞系，以便提供一个有用的研究工具来研究这种罕见的遗传代谢疾病的病理学。从一名15岁的PKU男性患者中分离出尿液来源的上皮细胞（UCs），并用携带OCT4、SOX2、KLF4和miR-302-367簇的无整合附加型载体进行重编程。使用碱性磷酸酶染色验证PKU-UiPSCs是否正确。通过实时PCR和流式细胞术检测多能性标记物。使用亚硫酸氢盐测序分析两个多能基因和的启动子甲基化。还进行了胚状体（EB）形成试验。从一名PKU患者尿液中的上皮细胞产生了诱导多能干细胞系（iPSC）。该细胞系干细胞生物标志物的表达增加，能有效形成EB，碱性磷酸酶（ALP）染色呈阳性，并且和基因的启动子甲基化显著降低。这里创建的PKU-UiPSCs具有典型特征，适合进一步分化。
