Generation of sepsis encephalopathy patient-specific inducible pluripotent stem cells with urine cells
10.3760/cma.j.issn.2095-4352.2019.12.003
- VernacularTitle: 利用尿液细胞建立脓毒症脑损伤患者来源的多能干细胞株实验
- Author:
Xinliang QIU
1
;
Ye PAN
;
Ning ZHAO
;
Kejian QIAN
;
Yian ZHAN
Author Information
1. Department of Critical Care Medicine, the First Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi, China
- Publication Type:Journal Article
- Keywords:
Sepsis encephalopathy;
Urine cell;
Induced pluripetent stem cell
- From:
Chinese Critical Care Medicine
2019;31(12):1445-1450
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To recombine the induced pluripotent stem cells (iPSC) derived from the urine of septic encephalopathy (SE) patients, and provided a specificity cell model to explore the mechanism of the neuronal damage and treatment for SE patients.
Methods:Urine of SE patient was collected, and tubular epithelial cells were isolated and cultured from the urine. iPSC were derived from SE patient by introducing 4 transcription factors OCT4, Klf4, Sox2, c-Myc (OKSM) into patient-specific urine cells by Millipore's Human STEMCCATM Constitutive Polycistronic (OKSM) Lentivirus Kit. Colony morphology, alkaline phosphatase (AKP) activity, immunofluorescence staining, quantitative reverse transcription-polymerase chain reaction (RT-qPCR), and differentiation ability were used to identify the pluripetency of these iPSC lines. In addition, neurons were derived from these iPSC by inhibiting transforming growth factor-β (TGF-β) pathway.
Results:The SE-iPSC exhibited morphological and growth characteristics of human embryonic stem cell (hES), showed positivity for AKP by histochemical staining, and expressed embryonic stem cell (ESC) marker genes. There was a significant statistical difference in ESC-marker mRNA expression between the SE-iPSC and the urine cells [NANOG mRNA (2-ΔΔCt): 1.153±0.142 vs. 0.126±0.024, t = -10.688; REX1 mRNA (2-ΔΔCt): 1.419±0.206 vs. 0.103±0.066, t = -14.245; OCT4 mRNA (2-ΔΔCt): 1.233±0.176 vs. 0.201±0.022, t = -9.028; Sox2 mRNA (2-ΔΔCt): 1.334±0.119 vs. 0.159±0.017, t = -12.653, all P < 0.01]. Subcutaneous injection of iPSC into NOD-SCID mice resulted in teratomas containing tissues from all the 3 germ layers. Furthermore, neurons were successfully induced from SE-iPSC.
Conclusion:The SE patient-specific iPSC could be generated from urine cells and differentiated into neurons, furthermore, the SE-iPSC cell line can be used as models for further elucidating the cellular pathology and developing therapeutic strategies for SE.
