机构:[1]Sun Yat Sen Univ, Zhongshan Ophthalm Ctr, State Key Lab Ophthalmol, Guangzhou, Guangdong, Peoples R China[2]Sun Yat Sen Univ, Canc Ctr, State Key Lab Oncol South China, Guangzhou, Guangdong, Peoples R China[3]Rutgers State Univ, Robert Wood Johnson Med Sch, Dept Pediat, Ctr Adv Biotechnol & Med, Piscataway, NJ 08854 USA
Objective: Establishing a practical procedure to generate induced pluripotent stem cells (iPSCs) and induced neural stem cells (iNSCs) from human urine cells (UCs). In this report, we optimized a non-integrative protocol to generate patient-specific iPSC and iNSC lines with high reprogramming efficiency. Methods: UCs were electroporated with the pEP4-EO2S-ET2K and pEP4-M2L plasmids containing the OCT4, SOX2, KLF4, SV40LT, c-MYC, and LIN28 genes, and then cultured with N2B27 medium plus four small molecule compounds (A83-01, PD0325901, Thiazovivin, and CHIR99021). When iPSC or iNSC clones emerged, the medium was replaced with mTeSR1 or neural growth medium. Morphological changes were seen at day 4-7. After day 10, the clones were picked up when the clone diameter exceeded 1 mm. Results: iPSCs and iNSCs were successfully derived from UCs with up to 80 clones/well. These iPSCs and iNSCs showed typical hESC or NSC morphology and were self-renewable. The iPSCs had pluripotency to differentiate into the three germinal layers and displayed high levels of expression of pluripotency markers SOX2, NANOG, OCT4, SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase (AP). They maintained normal karyotype and had no transgene expression or genomic integration. The iNSCs were positive for NSC markers NESTIN, PAX6, SOX2, and OLIG2. Conclusion: The optimized protocol is an easy and fast procedure to yield both iPSC and iNSC lines from a convenient source of human urine in a single experiment.
基金:
National Key R&D Program of China [2017YFA0104100]; National Basic Research Program (973 Program) of China [2015CB964600]; National Natural Science Foundation of China [81670862, 81603200]; China Postdoctoral Science Foundation [2015M580758]; Natural Science Foundation of Guangdong Province [2016A030310201]; Fundamental Research Funds of the State Key Laboratory of Ophthalmology, Sun Yat-sen University
第一作者机构:[1]Sun Yat Sen Univ, Zhongshan Ophthalm Ctr, State Key Lab Ophthalmol, Guangzhou, Guangdong, Peoples R China
通讯作者:
通讯机构:[1]Sun Yat Sen Univ, Zhongshan Ophthalm Ctr, State Key Lab Ophthalmol, Guangzhou, Guangdong, Peoples R China[*1]Sun Yat Sen Univ, Zhongshan Ophthalm Ctr, State Key Lab Ophthalmol, Guangzhou, Guangdong, Peoples R China[*2]Rutgers State Univ, Robert Wood Johnson Med Sch, Dept Pediat, Ctr Adv Biotechnol & Med, Piscataway, NJ 08854 USA[3]Rutgers State Univ, Robert Wood Johnson Med Sch, Dept Pediat, Ctr Adv Biotechnol & Med, Piscataway, NJ 08854 USA
推荐引用方式(GB/T 7714):
Cheng Lin,Lei Qiannan,Yin Chen,et al.Generation of Urine Cell-Derived Non-integrative Human iPSCs and iNSCs: A Step-by-Step Optimized Protocol[J].FRONTIERS IN MOLECULAR NEUROSCIENCE.2017,10:348.doi:10.3389/fnmol.2017.00348.
APA:
Cheng, Lin,Lei, Qiannan,Yin, Chen,Wang, Hui-Yun,Jin, Kangxin&Xiang, Mengqing.(2017).Generation of Urine Cell-Derived Non-integrative Human iPSCs and iNSCs: A Step-by-Step Optimized Protocol.FRONTIERS IN MOLECULAR NEUROSCIENCE,10,
MLA:
Cheng, Lin,et al."Generation of Urine Cell-Derived Non-integrative Human iPSCs and iNSCs: A Step-by-Step Optimized Protocol".FRONTIERS IN MOLECULAR NEUROSCIENCE 10.(2017)