利用商业上可获得的自我复制RNA和单电穿孔对尿源细胞进行重编程

Q2 Biochemistry, Genetics and Molecular Biology Current Protocols in Stem Cell Biology Pub Date : 2020-09-21 DOI:10.1002/cpsc.124

Marga J. Bouma, Christiaan H. Arendzen, Christine L. Mummery, Harald Mikkers, Christian Freund

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引用次数: 4

摘要

我们描述了一种利用含有重编程因子OCT3/4、SOX2、KLF4、GLIS1和c-MYC (ReproRNA-OKSGM)的自我复制RNA，从成人供体获得的尿源细胞(UDCs)高效生成人诱导多能干细胞(hiPSCs)的方案。电穿孔后，通过流式细胞术测量表达oct3 /4的UDCs来量化转染效率，转染效率应≥0.1%。hiPSC菌落在转染后3周内出现并表达多种多能性标记物。此外，udc衍生的hiPSCs能够分化为所有三种胚层的细胞，并显示正常的核型。ReproRNA-OKSGM在商业上是可用的，只需要一个转染步骤，因此该方案很容易获得，也很简单。除了使用ReproRNA-OKSGM从UDCs生成克隆hiPSC的详细步骤描述外，我们还提供了hiPSC系基本多能性表征的指导。©2020作者。基本方案:使用reprorna - oksgm对尿源性细胞进行重编程支持方案1:通过流式细胞术确定hipsc的多能性状态支持方案2:hipsc功能多能性的表征

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Reprogramming Urine-Derived Cells Using Commercially Available Self-Replicative RNA and a Single Electroporation

We describe a protocol for efficient generation of human-induced pluripotent stem cells (hiPSCs) from urine-derived cells (UDCs) obtained from adult donors using self-replicative RNA containing the reprogramming factors OCT3/4, SOX2, KLF4, GLIS1, and c-MYC (ReproRNA-OKSGM). After electroporation, transfection efficiency is quantified by measuring OCT3/4-expressing UDCs using flow cytometry and should be ≥0.1%. hiPSC colonies emerge within 3 weeks after transfection and express multiple pluripotency markers. Moreover, the UDC-derived hiPSCs are able to differentiate into cells of all three germ layers and display normal karyotypes. ReproRNA-OKSGM is available commercially and only requires a single transfection step so that the protocol is readily accessible, as well as straightforward. In addition to a detailed step-by-step description for generating clonal hiPSCs from UDCs using ReproRNA-OKSGM, we provide guidance for basic pluripotency characterization of the hiPSC lines. © 2020 The Authors.

Basic Protocol: Reprogramming of urine-derived cells using ReproRNA-OKSGM

Support Protocol 1: Determination of the pluripotency status of hiPSCs by flow cytometry

Support Protocol 2: Characterization of functional pluripotency of hiPSCs

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来源期刊

Current Protocols in Stem Cell Biology Biochemistry, Genetics and Molecular Biology-Cell Biology

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期刊介绍： Published in affiliation with the International Society for Stem Cell Research (ISSCR), Current Protocols in Stem Cell Biology (CPSC) covers the most fundamental protocols and methods in the rapidly growing field of stem cell biology. Updated monthly, CPSC will constantly evolve with thelatest developments and breakthroughs in the field. Drawing on the expertise of leading researchers from around the world, Current Protocols in Stem Cell Biology includes methods and insights that will enhance the progress of global research.