LUMC hiPSC Hotel, Leiden University Medical Center, Leiden, The Netherlands.
Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands.
Curr Protoc Stem Cell Biol. 2020 Dec;55(1):e124. doi: 10.1002/cpsc.124.
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.
我们描述了一种使用包含重编程因子 OCT3/4、SOX2、KLF4、GLIS1 和 c-MYC 的自我复制 RNA（ReproRNA-OKSGM）从成年供体的尿液来源细胞（UDC）中高效生成人诱导多能干细胞（hiPSC）的方案。电穿孔后，通过流式细胞术测量 OCT3/4 表达的 UDC 来定量转染效率，转染效率应≥0.1%。转染后 3 周内出现 hiPSC 集落，并表达多种多能性标志物。此外，源自 UDC 的 hiPSC 能够分化为三个胚层的细胞，并显示正常的核型。ReproRNA-OKSGM 可商购获得，仅需进行单次转染步骤，因此该方案易于实施，且操作简单。除了使用 ReproRNA-OKSGM 从 UDC 生成克隆 hiPSC 的详细分步说明外，我们还为 hiPSC 系的基本多能性特征提供了指导。©2020 作者。使用 ReproRNA-OKSGM 重编程尿液来源细胞的基本方案 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：表征 hiPSC 的功能多能性。
