
Reprogramming Urine‐Derived Cells Using Commercially Available Self‐Replicative RNA and a Single Electroporation
Author(s) -
Bouma Marga J.,
Arendzen Christiaan H.,
Mummery Christine L.,
Mikkers Harald,
Freund Christian
Publication year - 2020
Publication title -
current protocols in stem cell biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.658
H-Index - 28
eISSN - 1938-8969
pISSN - 1941-7322
DOI - 10.1002/cpsc.124
Subject(s) - reprogramming , biology , induced pluripotent stem cell , klf4 , sox2 , transfection , electroporation , flow cytometry , microbiology and biotechnology , germ layer , cell culture , cell , genetics , embryonic stem cell , gene
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