Reprogramming Urine‐Derived Cells Using Commercially Available Self‐Replicative RNA and a Single Electroporation
Current Protocols in Stem Cell Biology, vol. 55, pp. e124
Abstract
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.
Authors 5
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Marga J. Bouma corresponding Aachen Department of Anatomy and Embryology Leiden University Medical Center Leiden the Netherlands Department of Anatomy and Embryology LUMC hiPSC Hotel
Leiden University Medical Center
Affiliation as printed
Department of Anatomy and Embryology Leiden University Medical Center Leiden The Netherlands
LUMC hiPSC Hotel Leiden University Medical Center Leiden The Netherlands
Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands
LUMC hiPSC Hotel, Leiden University Medical Center, Leiden, The Netherlands
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Christiaan H. Arendzen Aachen Department of Anatomy and Embryology Leiden University Medical Center Leiden the Netherlands Department of Anatomy and Embryology LUMC hiPSC Hotel
Leiden University Medical Center
Affiliation as printed
Department of Anatomy and Embryology Leiden University Medical Center Leiden The Netherlands
LUMC hiPSC Hotel Leiden University Medical Center Leiden The Netherlands
Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands
LUMC hiPSC Hotel, Leiden University Medical Center, Leiden, The Netherlands
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Christine L. Mummery Aachen Department of Anatomy and Embryology Leiden University Medical Center Leiden the Netherlands Department of Anatomy and Embryology LUMC hiPSC Hotel
Leiden University Medical Center
Affiliation as printed
Department of Anatomy and Embryology Leiden University Medical Center Leiden The Netherlands
LUMC hiPSC Hotel Leiden University Medical Center Leiden The Netherlands
Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands
LUMC hiPSC Hotel, Leiden University Medical Center, Leiden, The Netherlands
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Harald M. M. Mikkers Aachen Department of Cell and Chemical Biology Leiden University Medical Center Leiden the Netherlands Department of Cell and Chemical Biology LUMC hiPSC Hotel
Leiden University Medical Center
Affiliation as printed
Department of Cell and Chemical Biology Leiden University Medical Center Leiden The Netherlands
LUMC hiPSC Hotel Leiden University Medical Center Leiden The Netherlands
Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, The Netherlands
LUMC hiPSC Hotel, Leiden University Medical Center, Leiden, The Netherlands
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Christian M.A.H. Freund Aachen Department of Anatomy and Embryology Leiden University Medical Center Leiden the Netherlands Department of Anatomy and Embryology LUMC hiPSC Hotel
Leiden University Medical Center
Affiliation as printed
Department of Anatomy and Embryology Leiden University Medical Center Leiden The Netherlands
LUMC hiPSC Hotel Leiden University Medical Center Leiden The Netherlands
Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands
LUMC hiPSC Hotel, Leiden University Medical Center, Leiden, The Netherlands
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