Research Paper Volume 14, Issue 10 pp 4445—4458
A real-time pluripotency reporter for the long-term and real-time monitoring of pluripotency changes in induced pluripotent stem cells
- 1 Cancer Research Institute, School of Basic Medical Science, Southern Medical University, Guangzhou 510515, China
- 2 Laboratory Animal Center, Southern Medical University, Guangzhou 510515, China
- 3 Guangzhou Southern Medical Laboratory Animal Sci. and Tech. Co., Ltd., Guangzhou 510515, China
- 4 The Third People’s Hospital of Kunming, The Sixth Affiliated Hospital of Dali University, Kunming 650041, China
- 5 Akeso Biopharma, Inc., Zhongshan 528400, China
- 6 Yue Bei People’s Hospital, Shaoguan 512025, China
- 7 Department of Oncology, The First People’s Hospital of Chenzhou, Chenzhou 423000, China
- 8 Cancer Center, Integrated Hospital of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510315, China
- 9 Department of Gastrointestinal Oncology, The Third Affiliated Hospital of Kunming Medical University, Yunnan Cancer Hospital, Yunnan Cancer Center, Kunming 650118, China
- 10 Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou 510080, China
- 11 National Demonstration Center for Experimental Education of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
Received: December 23, 2021 Accepted: February 15, 2022 Published: May 15, 2022
https://doi.org/10.18632/aging.204083How to Cite
Copyright: © 2022 Shen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Abstract
To master the technology of reprogramming mouse somatic cells to induced pluripotent stem cells (iPSCs), which will lay a good foundation for setting up a technology platform on reprogramming human cancer cells into iPSCs. Mouse iPSCs (i.e., Oct4-GFP miPSCs) was successfully generated from mouse embryonic fibroblasts (MEFs) harboring Oct4-EGFP transgene by introducing four factors, Oct4, Sox2, c-Myc and Klf4, under mESC (Murine embryonic stem cells) culture conditions. Oct4-GFP miPSCs were similar to mESCs in morphology, proliferation, mESC-specific surface antigens and gene expression. Additionally, Oct4-GFP miPSCs could be cultured in suspension to form embryoid bodies (EBs) and differentiate into cell types of the three germ layers in vitro. Moreover, Oct4-GFP miPSCs could develop to teratoma and chimera in vivo. Unlike cell cycle distribution of MEFs, Oct4-GFP miPSCs are similar to mESCs in the cell cycle structure which consists of higher S phase and lower G1 phase. More importantly, our data demonstrated that MEFs harboring Oct4-EGFP transgene did not express GFP, until they were reprogrammed to the pluripotent stage (iPSCs), while the GFP expression was progressively lost when these pluripotent Oct4-GFP miPSCs exposed to EB-mediated differentiation conditions, suggesting the pluripotency of Oct4-GFP miPSCs can be real-time monitored over long periods of time via GFP assay. Altogether, our findings demonstrate that Oct4-GFP miPSC line is successfully established, which will lay a solid foundation for setting up a technology platform on reprogramming cancer cells into iPSCs. Furthermore, this pluripotency reporter system permits the long-term real-time monitoring of pluripotency changes in a live single-cell, and its progeny.
Abbreviations
ALP: alkaline phosphatase; BSA: bovine serum albumin; EBs: embryoid bodies; EGFP: enhanced green fluorescent protein; FACS: fluorescence activating cell sorter; h: hour; HE: hematoxylin-eosin; iPSCs: induced pluripotent stem cells; LIF: leukemia inhibitory factor; MEFs: mouse embryonic fibroblasts; mESCs: murine/mouse embryonic stem cells; min: minute; miPSCs: mouse iPSCs; NPC: nasopharyngeal carcinoma; PBS: phosphate-buffered saline; qRT-PCR: quantitative real time polymerase chain reaction; RT: room temperature; RT-PCR: reverse transcription-polymerase chain reaction.