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- Title
A cytokine screen using CRISPR-Cas9 knock-in reporter pig iPS cells reveals that Activin A regulates NANOG.
- Authors
Xu, Junjun; Zheng, Zheng; Du, Xuguang; Shi, Bingbo; Wang, Jichang; Gao, Dengfeng; Zhu, Qianqian; Chen, Xinze; Han, Jianyong
- Abstract
Background: NANOG functions as the gateway for the generation of pluripotent stem cells (PSCs) in mice and humans. NANOG is a transcription factor highly expressed in pig pre-implantation embryos, indicating that it is a conserved pluripotency-associated factor. However, pig NANOG reporter PSCs have yet to be established, and the regulation of pluripotency by NANOG is not fully understood in this animal. Methods: In this study, pig NANOG tdTomato knock-in reporter positive PC-iPS cells were established using CRISPR/Cas9. The resulting cell line was treated with several cytokines and their corresponding inhibitors to identify pathways that regulate NANOG expression. The pathways examined were LIF (leukemia inhibitory factor)/IL6 (interleukin 6)-STAT3, FGF (fibroblast growth factor)/ERK, IGF1 (insulin-like growth factor 1)/PIP3 (phosphoinositide 3-kinase)-AKT, Activin A/SMAD, and BMP4 (bone morphogenetic proteins)/SMAD. Results: Our experiments showed that the Activin A/SMAD pathway is directly associated with activation of NANOG expression in the pig, as is also the case in mice and humans. Activin A directly regulates the expression of pig NANOG via SMAD2/3; inhibition of this pathway by SB431542 resulted in inhibition of NANOG expression. Conclusions: Our results show that Activin A plays an important regulatory role in NANOG-mediated pluripotency in pig iPS cells. Activin A treatment may be therefore an effective method for de novo derivation of authentic embryonic stem cells (ESCs) from pig pre-implantation embryos.
- Subjects
ACTIVIN; INDUCED pluripotent stem cells; SOMATOMEDIN C; LEUKEMIA inhibitory factor; BONE morphogenetic proteins; FIBROBLAST growth factors
- Publication
Stem Cell Research & Therapy, 2020, Vol 11, Issue 1, p1
- ISSN
1757-6512
- Publication type
Article
- DOI
10.1186/s13287-020-1588-z