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32070424
A cytokine screen using CRISPR-Cas9 knock-in reporter pig iPS cells reveals that Activin A regulates NANOG.

Stem Cell Res Ther, 2020/02/18;11(1):67.

Xu J[1, 2], Zheng Z[2], Du X[1, 2], Shi B[1, 2], Wang J[3, 4], Gao D[1, 2], Zhu Q[1, 2], Chen X[1, 2], Han J[5, 6]

Affiliations

PMID: 32070424DOI: 10.1186/s13287-020-1588-z

Impact factor: 8.079

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.

Keywords: Activin A; CRISPR/Cas9; Cytokine screen; NANOG reporter; Pluripotency

MeSH terms
Activins; Animals; CRISPR-Cas Systems; Cell Differentiation; Humans; Induced Pluripotent Stem Cells; Nanog Homeobox Protein; Pluripotent Stem Cells; Signal Transduction; Swine
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