Medaka insulin-like growth factor-2 supports self-renewal of the embryonic stem cell line and blastomeres in vitro

Yongming Yuan,Yunhan Hong

doi:10.1038/s41598-017-00094-y

Yongming Yuan, Yunhan Hong

Open Access

https://doi.org/10.1038/s41598-017-00094-y

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Journal: Scientific Reports	Publication Date: Mar 6, 2017
Citations: 24	License type: open-access

Affiliation: National University of Singapore

Abstract

Insulin-like growth factors (IGFs) regulate diverse processes including energy metabolism, cell proliferation and embryonic development. They activate the IGF signaling pathway via binding to cell surface receptors. Here we report an essential role of IGF2 in maintaining the pluripotency of embryonic stem (ES) cell from medaka (Oryzias latipes). The medaka igf2 gene was cloned for prokaryotically expression of IGF2 ligand and green fluorescent protein-tagged IGF2 namely IGF2:GFP. With flow cytometry analysis, we demonstrated that the IGF2:GFP can bind to the cultured ES cells from medaka and zebrafish respectively. We also verified that IGF2 is able to activate the phosphorylation of Erk1/2 and Akt, and sustain the viability and pluripotency of medaka ES cells in culture. Furthermore, we characterized the binding of IGF2:GFP to freshly isolated blastomeres by fluorescence microscopy and electron microscopy. Most importantly, we revealed the important role of IGF2 in supporting the derivation of blastomeres in short-term culture. Therefore, Medaka IGF2 is essential for the self-renewal of cultured ES cells and blastomeres from fish embryos. This finding underscores a conserved role of the IGF signaling pathway in stem cells from fish to mammals.

Highlights

Health and sustain the pluripotency of medaka ES cells
With primer pair of IGF2F plus IGF2R, a 636 bp DNA fragment was PCR-amplified from the medaka fish cDNA template
The homologies of B and A domains of IGF2 among bony fish are very high, which can be explained that most residues of these domains are involved in the binding of its receptor or IGFBP reported in mammals, such as the Arg[21] and Phe23-Tyr24-Phe[25] motif in domain B and Phe[51] and Ser[53] in domain A23, 24

Summary

Introduction

Health and sustain the pluripotency of medaka ES cells. we detected IGF2 binding to the blastomeres under fluorescence microscopy and transmission electron microscopy (TEM). To test the bioactivity of IGF2, HX1 cell were first starved in the basic medium for 8 h to eliminate the effects of existing growth factors, and they were subsequently cultured for 2 days in the medium containing IGF2 added at different concentrations. The change of cell morphology was found in the basic medium containing IGF2:GFP or human IGF2 at the concentration of 100 nM and 200 nM respectively (Fig. S1).

Results

Conclusion