Simultaneous binding of bFGF to both FGFR and integrin maintains properties of primed human induced pluripotent stem cells

Abstract

Introduction

Basic fibroblast growth factor (bFGF, FGF2) and integrin α6β1 are important for maintaining the pluripotency of human pluripotent stem cells (hPSCs). Although bFGF-integrin binding contributes to biofunctions in cancer cells, the relationship in hPSCs remains unclear.

Methods

To investigate the relationship between bFGF and integrin in human induced pluripotent stem cells (hiPSCs), we generated recombinant human bFGF wild-type and mutant proteins, that do not bind to integrin, FGFR, or both. We then cultured hiPSCs with these recombinant bFGF proteins. To evaluate the abilities of recombinant bFGF proteins in maintaining hPSC properties, pluripotent markers, ERK activity, and focal adhesion structure were analyzed through flow cytometry, immunofluorescence (IF), and immunoblotting (IB).

Result

We identified an interaction between bFGF and integrin α6β1 in vitro and in hiPSCs. The integrin non-binding mutant was incapable of inducing the hPSC properties, such as proliferation, ERK activity, and large focal adhesions at the edges of hiPSC colonies. Signaling induced by bFGF-FGFR binding was essential during the first 24 h after cell seeding for maintaining the properties of hPSCs, followed by a shift towards intracellular signaling via the bFGF-integrin interaction. The mixture of the two bFGF mutants also failed to maintain hPSC properties, indicating that bFGF binds to both FGFR and integrin.

Conclusion

Our study demonstrates that the integrin-bFGF-FGFR ternary complex maintains the properties of hPSCs via intracellular signaling, providing insights into the functional crosstalk between bFGF and integrins in hiPSCs.

Graphical abstract

Highlights

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  bFGF binds integrin α6β1 and FGFR in vitro and in hiPSCs.

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  bFGF-FGFR binding is critical for ERK activation after the start of culture.

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  bFGF-integrin binding is essential for the long-term maintenance of hiPSCs.

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  Integrin-bFGF-FGFR ternary complex is crucial for the maintenance of hPSCs.