Oriented immobilization of basic fibroblast growth factor: Bioengineered surface design for the expansion of human mesenchymal stromal cells

Ajay Shakya,Tamamo Matsuyama,Phuong Kim Nguyen,Isao Hirata,Eiji Imado,Koichi Kato,Kotaro Horimoto

doi:10.1038/s41598-020-65572-2

Abstract

E. coli expressed recombinant basic fibroblast growth factor (bFGF) with histidine-tag (bFGF-His) was immobilized onto the surface of a glass plate modified with a Ni(II)-chelated alkanethiol monolayer. The immobilization is expected to take place through the coordination between Ni(II) and His-tag. The bFGF-immobilized surface was exposed to citrate buffer solution to refold in situ the surface-immobilized bFGF. The secondary structure of immobilized bFGF-His was analyzed by solid-phase circular dichroism (CD) spectroscopy. Immortalized human mesenchymal stromal cells (hMSCs) were cultured on the bFGF-His-immobilized surface to examine their proliferation. CD spectroscopy revealed that the immobilized bFGF initially exhibited secondary structure rich in α-helix and that the spectrum was gradually transformed to exhibit the formation of β-strands upon exposure to citrate buffer solution, approaching to the spectrum of native bFGF. The rate of hMSC proliferation was 1.2-fold higher on the bFGF-immobilized surface treated with in situ citrate buffer, compared to the polystyrene surface. The immobilized bFGF-His treated in situ with citrate buffer solution seemed to be biologically active because its secondary structure approached its native state. This was well demonstrated by the cell culture experiments. From these results we conclude that immobilization of bFGF on the culture substrate serves to enhance proliferation of hMSCs.

Highlights

In this study, attempts were made to develop a novel culture substrate for an efficient expansion of Human mesenchymal stromal cells (hMSCs)
Preparation of bFGF-His. bFGF-His was expressed in bacteria which were newly transformed with the previously-constructed plasmid DNA19 and subjected to step-wise dialysis under conditions shown in Supplementary Table S1
The SDS-PAGE analysis of three types of bFGF-His showed clear single bands at approximately 18 kDa, in agreement with the molecular weight predicted for bFGF-His. These results suggest that purified proteins of a correct amino acid sequence were reproducibly obtained using the new transformants

Summary

Introduction

Attempts were made to develop a novel culture substrate for an efficient expansion of hMSCs. This ligand binding is expected to activate FGFR signaling, leading to the promotion of cell proliferation, while maintaining the stemness and delaying senescence. According to our previous study[23], the surface immobilization of growth factor can be suitably achieved through the chelate linkage formed between surface-bound nickel ions and a hexahistidine peptide fused with the terminus of growth factor Such an immobilization chemistry provides structural integrity and firm attachment of the growth factor on the surface, helping in the efficient expansion of cells with specific receptors. We aimed to demonstrate that hMSCs can effectively proliferate on a substrate with immobilized bFGF-His in the active form, while holding potentials to differentiate into osteogenic, chondrogenic, and adipogenic lineages

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Conclusion