A comparative in vitro study of the effect of biospecific integrin recognition processes and substrate nanostructure on stem cell 3D spheroid formation

Valeria Perugini,Matteo Santin

doi:10.1007/s10856-020-06373-x

Valeria Perugini, Matteo Santin

Open Access

https://doi.org/10.1007/s10856-020-06373-x

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Abstract

The in vitro study of the properties of the human mesenchymal stem cells as well as their manipulation in culture for clinical purposes depends on the elimination of artefacts caused by the lack of their natural environment. It is now widely accepted that mesenchymal stem cells should be studied when they are organised as 3D spheroids rather than fibroblast-like colonies. Although this can be achieved with the use of some extracellular matrix proteins or by non-adherent conditions these suffer of significant limitations. The recent development of synthetic substrates resembling the physicochemical and biochemical properties of the adult stem cell niche has prompted questions about the role played by nanotopography and receptor-mediated adhesion. In the present paper, the influence of two types of substrates bearing the same nanostructure, but exposing either a non-specific or an integrin-specific binding motif was studied. Carboxybetaine-tethered hyperbranched poly(ɛ-lysine) dendrons showed that the hyperbranched structure was fundamental to induce spheroid formation, but these were forming more slowly, were of reduced size and less stable than those growing on substrates based on the same hyperbranched structures that had been functionalised at their uppermost branching generation by a laminin amino acid sequence, i.e. YIGSR. The study shows that both nanostructure and biorecognition need to be combined to achieve a substrate for stem cell spheroid formation as that observed in vivo in the adult stem cell niche.

Highlights

A consensus has been building on the advantage of growing stem cells, such as the human adult bone marrow mesenchymal stem cells, as 3D spheroids rather than as fibroblast-like colonies [1]
This substrate was shown to be able to induce the formation of 3D human adult bone marrow mesenchymal stem cells (hMSCs) spheroids through the combination of the bio-specific features of the laminin with those of the mesh-like structure of Collagen Type IV, another key component of adult stem cell niches
The systematic study highlighted a different clustering of the cell surface integrins induced by the orderly-spaced presentation of a laminin-specific amino acid sequence, the YIGSR, that led to an activation of the intracellular biochemical pathway, Rho-A that is known to inhibit the formation of the cytoskeleton and, as a consequence, of the cell spreading

Summary

Introduction

A consensus has been building on the advantage of growing stem cells, such as the human adult bone marrow mesenchymal stem cells (hMSCs), as 3D spheroids rather than as fibroblast-like colonies [1]. The research group at the Centre for Regenerative Medicine and Devices, University of Brighton has developed a synthetic substrate where hyperbranched poly(ɛ-lysine) dendrons expose the amino acid sequence, YIGSR, that is present in laminin (i.e. PhenoDrive, Tissue Click Ltd, UK). This substrate was shown to be able to induce the formation of 3D hMSC spheroids through the combination of the bio-specific features of the laminin (the YIGSR sequence) with those of the mesh-like structure of Collagen Type IV, another key component of adult stem cell niches (the hyperbranched structure of the dendrons). The results showed that a reduction of the YIGSR density, and their spacing, led to the more familiar spreading of the cells and their proliferation into fibroblast-like colony [6]

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