Cluster-assembled zirconia substrates promote long-term differentiation and functioning of human islets of Langerhans

Abstract

Ex vivo expansion and differentiation of human pancreatic β-cell are enabling steps of paramount importance for accelerating the development of therapies for diabetes. The success of regenerative strategies depends on their ability to reproduce the chemical and biophysical properties of the microenvironment in which β-cells develop, proliferate and function. In this paper we focus on the biophysical properties of the extracellular environment and exploit the cluster-assembled zirconia substrates with tailored roughness to mimic the nanotopography of the extracellular matrix. We demonstrate that β-cells can perceive nanoscale features of the substrate and can convert these stimuli into mechanotransductive processes which promote long-term in vitro human islet culture, thus preserving β-cell differentiation and function. Proteomic and quantitative immunofluorescence analyses demonstrate that the process is driven by nanoscale topography, via remodelling of the actin cytoskeleton and nuclear architecture. These modifications activate a transcriptional program which stimulates an adaptive metabolic glucose response. Engineered cluster-assembled substrates coupled with proteomic approaches may provide a useful strategy for identifying novel molecular targets for treating diabetes mellitus and for enhancing tissue engineering in order to improve the efficacy of islet cell transplantation therapies.

Shaping Pancreatic β-Cell Differentiation and Functioning: The Influence of Mechanotransduction

“…We demonstrated that human islets of Langerhans perceive the nanoscale properties of the substrate and activate a mechanotransductive pathway (discussed below), which promotes long-term β-cell survival and function. Interestingly, human islets grown on the nanotopographical substrates (of a certain roughness) maintain glucose-stimulated calcium currents and insulin secretion comparable to those observed in freshly isolated islet, suggesting a crucial role of nanometric topographical signals in shaping β-cell fate in in vitro cultures [64].…”

“…Softer matrices such as hydrogels, chitosan, polylactic-coglycolid (PLGA), and poly-L-lactide (PLA) acids, which better mimic the physiological conditions, instead preserve the islet clustering organization and the β-cell function (reviewed in [54,62,63]). Indeed, when human islets are cultured on soft substrates cell-cell interactions dominate, favoring cell coalescence and preserving the cluster-like organization of the native islet; in contrast, when they are cultured on stiff supports, the extracellular-cell interactions are much stronger, causing cell scattering and the loss of islet-like structure [64].…”

“…Generally, if the spacing of the integrin ligands is appropriate, the stiffer the substrate is, the larger and thicker the IAC and stress fibers are [84]. This holds true also for β-cells, although well-structured stress fibers are seldom observed in these cells [64].…”

“…Even if they are initially organized in clusters, over time in culture, the cells spread and tend to dissociate. β-cells on nanostructured substrates, instead develop tiny focal contacts (i.e., focal complexes) and assume a more relaxed, round shape [64]. This geometry favors the formation and maintenance of cell-cell contacts, which drives the cell organization in islet-like structures (Figure 3).…”

“…We recently demonstrated that both human and clonal β-cells can activate a fast mechanotransduction pathway in response to nanotopography [64] (Figure 3). Indeed, we found reorganization of the actin cytoskeleton and modification of the nuclear shape in cells grown on nanostructured substrates with a roughness of 15 nm root-mean-square compared to flat surfaces.…”