A Substrate-Mimicking Basement Membrane Drives the Organization of Human Mesenchymal Stromal Cells and Endothelial Cells Into Perivascular Niche-Like Structures.

Valeria Perugini,Matteo Santin

doi:10.3389/fcell.2021.701842

Valeria Perugini, Matteo Santin

Open Access

https://doi.org/10.3389/fcell.2021.701842

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Abstract

Extracellular matrix-derived products (e.g. Matrigel) are widely used for in vitro cell cultures both as two-dimensional (2D) substrates and as three-dimensional (3D) encapsulation gels because of their ability to control cell phenotypes through biospecific cues. However, batch-to-batch variations, poor stability, cumbersome handling, and the relatively high costs strictly limit their use. Recently, a new substrate known as PhenoDrive-Y has been used as 2D coating of tissue culture plastic showing to direct the bone marrow mesenchymal stromal cells (MSCs) toward the formation of 3D spheroids. When organized into 3D spheroids, the MSCs expressed levels of pluripotency markers and of paracrine angiogenic activity higher than those of the MSCs adhering as fibroblast-like colonies on tissue culture plastic. The formation of the spheroids was attributed to the properties of this biomaterial that resemble the main features of the basement membrane by mimicking the mesh structure of collagen IV and by presenting the cells with orderly spaced laminin bioligands. In this study, PhenoDrive-Y was compared to Matrigel for its ability to drive the formation of perivascular stem cell niche-like structures in 2D co-culture conditions of human endothelial cells and adult bone marrow MSCs. Morphological analyses demonstrated that, when compared to Matrigel, PhenoDrive-Y led endothelial cells to sprout into a more consolidated tubular network and that the MSCs nestled as compact spheroids above the anastomotic areas of this network resemble more closely the histological features of the perivascular stem cell niche. A study of the expressions of relevant markers led to the identification of the pathways linking the PhenoDrive-Y biomimicking properties to the acquired histological features, demonstrating the enhanced levels of stemness, renewal potential, predisposition to migration, and paracrine activities of the MSCs.

Highlights

The bone marrow is a source of progenitor cells including the mesenchymal stromal cells (MSCs) (Charbord, 2010)
MSC monoculture on the type of Matrigel used in this study showed increasing formation of spheroidal MSC clusters (Supplementary Figure 1B)
The recent discovery that MSCs are organized as spheroids or “mesenspheres” within their niche has led to reconsideration of the use of in vitro culture systems for engineering the perivascular niche (Krock et al, 2011)

Summary

Introduction

The bone marrow is a source of progenitor cells including the mesenchymal stromal cells (MSCs) (Charbord, 2010). Despite the progress achieved using these substrates, the uncontrolled spacing and density of these motifs have currently prevented them to instruct MSCs in assuming an abluminal position akin to their perivascular organization in vivo (Ehninger and Trumpp, 2011) Starting from these observations, PhenoDrive-Y, a novel substrate that has been demonstrated to mimic in vitro both the biospecific and physical features driving MSC interactions with the BM in vivo (Perugini and Santin, 2017), was considered a suitable substrate to study the MSC phenotype and paracrine activities when sitting in the perivascular niche.

Methods

Results

Conclusion