Characterization of Mesenchymal Stem Cell Differentiation within Miniaturized 3D Scaffolds through Advanced Microscopy Techniques.

Valentina Parodi,Benedetta Talone,Roberto Osellame,Carlo M Valensise,Arianna Bresci,Dario Polli,Manuela T Raimondi,Emanuela Jacchetti,Giulio Cerullo

doi:10.3390/ijms21228498

Abstract

Three-dimensional culture systems and suitable substrates topographies demonstrated to drive stem cell fate in vitro by mechanical conditioning. For example, the Nichoid 3D scaffold remodels stem cells and shapes nuclei, thus promoting stem cell expansion and stemness maintenance. However, the mechanisms involved in force transmission and in biochemical signaling at the basis of fate determination are not yet clear. Among the available investigation systems, confocal fluorescence microscopy using fluorescent dyes enables the observation of cell function and shape at the subcellular scale in vital and fixed conditions. Contrarily, nonlinear optical microscopy techniques, which exploit multi-photon processes, allow to study cell behavior in vital and unlabeled conditions. We apply confocal fluorescence microscopy, coherent anti-Stokes Raman scattering (CARS), and second harmonic generation (SHG) microscopy to characterize the phenotypic expression of mesenchymal stem cells (MSCs) towards adipogenic and chondrogenic differentiation inside Nichoid scaffolds, in terms of nuclear morphology and specific phenotypic products, by comparing these techniques. We demonstrate that the Nichoid maintains a rounded nuclei during expansion and differentiation, promoting MSCs adipogenic differentiation while inhibiting chondrogenesis. We show that CARS and SHG techniques are suitable for specific estimation of the lipid and collagenous content, thus overcoming the limitations of using unspecific fluorescent probes.

Highlights

IntroductionSeveral studies demonstrated the role of the cytoskeleton in driving stem cell fate [10,11,12] by so-called mechanotransduction process, translating mechanical stimuli into biochemical signals: for example, a force transmission between the external and the internal cellular compartments alters cell shape and, the import of gene-regulating transcription factors that, in turn, determine gene expression [13,14]
We studied mesenchymal stem cells (MSCs) adipogenic and chondrogenic differentiation inside the Nichoid scaffold by comparing confocal fluorescence microscopy with nonlinear optical microscopy techniques, such as coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG)
We demonstrated that the MSCs nuclear shape inside Nichoids remained roundish with respect to cells grown on flat glass substrates, suggesting that mechanical conditioning prevails on chemical conditioning in this regard

Summary

Introduction

Several studies demonstrated the role of the cytoskeleton in driving stem cell fate [10,11,12] by so-called mechanotransduction process, translating mechanical stimuli into biochemical signals: for example, a force transmission between the external and the internal cellular compartments alters cell shape and, the import of gene-regulating transcription factors that, in turn, determine gene expression [13,14]. The research in this novel field focused on the development of engineered systems capable to control cell stemness in vitro through mechanical conditioning [15]. These studies highlight the need for label-free and non-destructive methods for MSCs phenotypic characterization in the field of tissue engineering

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Results

Discussion

Conclusion