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MyoD is reduced in mesenchymal stem cells grown in a 3D micro‑engineered niche

The nuclear import of the transcription factor MyoD is reduced in mesenchymal stem cells grown in a 3D micro‑engineered niche

Smart biomaterials are increasingly being used to control stem cell fate in vitro by the recapitulation of the native niche microenvironment. By integrating experimental measurements with numerical models, they show that in mesenchymal stem cells grown inside a 3D synthetic niche both nuclear transport of a myogenic factor and the passive nuclear diffusion of a smaller inert protein is reduced. their results also suggest that cell morphology modulates nuclear proteins import through a partition of the nuclear envelope surface, which is a thin but extremely permeable annular portion in cells cultured on 2D substrates. Therefore, their results support the hypothesis that in stem cell differentiation, the nuclear import of gene-regulating transcription factors is controlled by a strain-dependent nuclear envelope permeability, probably related to the reorganization of stretch-activated nuclear pore complexes.

In the last 2 decades, stem cells have generated considerable interest in biomedicine with many potential applications such as regenerative and personalized medicine. Mesenchymal stem cells (MSCs) are the most commonly studied due to their ready isolation for autologous use, extensive in vitro expansion capability, and the ability to be differentiated into a wide number of tissue-specific lineages.

However, efcient culture systems that allow the large-scale expansion of MSCs while maintaining control of their function are still lacking. Today, much is known about the efect of chemical signals on the activation of cellular biochemical processes. Several studies have shown that also physical signals from the cellular environment, such as substrate stifness , topography and dynamic mechanical stimuli , can infuence gene regulation and, therefore, cell fate decisions . In physiological environments, cells transmit forces from the focal adhesions at their periphery to the nucleus via the actin cytoskeleton, where the nucleus acts as a mechanosensor afecting chromatin organization and gene expression . While the efect of cytoskeletal deformation on cell fate has been extensively characterised, only recent research has focused on the efects of cell nuclear deformation . For example, it is now accepted that there are mechanosensitive ion channels on the nuclear membrane, which, coupled to the mechanosensitive cytoskeleton, promote ion infux and associated gene transcription . Other structures that may link cellular function to nuclear plasticity/deformation are being searched for. Current methods to investigate the cell nucleus as a mechanosensor aim to control nuclear deformation, for example by micropipette aspiration, nanoindentation and microfuidic flow. Other techniques control cell adhesion on novel materials, either by modifying the material’s stifness or by patterning the substrate with engineered micro/nanoscale features such as pits, protrusions, channels, and pillars. However, these substrates do not surround the cells but stand as an underlying support; thus, they are not fully able to mimic the architectural cue that regulates the fate of stem cells in situ, i.e. their three-dimensional (3D) spatial organisation.

Te innovative approach of their study is to use a scafold enabling the cells to self-organize into a three-dimensional confguration allowing a more realistic study of the nuclear import fows than in any 2D cell adhesion confguration. Tis scafold is a micro-fabricated substrate, produced by two-photon polymerization technique of a biocompatible, inert and mechanically stable photoresin.

Tanks to this optical lithographic technique, it is possible to create three-dimensional scafolds with a resolution up to 100 nm. In their case, the scafolds (30 μm high and 450 μm×450 μm in transverse dimensions) are a lattice of interconnected lines forming pores of graduated size, spaced between 10 and 30 μm transversely and with a uniform spacing of 15 μm vertically. From now, in this paper, these substrates will be called “Nichoid” for convenience.

Despite today, as demonstrated from the research of Discher and Engler groups , it is well known that substrate stifness correlates with stem cell fate, another factor infuencing cellular functionality is the architecture of the surrounding environment. Bao et al., for example, demonstrated that substrate architecture plays an important role in generating specifc cell adhesion confguration and in remodelling actin cytoskeletal organization or the shape of the cell nucleus. Another example is the Nichoid, which is able to modulate cell morphology and gene activation in several types of cells, based on purely architectural cues. In this paper, their modelling approach, based on experimental results, aims at providing a mechanistic interpretation of nucleocytoplasmic protein transport and protein localization. their interesting fndings are that in MSCs grown inside the Nichoid, a transcriptional activator of myogenesis fused with a GFP (MyoD-GFP) is retained in the cytoplasm and that its nuclear import fow is reduced. In addition, this reduction occurs also for GFP proteins that translocate into the cell nucleus by passive difusion. Finally,their results suggest that cell morphology modulates the protein nuclear import through a partition of the nuclear envelope (NE) surface1.

Efective NE surface for nucleo-cytoplasmic protein fuxes. (a) 3D rendering of MSCs grown in the Nichoid. Actin fbers are in green and the cell nuclei are in blue. Te cell nucleus is surrounded by the cell cytosol in each direction. (b) 3D rendering of z-stack images: actin (green) and nuclei (blue). Te image shows that on a 2D fat control substrate, MSCs are very thin and the volume of the cell cytosol at the top and the bottom of the nucleus is negligible. (c) Collection of explanatory images of the frst seconds afer the photobleaching. Tese results show that, fuorescence recovery starts at the nuclear periphery and gradually difuses towards the nuclear centrum. In fact, the fuorescence recovery in the nuclear slice is more uniform in the Nichoid than in the Control, where a fuorescence gradient towards the center of the nucleus is clearly visible. On the lef there are representative images of MSCs expressing GFP protein. Te red ROI highlights the cell nucleus. Te white ROI shows the rectangle on which the pixel-by-pixel fuorescence intensity plots were made. From lef to right surface plots are shown at diferent times: 200 ms, 4 s, 35 s. (d) Scheme of the nuclear surface (in blue) exposed for protein transport in MSCs grown into the Nichoid (up) and on a fat glass substrate (down). Te sofware used to create the images is Ansys 2019 R2 ( (e) Comparison of recovery curve and mean fux within frst 22 s afer photobleaching for cells on a fat control substrate considering the efective surface of transport: numerical simulation (gray), FRAP experiments (blue).
Efective NE surface for nucleo-cytoplasmic protein fuxes

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