Dissecting the Effect of a 3D Microscaffold on the Transcriptome of Neural Stem Cells with Computational Approaches: A Focus on Mechanotransduction

Abstract

3D cell cultures are becoming more and more important in the field of regenerative medicine due to their ability to mimic the cellular physiological microenvironment. Among the different types of 3D scaffolds, we focus on the Nichoid, a miniaturized scaffold with a structure inspired by the natural staminal niche. The Nichoid can activate cellular responses simply by subjecting the cells to mechanical stimuli. This kind of influence results in different cellular morphology and organization, but the molecular bases of these changes remain largely unknown. Through RNA-Seq approach on murine neural precursors stem cells expanded inside the Nichoid, we investigated the deregulated genes and pathways showing that the Nichoid causes alteration in genes strongly connected to mechanobiological functions. Moreover, we fully dissected this mechanism highlighting how the changes start at a membrane level, with subsequent alterations in the cytoskeleton, signaling pathways, and metabolism, all leading to a final alteration in gene expression. The results shown here demonstrate that the Nichoid influences the biological and genetic response of stem cells thorough specific alterations of cellular signaling. The characterization of these pathways elucidates the role of mechanical manipulation on stem cells, with possible implications in regenerative medicine applications.

Neural Precursor Cells Expanded Inside the 3D Micro-Scaffold Nichoid Present Different Non-Coding RNAs Profiles and Transcript Isoforms Expression: Possible Epigenetic Modulation by 3D Growth

“…Indeed, ncRNAs could possibly influence numerous coding genes found altered via RNAseq, thus suggesting an involvement in the altered pathways in mechanotransduction, stemness, and neural differentiation that were investigated via KEGG, REACTOME, and WikiPathways, along with GO analysis. The deregulation of pathways related to membrane alteration (e.g., Axon guidance, Extracellular matrix organization, Focal adhesion) as well as cell-cell interaction (e.g., Tight junction) revealed a role for both lncRNAs and small ncR-NAs in altering expression of coding genes implicated in mechanotransduction, supporting previous studies [32], but also highlighted their importance in biological processes. Noncoding RNAs were also shown to influence metabolic processes of coding genes, resulting in the overall deregulation of metabolic pathways observed.…”

“…Lastly, 130 significant terms from turquoise module and 73 from the blue one emerged in GO CC analysis, and the top 10 are displayed as dotplot graph respectively in Figure 4E,F. Both the up-and down-regulated genes are implicated in terms such as "cellular anatomical entity", "intracellular", "cytoplasm", "membrane", "extracellular region", and "organelle" highlighting the Nichoid ability to transcriptionally deregulate ncRNAs related to multiple cellular levels, integrating what has been previously reported for coding transcripts [32]. Interestingly, only the up-regulated terms appear to be correlated with neuronal-specific phenotypes, such as "neuronal cell body", "synapse", and "somatodendritic compartment" (Figure 4E).…”

“…Altered expression of a similar gene in human has been associated with a susceptibility to myocardial infarction, and is involved in pathways that may contribute to the pathophysiology of schizophrenia. As already demonstrated, NPCs' growth inside the Nichoid present altered expression of genes that regulate mechanotransduction and stem cells fate [30,32]. Moreover, as there is a specific alteration of the lncRNAs expression as result of cells' expansion inside the Nichoid, we investigated whether this could be due to mechanotransduction or stemness-TF regulation.…”

“…Moreover, 1% concentration of Irg photoinitiator (Irgacure 369, 2-Benzyl-2-dimethylamino-1-(4-morpholinophenyl)-butanone-1) allowed to start the photopolymerization process [34]. SZ2080 offers biocompatibility, good optical transmission, chemical and electrochemical inertia, and long-term stability [32].…”

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

“…The Nichoid remodels the cytoskeleton and conditions cells to maintain a more spherical shape of the nuclei during proliferation with respect to cells grown on flat substrates [ 28 ]. Furthermore, we demonstrated that this scaffold maintains MSCs multipotency in vitro [ 31 , 32 , 33 ]. The architecture of the Nichoids allows optical inspection being composed of micrometric pores (tens of microns in size) and has a thickness of only 30 μm [ 24 ].…”