Testa Carolina, Oliveto Stefania, Jacchetti Emanuela, Donnaloja Francesca, Martinelli Chiara, Pinoli Pietro, Osellame Roberto, Cerullo Giulio, Ceri Stefano, Biffo Stefano, Raimondi Manuela T
Department of Electronics, Information and Bioengineering, Politecnico di Milano, Milano, Italy.
Department of Chemistry, Materials and Chemical Engineering "Giulio Natta", Politecnico di Milano, Milano, Italy.
Front Bioeng Biotechnol. 2023 Jan 6;10:945474. doi: 10.3389/fbioe.2022.945474. eCollection 2022.
Mesenchymal stem cells (MSCs) are known to be ideal candidates for clinical applications where not only regenerative potential but also immunomodulation ability is fundamental. Over the last years, increasing efforts have been put into the design and fabrication of 3D synthetic niches, conceived to emulate the native tissue microenvironment and aiming at efficiently controlling the MSC phenotype . In this panorama, our group patented an engineered microstructured scaffold, called Nichoid. It is fabricated through two-photon polymerization, a technique enabling the creation of 3D structures with control of scaffold geometry at the cell level and spatial resolution beyond the diffraction limit, down to 100 nm. The Nichoid's capacity to maintain higher levels of stemness as compared to 2D substrates, with no need for adding exogenous soluble factors, has already been demonstrated in MSCs, neural precursors, and murine embryonic stem cells. In this work, we evaluated how three-dimensionality can influence the whole gene expression profile in rat MSCs. Our results show that at only 4 days from cell seeding, gene activation is affected in a significant way, since 654 genes appear to be differentially expressed (392 upregulated and 262 downregulated) between cells cultured in 3D Nichoids and in 2D controls. The functional enrichment analysis shows that differentially expressed genes are mainly enriched in pathways related to the actin cytoskeleton, extracellular matrix (ECM), and, in particular, cell adhesion molecules (CAMs), thus confirming the important role of cell morphology and adhesions in determining the MSC phenotype. In conclusion, our results suggest that the Nichoid, thanks to its exclusive architecture and 3D cell adhesion properties, is not only a useful tool for governing cell stemness but could also be a means for controlling immune-related MSC features specifically involved in cell migration.
间充质干细胞（MSCs）被认为是临床应用的理想候选者，在这些应用中，不仅再生潜力，而且免疫调节能力都是至关重要的。在过去几年中，人们越来越致力于3D合成微环境的设计和制造，其旨在模拟天然组织微环境，并旨在有效控制MSC表型。在这种情况下，我们的团队获得了一种名为Nichoid的工程化微结构支架的专利。它是通过双光子聚合制造的，该技术能够创建3D结构，在细胞水平上控制支架几何形状，并且空间分辨率超出衍射极限，低至100纳米。与2D底物相比，Nichoid在无需添加外源性可溶性因子的情况下维持更高水平干性的能力，已经在MSCs、神经前体细胞和小鼠胚胎干细胞中得到证实。在这项工作中，我们评估了三维性如何影响大鼠MSCs的整个基因表达谱。我们的结果表明，在细胞接种仅4天后，基因激活就受到显著影响，因为在3D Nichoid中培养的细胞和2D对照中培养的细胞之间，有654个基因似乎存在差异表达（392个上调和262个下调）。功能富集分析表明，差异表达的基因主要富集在与肌动蛋白细胞骨架、细胞外基质（ECM），特别是细胞粘附分子（CAMs）相关的途径中，从而证实了细胞形态和粘附在决定MSC表型中的重要作用。总之，我们的结果表明，Nichoid由于其独特的结构和3D细胞粘附特性，不仅是控制细胞干性的有用工具，而且还可能是控制与免疫相关的、特别参与细胞迁移的MSC特征的一种手段。
