Effect of the 3D micro-scaffold Nichoid on the transcriptome of Neural Stem Cells

3D scaffolds are becoming more and more important in the field of regenerative medicine due to their ability to mimic the physiological microenvironment. In this field, Raimondi and colleagues applied two-photon polymerization (2PP) to fabricate the “Nichoid”, an engineered stem niche able to mimic the physiological environment where stem cells reside [1]. To investigate the Nichoid’s influence on cellular response in terms of global gene expression, murine neural stem cells (NSCs) where expanded inside the micro-scaffold [2] and analysed through a whole transcriptomic approach [3]. Nichoids were fabricated by femtosecond laser 2PP using an organic-inorganic photoresist. 104 NSCs were expanded inside the engineered niches and in control conditions for 7 days and then analysed through RNA-seq to evaluate how the NSCs’ expansion in the bioengineered 3D micro-scaffold may influence the cellular response. The outcomes of RNA-Seq were confirmed through Real Time PCR and immunofluorescence analysis. Here we demonstrate that the Nichoid impacts the biological and genetic response of stem cells, as genes strongly connected to mechanobiological functions emerged. 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. Moreover, NSCs grown inside the Nichoid show also a significant increase in the expression of pluripotency genes, as a result of the changes in membrane, cytoskeleton and nucleus. Thus, RNA-Seq highlights the Nichoid’s ability to induce changes in cultured stem cells at any molecular level without any other chemical agents, allowing recreating the stem cells adhesion, migration, differentiation, proliferation and cell signaling that best mimics physiological conditions. Here we provide evidences that this capacity is essential for regenerative medicine approaches based on the development of stem cell therapies. References [1] Remuzzi A, Bonandrini B, Tironi M, Longaretti L, Figliuzzi M, Conti S, Zandrini T, Osellame R, Cerullo G, Raimondi MT. Effect of the 3D Artificial Nichoid on the Morphology and Mechanobiological Response of Mesenchymal Stem Cells Cultured In Vitro. Cells. 2020 Aug 11;9(8):1873. doi: 10.3390/cells9081873. [2] Carelli S, Giallongo T, Rey F, Barzaghini B, Zandrini T, Pulcinelli A, Nardomarino R, Cerullo G, Osellame R, Cereda C, Zuccotti GV, Raimondi MT. Neural precursors cells expanded in a 3D micro-engineered niche present enhanced therapeutic efficacy in vivo. Nanotheranostics 2021; 5(1):8-26. doi:10.7150/ntno.50633. Available from http://www.ntno.org/v05p0008.htm [3] Rey F, Pandini C, Barzaghini B, Messa L, Giallongo T, Pansarasa O, Gagliardi S, Brilli M, Zuccotti GV, Cereda C, Raimondi MT, Carelli S. Dissecting the Effect of a 3D Microscaffold on the Transcriptome of Neural Stem Cells with Computational Approaches: A Focus on Mechanotransduction. Int J Mol Sci. 2020 Sep 15;21(18):6775. doi: 10.3390/ijms21186775. Acknowledgements This research has received funding from: the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (G.A. No. 646990 – NICHOID, No. 754467 - NICHOIDS and No. 825159 – MOAB); the Italian Ministry of University and Research (MIUR) under the grant program MIUR-FARE-2016 (G.A. No. R16ZNN2R9K – BEYOND); the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) under the CleanCut 2020 Challenge (G.A. No. NC/C01903/1 – MOAB).