Neural precursors cells expanded in a 3D micro-engineered niche present enhanced therapeutic efficacy in vivo

Abstract

Rationale: Stem Cells (SCs) show a great potential in therapeutics for restoring and regenerating native tissues. The clinical translation of SCs therapies is currently hindered by the inability to expand SCs in vitro in large therapeutic dosages, while maintaining their safety and potency. The use of biomaterials allows for the generation of active biophysical signals for directing SCs fate through 3D micro-scaffolds, such as the one named "Nichoid", fabricated with two-photon laser polymerization with a spatial resolution of 100 nm. The aims of this study were: i) to investigate the proliferation, differentiation and stemness properties of neural precursor cells (NPCs) following their cultivation inside the Nichoid micro-scaffold; ii) to assess the therapeutic effect and safety in vivo of NPCs cultivated in the Nichoid in a preclinical experimental model of Parkinson's Disease (PD). Methods: Nichoids were fabricated by two photon laser polymerization onto circular glass coverslips using a home-made SZ2080 photoresist. NPCs were grown inside the Nichoid for 7 days, counted and characterized with RNA-Seq, Real Time PCR analysis, immunofluorescence and Western Blot. Then, NPCs were transplanted in a murine experimental model of PD, in which parkinsonism was induced by the intraperitoneal administration of the neurotoxin MPTP in C57/bl mice. The efficacy of engrafted Nichoid-expanded NPCs was evaluated by means of specific behavioral tests and, after animal sacrifice, with immunohistochemical studies in brain slices. Results: NPCs grown inside the Nichoid show a significantly higher cell viability and proliferation than in standard culture conditions in suspension. Furthermore, we report the mechanical conditioning of NPCs in 3D micro-scaffolds, showing a significant increase in the expression of pluripotency genes. We also report that such mechanical reprogramming of NPCs produces an enhanced therapeutic effect in the in vivo model of PD. Recovery of PD symptoms was significantly increased when animals were treated with Nichoid-grown NPCs, and this is accompanied by the recovery of dopaminergic markers expression in the striatum of PD affected mice. Conclusion: SCs demonstrated an increase in pluripotency potential when expanded inside the Nichoid, without the need of any genetic modification of cells, showing great promise for large-scale production of safe and functional cell therapies to be used in multiple clinical applications.

Multimodality Approach to Characterizing the Distinctive Hallmarks of Lung Fibrosis: A Mouse Model with Bleomycin and Indocyanine Green

“…Nonetheless, in our study the combination of BLM and ICG triggered a disease that was clearly different from the typical BLM-induced lung brosis with a single administration corresponding to half of the dose we routinely use [16.] ICG is known to have a very short half-life and to be rapidly eliminated by the liver [25] upon intravenous administration, which seems to be in contrast with our ndings, as we detected a remarkable uorescence signal in lungs up to 21 days (Fig. 1).…”

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

“…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.…”

“…Here, we focused on the potential of the scaffold called “Nichoid” [ 24 , 25 ], engineered in the form of a 3D micro-grid in order to mimic the 3D microscopic architecture of a stem cells niche. The Nichoid already demonstrated able to maintain the stemness capacity of primary rat mesenchymal stem cells (MSCs), human bone marrow-derived MSCs, mouse ES cells [ 26 , 27 , 28 , 29 ] and NPCs [ 30 , 31 ].…”

“…In this context, we have already investigated how the 3D spatial structure of the Nichoid scaffold can influence gene expression through the alteration of mechanotransduction-related processes, subsequently influencing the stemness and therapeutic potentials of NPCs [ 30 , 31 , 32 ]. Specifically, the deregulated coding genes and subsequent pathways analysis showed that the Nichoid causes alterations in genes starting at a membrane level, through an impact on signaling transduction and cellular metabolism, which ultimately leads to altered nuclear activity and thus changes in cellular functions.…”

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].…”