After read more: Spinal cord injury (SCI) represents an extremely invalidating condition for patients and caregivers. Effective treatments for SCI are lacking, due to the complex pathophysiology. In the last decade, neural stem/progenitor cell transplantations showed exciting results in replacing the damaged tissues and promoting functional recovery. At the same time, nanomedicine takes advantage of advanced nanotechnologies to deliver new therapeutic tools or enhance the efficacy of potential therapies. In this Ph.D. project, we explored through in vitro models an innovative approach for SCI treatment, based on the combination of neural stem cells (NSCs) – specifically spinal cord neuroepithelial stem (SC-NES) cells - and manipulation of cell mechano-transduction. The latter has been performed through magnetic nano-pulling, a nano-technology based on the generation of low forces on cells loaded with magnetic nanoparticles (MNPs) in presence of an external magnetic field.
The first part of the work aimed to optimize appropriate in vitro models for validating the proposed combinatorial approach. We decided to evaluate the effectiveness of this approach using NSCs cultured in both monolayer conditions and after transplantation into to ex vivo model of spinal cord and in a human 3D platform resembling the cortico-spinal tract in vitro. To this purpose, we optimized in parallel a long-term ex vivo culture of mouse spinal cord and human cortico-spinal assembloids, a 3D platform obtained by the fusion of cortical and spinal cord organoids in order to model human cortico-spinal tract in vitro. The organotypic model retained cytoarchitectural features of the spinal cord in vivo until day 90 in vitro, providing a system also useful to study critical transplanted cell parameters that require long-term time points to be evaluated, such as integration, differentiation and maturation. The human 3D assembloid platform required firstly to optimize the generation of cortical and spinal cord organoids, separately. Each type of organoid displayed the expression over time in culture of key developmental markers, attesting for the successfulness of the chosen protocol. Then, cortico-spinal assembloids were obtained effectively by fusing together cortical and spinal cord organoids.
The second part of this Ph.D. project aimed to assess the effectiveness of the combined approach on monolayer culture of NSCs and on the same cells transplanted into the optimized ex vivo models. We decided to use SC-NES cells as NSCs, because they already demonstrated efficacy in spinal cord recovery after transplantation into in vivo injury models. Firstly, we evaluated cell parameters such as cell process elongation, caliber, neurite network and maturation of SC-NES cells loaded with MNPs after mechanical stimulation through nano-pulling for short-term (2 days) and long term (52 days) time points. We observed more elongated cell processes in the stimulated samples and stable caliber instead of thinning, thus attesting for mass addition during elongation. Stimulated cells displayed also an increased cell networking and maturation after longer period of magnetic field application. Secondly, we validated the effectiveness of the nano-pulling on SC-NES cells after transplantation into mouse SC organotypic slices. SC-NES cells survived well after transplant into the tissue and cells were short-term stimulated. We assessed longer cell processes after mechanical stimulation. These processes were also preferentially oriented towards a specific direction, corresponding to the applied force direction. In the end, we optimized the transplantation of NES cells into the cortico-spinal assembloid model in order to further validate the combinatorial approach also in this human 3D in vitro platform.
In the end, since promising results were obtained in the in vitro and in the ex vivo models, in the third part of this Ph.D. work, we designed and produced magnetic applicators for in vitro and in vivo applications on murine models, in collaboration with the company eSPress3D (spin-off of the University of Pisa and Istituto Superiore Sant’Anna) with the purpose to translate and validate also in animal models the proposed combinatorial approach. This third part of the project has relied on engineering design research techniques and helped to gain new insights on the translation to in vivo models (mice) of nano-pulling technology applied to cell therapies. This step is at the basis for further in vivo evaluation of the proposed combinatorial approach.
The work illustrated in this Ph.D. thesis provided novel model systems useful to investigate more deeply cell processes after cell transplantation and related to mechano-transduction. It provided proof-of-concept evidences of the effectiveness of the nano-pulling applied to NSCs, in both in vitro and ex vivo conditions. Moreover, it pushed towards the validation of the proposed approach also in in vivo models. Overall, this work established a solid basis for further investigations, in the challenging avenue for finding more effective solutions to manage spinal cord lesions.