Nuno André Silva

Cell-free therapies based on the mesenchymal stem cells (MSCs) secretome are being widely explored for spinal cord injury (SCI). Induced pluripotent stem cells (iPSCs) provide a scalable source for generating mesenchymal stem cells (iMSCs), but limited studies have evaluated the modulatory capacity of iMSCs for CNS applications. Due to the importance of the glial and vascular responses to SCI progression, this study evaluated the activity of the iMSCs secretome on glial and endothelial cells in SCI-relevant paradigms. In primary mixed spinal cord cultures, the secretome was tested under supplement withdrawal and hyperosmotic injury conditions, while endothelial network formation was assessed in a morphogenesis assay. In vitro, the iMSCs secretome promoted a modest protective effect on cell survival, modulated astrocyte morphology and preserved myelin basic protein expression within the oligodendroglial lineage, without clear alterations in microglial morphometric readouts. Secretome treatment also increased Arg1 expression in mixed spinal cord cultures. In endothelial cultures, the secretome promoted a denser and more interconnected endothelial network. In a mouse thoracic SCI model, the secretome was administered intravenously, and tissue was analyzed one week after injury. In vivo, the secretome reduced perilesional GFAP coverage and increased selected vascular morphometric parameters without altering lesion size or microglia morphological readouts. Overall, these findings indicate that the iMSCs secretome produced context-dependent changes in glial and vascular cells, mainly regarding morphological parameters, supporting further studies of iPSC-derived MSCs as a scalable source for secretome-based strategies.

Introduction: Biological sex has been shown to influence immune function and inflammatory responses. However, most preclinical studies in spinal cord injury (SCI) have predominantly used female animals, potentially introducing a sex bias in our understanding of post-injury inflammation. Given the central role of inflammation in secondary pathology and repair after SCI, males and females are likely to respond differently to this condition, with sex-dependent immune differences potentially shaping injury outcomes. In this study, we explored how biological sex shapes the course of inflammation after SCI and its potential impact on functional recovery. Methods: Locomotor recovery in the SCI mouse model was assessed using the Basso Mouse Scale (BMS). Autonomic recovery was analyzed through the spontaneous void spot assay. Peripheral and local immune responses were characterized by flow cytometry of blood and spinal cord samples, respectively, to track the temporal dynamics of distinct immune cell populations across acute and chronic stages of injury. Results: We found that, acutely after SCI, males exhibit higher frequencies of circulating myeloid cells, whereas females show higher numbers of these cells in the spinal cord, suggesting delayed myeloid infiltration in males. At this timepoint, females initiate an early attempt to direct some cells toward a phenotype more favorable for tissue repair, with their infiltrative monocytes expressing both pro-inflammatory and alternative activation markers. Sex differences in blood CD54 expression at 4 weeks post-injury suggest that sex-specific immune responses may continue to develop at later stages after SCI. Post-injury welfare outcomes differ between sexes, with males exhibiting delayed post-operative recovery. However, spontaneous long-term functional recovery is largely comparable between sexes after SCI. Conclusion: Overall, our data identify biological sex as a key determinant of immune and neuroinflammatory responses after SCI while suggesting a limited impact on long-term recovery. These findings highlight the lack of rationale for the underrepresentation of males in preclinical studies and emphasize the importance of sex-inclusive research to better inform personalized therapeutic strategies.

Innovations in spinal cord injury (SCI) models are crucial for developing effective therapies. This study introduces a novel in vitro SCI model using cultures of primary mixed spinal cord cells from rat pups, featuring key spinal cord cell types. This model offers distinct advantages in terms of feasibility, reproducibility, and cost-effectiveness, requiring only basic cell culture equipment. Following hyperosmotic stress via sorbitol treatment, the model recapitulated SCI pathophysiological hallmarks, with a 65% reduction in cell viability and gradual cell death over 48 h, making it ideal for evaluating neuroprotective agents. Notably, the human adipose tissue stem cell (hASC) secretome provided significant protection: it preserved metabolic viability, reduced β amyloid precursor protein (β-APP) expression in surviving neurons, and modulated the shift in the astrocytic morphotype. A transcriptomic profile of the effect of the hASC secretome treatment showed significant functional enrichments related to cell proliferation and cycle progression pathways. In addition to supporting the use of the hASC secretome as a therapy for SCI, this study is the first to use sorbitol as a hyperosmolar stressor to recapitulate key aspects of SCI pathophysiology. Thereby, this model can be used as a promising platform for evaluating therapeutic agents targeting neuroprotection and neuroregeneration, offering outputs related to cell death, neuronal stress, and protection, as well as induction of glial reactivity.

Exposure to electric fields affects cell membranes impacting their potential and altering cellular excitability, nerve transmission, or muscle contraction. Furthermore, electric stimulation influences cell communication, migration, proliferation, and differentiation, with potential therapeutic applications. In vitro platforms for electrical stimulation are valuable tools for studying these effects and advancing medical research. In this study, we developed and tested a novel multi-channel in vitro electrical stimulator designed for cellular applications. The device aims to facilitate research on the effects of electrical stimulation (ES) on cellular processes, providing a versatile platform that is easy to reproduce and implement in various laboratory settings. The stimulator was designed to be simple, cost-effective, and versatile, fitting on standard 12-well plates for parallel experimentation. Extensive testing was conducted to evaluate the performance of the stimulator, including 3D finite element modelling to analyse electric field distribution. Moreover, the stimulator was evaluated in vitro using neuronal and stem cell cultures. Finite element modelling confirmed that the electric field was sufficiently homogeneous within the stimulation zone, though liquid volume affected field strength. A custom controller was developed to program stimulation protocols, ensuring precise and adjustable current delivery up to 160 V/m. ES promoted neurite outgrowth when applied to SH-SY5Y neural cells or to primary spinal cord-derived cells. In human neuronal progenitor cells (hNPCs), ES enhanced neurite growth as well as differentiation into neurons. In adipose stem cells (ASCs), ES altered the secretome, enriching it in molecules that promoted hNPC differentiation into neurons without enhancing neurite growth. Our results highlight the potential of this multi-channel electrical stimulator as a valuable tool for advancing the understanding of ES mechanisms and its therapeutic applications. The simplicity and adaptability of this novel platform make it a promising addition to the toolkit of researchers studying electrical stimulation in cellular models.

Background: Priming strategies that improve the functionality of MSCs may be required to address issues limiting successful clinical translation of MSC therapies. For conditions requiring high trophic support such as brain and spinal cord injuries, priming MSCs to produce higher levels of trophic factors may be instrumental to facilitate translation of current MSC therapies. We developed and tested a novel molecular priming paradigm using docosahexaenoic acid (DHA) to prime adipose tissue-derived mesenchymal stromal cells (ASCs) to enhance the secretome neuroregulatory potential. Methods: Comprehensive dose–response and time-course assays were carried to determine an optimal priming protocol. Secretome total protein measurements were taken in association with cell viability, density and morphometric assessments. Cell identity and differentiation capacity were studied by flow cytometry and lineage-specific markers. Cell growth was assessed by trypan-blue exclusion and senescence was probed over time using SA-β-gal, morphometry and gene expression. Secretomes were tested for their ability to support differentiation and neurite outgrowth of human neural progenitor cells (hNPCs). Neuroregulatory proteins in the secretome were identified using multiplex membrane arrays. Results: Priming with 40 µM DHA for 72 h significantly enhanced the biosynthetic capacity of ASCs, producing a secretome with higher protein levels and increased metabolic viability. DHA priming enhanced ASCs adipogenic differentiation and adapted their responses to replicative senescence induction. Furthermore, priming increased concentrations of neurotrophic factors in the secretome promoting neurite outgrowth and modulating the differentiation of hNPCs. Conclusions: These results provide proof-of-concept evidence that DHA priming is a viable strategy to improve the neuroregulatory profile of ASCs.

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The Spinal Cord Injury (SCI) pathophysiology is highly complex, contributing to a poor prognosis and lack of effective treatments. Previously, we demonstrated that Roflumilast (Rof), leads to functional recovery when tested in a SCI contusion model. However, it is unlikely that Rof treatment on its own could fully restore the spinal cord. Therefore, our objective was to scrutinize the synergistic effects of combining Rof with neuroprotective approaches. Herein we tested two therapies, firstly, Rof combined with Levetiracetam (Lev), and in a second phase, the complementary interplay between Rof and Adipose Stem Cells secretome (Sec). We induced SCI using a weight drop trauma model at the T8 level. Functional recovery was assessed according to the Basso, Beattie, and Bresnahan scale, Activity Box Test, Motor Swimming Test, and Von Frei test. Results indicate that the unilateral use of Rof, Lev, or Sec was effective in promoting functional recovery. However, the combination of Rof + Lev or Rof + Sec did not lead to an improvement in functional outcomes when compared to standalone treatments. Moreover, the combination of Rof + Sec actually led to worst functional outcome than the single treatments. Further studies are needed to find a combinatorial treatment that can lead to superior therapeutic effects with potential clinical application.

Spinal cord injuries have profound detrimental effects on individuals, regardless of whether they are caused by trauma or non-traumatic events. The compromised regeneration of the spinal cord is primarily attributed to damaged neurons, inhibitory molecules, dysfunctional immune response, and glial scarring. Unfortunately, currently, there are no effective treatments available that can fully repair the spinal cord and improve functional outcomes. Nevertheless, numerous pre-clinical approaches have been studied for spinal cord injury recovery, including using biomaterials, cells, drugs, or technological-based strategies. Combinatorial treatments, which target various aspects of spinal cord injury pathophysiology, have been extensively tested in the last decade. These approaches aim to synergistically enhance repair processes by addressing various obstacles faced during spinal cord regeneration. Thus, this review intends to provide scientists and clinicians with an overview of pre-clinical combinatorial approaches that have been developed toward the solution of spinal cord regeneration as well as update the current knowledge about spinal cord injury pathophysiology with an emphasis on the current clinical management.

Astrocytes are ubiquitous in the brain and spinal cord and display a complex morphology important for the local interactions with neighboring cells, resulting in the modulation of circuit function. Thus, studies focusing on astrocyte physiology in the healthy and diseased brain generally present analyses of astrocytic structure. The labeling method used to visualize the astrocytic structure defines the morphological level to observe and may vary depending on the anatomical sub-regions. The method choice may significantly affect our understanding of their structural diversity. The main goal of this work was to identify a straightforward and efficient protocol for labeling and reconstructing a detailed astrocytic structure to apply and validate in different brain tissue preparations across laboratories. For that, we explored different tissue processing protocols before GFAP labeling to determine the most effective method for reconstructing astrocytic backbones in the mouse hippocampus. Our results show that the reconstruction of astrocytic structure in vibratome sections labeled by free-floating immunofluorescence protocol provides a more practical method to achieve a higher level of detail and arbor complexity in astrocyte backbone reconstruction. Free-floating immunofluorescence labeling is the most reliable method for obtaining better antibody penetration and more detailed astrocyte structure. Finally, we also show that introducing an antigen retrieval step appears useful for visualizing more complete structural details.