Assunção-Silva RC , Pinho A , Cibrão JR , Pereira IM , Monteiro S , Silva NA , Campos J , Rebelo AL , Schlosser G , Pinto L , Pandit A , Salgado AJ .

	Figure 1. Therapeutic effects of ASC secretome on Xenopus laevis tadpoles after complete transection on swimming recovery, axonal growth and regeneration, in the refractory stage of development. (a and b) Swimming pattern and quantification of the distance traveled by the refractory animals after SCI, at 2, 3, and 5 days after treatment with ASC secretome (CM group) or neurobasal medium (NB). Tadpoles with no SCI and treated with neurobasal medium (SH group) were used as controls for healthy animals. ASC secretome promoted significant functional recovery (868.861 ± 223.901 mm) of Xenopus laevis tadpoles after SCI from the refractory period, 5 days post-treatment, compared to neurobasal-treated animals (NB group; 283.015 ± 88.471 mm). (c) Representative confocal images of longitudinal cross sections of Xenopus laevis spinal cord after immunostaining for βIII-tubulin (axonal sprouting) and GAP-43 (axonal regeneration), at the refractory stage. Quantification of the percentage of (d) βIII-tubulin and (e) GAP-43 immunoreactivity in the spinal cord of Xenopus laevis tadpoles, at the refractory stage. At this stage, the ASC secretome group (CM) showed a clear gap closure and the formation of a robust axonal bridge at the lesion core (LC), between the two stumps of the spinal cord, 5 days post-treatment. This was confirmed by elevated, but not statistically significant, expression of βIII-tubulin in CM group (81.171 ± 1.679) when compared to NB group (54.529 ± 6.015). GAP-43 positive regenerating cells were present in the spinal cord tissue of these animals, with significant differences observed at 2 days post-treatment (CM group: 10.879 ± 1.071 vs NB group: 0.219 ± 0.037). Mean ± SEM; n = 12 for locomotor assessment; n = 5 for histological evaluation *p < 0.05; **p < 0.01; ***p < 0.001.
	Figure 2. Recovery of motor and sensorial function of mice with complete spinal cord transection after ASC secretome treatment. (a) BMS test was performed up to 6 weeks after treatment. ASC secretome treatment (CM) significantly improved the locomotor function of the transected animals, when compared to NB treatment CM-group at 6 weeks (3.778 ± 0619) versus NB-group (0.357 ± 0.210). Animals with no SCI treated with NB medium (SH group) showed completely normal locomotor performance. (b) Von-Frey Trial was performed at 2 and 6-week after ASC secretome or NB treatment as measure of sensitivity regain. Although no statistical differences were found between groups at both time-points, CM group show a trend of recovery of the sensorial function (0.237 ± 0.351) early at 2 weeks post-injury, when compared to NB group (0.022 ± 0.025). Data is presented as Mean ± SEM; n = 8 (SH), n = 7 (NB), n = 9 (CM); **p < 0.01; ***p < 0.001; ****p < 0.0001.
	Figure 3. Therapeutic effects of ASC secretome in mice spinal cord 6 weeks after complete transection on neuroinflammation and white matter injury. (a–d) Representative confocal images of longitudinal cross sections of mice spinal cord after immunostaining for Iba-1 ((a), neuroinflammation), fluoromyelin (b), white matter injury. Quantification of the (c) percentage of Iba-1 reactivity, (d) area of injured white matter. ASC secretome group (CM) presented statistically significant effect in the modulation of microglial response, decreasing the area of inflammatory microglial cells (21.124 ± 2.060) while increasing the area of homeostatic ones (96.396 ± 7.370) when compare to NB group (39.554 ± 4.536) and (61.105 ± 5.944), respectively. Similar effects were produced toward the area of injured white matter as CM-group had lower (23.933 ± 2.764), in comparison to the (NB) (39.554 ± 4.536) white matter degeneration. Dashed Lines denotes area coverage of ameboid microglia in (a) and epicenter regions in (b). Data is presented as Mean ± SEM; n = 5; *p < 0.05; **p < 0.01; ***p < 0.001.
	Figure 4. Therapeutic effects of ASC secretome in mice spinal cord 6 weeks after complete transection on axonal growth and regeneration. (a–e) Representative confocal images of longitudinal cross sections of mice spinal cord after immunostaining for ßIII-tubulin (axonal growth) and GAP-43 (axonal regeneration). Axonal outgrowth at the lesion site, as measured by a significant increase of ßIII-tubulin for ASC secretome group (69.106 ± 19.013), in comparison to NB group (22.677 ± 5.535) (e). Regeneration fibers were also clearly observed in CM group, as denoted by a significant increase GAP-43 expression at the lesion site (65.695 ± 16.990), when compared to NB group (23.193 ± 4.423) (f). Data is presented as Mean ± SEM; n = 5; ****p < 0.0001. White arrows point to ßIII-tubulin and GAP-43 immuno reactive fibers in (b and d) single channel images respectively. Scale bars are 200 and 50 µm in (a, c) and (b, d) respectively.
	Figure 5. Concentration of cytokines in the blood serum of mice at 6 weeks post-injury, and their interaction with other molecules present in ASC secretome. (a) The concentration (pg/ml) of pro-inflammatory—IL-1β, IL-6, IFN-γ and anti-inflammatory—IL-4 cytokines in the blood serum of SCI mice was assessed using multiplex-based ELISA. Secretome group (CM) showed decreased levels of pro-inflammatory cytokines (IL1-β: 6.463 ± 2.383; IL-6: 2.286 ± 1.460; IFN-γ: 1.573 ± 0.704), and increased levels of the anti-inflammatory cytokine (IL-4: 0.696 ± 0.247), when compared to NB- (IL-4: 0.390 ± 0.256; IL1-β: 10.864 ± 1.026; IL-6: 84.702 ± 38.767; IFN-γ: 9.976 ± 3.174) and SH- (IL-4: 0.606 ± 0.363; IL1-β: 4.232 ± 1.106; IL-6: 1.143 ± 1.143; IFN-γ: 2.871 ± 1.521) treated groups. (b) Representative networks of molecules present in ASC secretome, and respective receptors and signaling molecules, based on their role on inflammation and immune response (in green), and neurogenesis and axonogenesis (in blue), identified using STRINGS bioinformatics tools. in Mean ± SEM; n = 7 for SH and CM groups; n = 4 for NB group; *p < 0.05
	Figure 6. Schematics of the experimental paradigm employed for the Xenopus laevis model of transection SCI.
	Figure 7. Schematics of the experimental paradigm employed for the mouse model of transection SCI.5.5. Secretome injection in the Xenopus tadpole’s spinal cord after injury.
	Graphical abstract.

Adams, H+ pump-dependent changes in membrane voltage are an early mechanism necessary and sufficient to induce Xenopus tail regeneration. 2007, Pubmed, Xenbase

Adams, H+ pump-dependent changes in membrane voltage are an early mechanism necessary and sufficient to induce Xenopus tail regeneration. 2007, Pubmed , Xenbase
Alizadeh, Traumatic Spinal Cord Injury: An Overview of Pathophysiology, Models and Acute Injury Mechanisms. 2019, Pubmed
Andreu-Agulló, Vascular niche factor PEDF modulates Notch-dependent stemness in the adult subependymal zone. 2009, Pubmed
Ashwini, Cyclosporine A-Mediated IL-6 Expression Promotes Neural Induction in Pluripotent Stem Cells. 2018, Pubmed
Assinck, Cell transplantation therapy for spinal cord injury. 2017, Pubmed
Assunção-Silva, Exploiting the impact of the secretome of MSCs isolated from different tissue sources on neuronal differentiation and axonal growth. 2018, Pubmed
Badner, Spinal cord injuries: how could cell therapy help? 2017, Pubmed
Barker, Overexpression of the transcription factor Msx1 is insufficient to drive complete regeneration of refractory stage Xenopus laevis hindlimbs. 2009, Pubmed , Xenbase
Basso, Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains. 2006, Pubmed
Beck, Molecular pathways needed for regeneration of spinal cord and muscle in a vertebrate. 2003, Pubmed , Xenbase
Beck, Beyond early development: Xenopus as an emerging model for the study of regenerative mechanisms. 2009, Pubmed , Xenbase
Bermudez, Corneal epithelial wound healing and bactericidal effect of conditioned medium from human uterine cervical stem cells. 2015, Pubmed
Bermudez, Anti-inflammatory effect of conditioned medium from human uterine cervical stem cells in uveitis. 2016, Pubmed
Blight, Effects of silica on the outcome from experimental spinal cord injury: implication of macrophages in secondary tissue damage. 1994, Pubmed
Blight, Morphometric analysis of a model of spinal cord injury in guinea pigs, with behavioral evidence of delayed secondary pathology. 1991, Pubmed
Bloom, Non-mammalian model systems for studying neuro-immune interactions after spinal cord injury. 2014, Pubmed
Boruch, Neurotrophic and migratory properties of an olfactory ensheathing cell line. 2001, Pubmed
Brennan, Microglia coordinate cellular interactions during spinal cord repair in mice. 2022, Pubmed
Bunge, Observations on the pathology of human spinal cord injury. A review and classification of 22 new cases with details from a case of chronic cord compression with extensive focal demyelination. 1993, Pubmed
Caliezi, C1-Esterase inhibitor: an anti-inflammatory agent and its potential use in the treatment of diseases other than hereditary angioedema. 2000, Pubmed
Cameron, Cellular proliferation and neurogenesis in the injured retina of adult zebrafish. 2000, Pubmed
Chaplan, Quantitative assessment of tactile allodynia in the rat paw. 1994, Pubmed
Cizkova, Localized Intrathecal Delivery of Mesenchymal Stromal Cells Conditioned Medium Improves Functional Recovery in a Rat Model of Spinal Cord Injury. 2018, Pubmed
Cízková, The expression of B-50/GAP-43 and GFAP after bilateral olfactory bulbectomy in rats. 1997, Pubmed
Cízková, Transplants of human mesenchymal stem cells improve functional recovery after spinal cord injury in the rat. 2006, Pubmed
Colton, Assessing activation states in microglia. 2010, Pubmed
Colton, Heterogeneity of microglial activation in the innate immune response in the brain. 2009, Pubmed
Cui, Expanded adipose-derived stem cells suppress mixed lymphocyte reaction by secretion of prostaglandin E2. 2007, Pubmed
Czopik, Semaphorin 7A is a negative regulator of T cell responses. 2006, Pubmed
Davis, C1 inhibitor, a multi-functional serine protease inhibitor. 2010, Pubmed
Deczkowska, Microglial immune checkpoint mechanisms. 2018, Pubmed
DelaRosa, Requirement of IFN-gamma-mediated indoleamine 2,3-dioxygenase expression in the modulation of lymphocyte proliferation by human adipose-derived stem cells. 2009, Pubmed
de Sousa, Acute baclofen administration promotes functional recovery after spinal cord injury. 2023, Pubmed
Deuis, Methods Used to Evaluate Pain Behaviors in Rodents. 2017, Pubmed
Dixon, Efficient analysis of experimental observations. 1980, Pubmed
Donnelly, Inflammation and its role in neuroprotection, axonal regeneration and functional recovery after spinal cord injury. 2008, Pubmed
Ducker, Experimental treatments of acute spinal cord injury. 1969, Pubmed
Eckert, Trauma: Spinal Cord Injury. 2017, Pubmed
Edwards-Faret, Spinal cord regeneration in Xenopus laevis. 2017, Pubmed , Xenbase
Farmer, Glia-derived nexin potentiates neurite extension in hippocampal pyramidal cells in vitro. 1990, Pubmed
Flanagan, Early Tracheostomy in Patients With Traumatic Cervical Spinal Cord Injury Appears Safe and May Improve Outcomes. 2018, Pubmed
Foshay, Regulation of Sox2 by STAT3 initiates commitment to the neural precursor cell fate. 2008, Pubmed
Francos-Quijorna, IL-4 drives microglia and macrophages toward a phenotype conducive for tissue repair and functional recovery after spinal cord injury. 2016, Pubmed
Fukazawa, Suppression of the immune response potentiates tadpole tail regeneration during the refractory period. 2009, Pubmed , Xenbase
Furlan, Timing of decompressive surgery of spinal cord after traumatic spinal cord injury: an evidence-based examination of pre-clinical and clinical studies. 2011, Pubmed
Gaete, Spinal cord regeneration in Xenopus tadpoles proceeds through activation of Sox2-positive cells. 2012, Pubmed , Xenbase
Garcia, Cytokine and Growth Factor Activation In Vivo and In Vitro after Spinal Cord Injury. 2016, Pubmed
Giulian, Inhibition of mononuclear phagocytes reduces ischemic injury in the spinal cord. 1990, Pubmed
Godwin, Scar-free wound healing and regeneration in amphibians: immunological influences on regenerative success. 2014, Pubmed , Xenbase
Gordon, Alternative activation of macrophages. 2003, Pubmed
Guerrero, Blockade of interleukin-6 signaling inhibits the classic pathway and promotes an alternative pathway of macrophage activation after spinal cord injury in mice. 2012, Pubmed
Guimarães, Evidence for lack of direct causality between pain and affective disturbances in a rat peripheral neuropathy model. 2019, Pubmed
Gur-Wahnon, The plasminogen activator system: involvement in central nervous system inflammation and a potential site for therapeutic intervention. 2013, Pubmed
Gveric, Plasminogen activators in multiple sclerosis lesions: implications for the inflammatory response and axonal damage. 2001, Pubmed
Harper, Role of transforming growth factor beta and decorin in controlling fibrosis. 1994, Pubmed
Hatakeyama, Cadherin-based adhesions in the apical endfoot are required for active Notch signaling to control neurogenesis in vertebrates. 2014, Pubmed
Hatta-Kobayashi, Acute phase response in amputated tail stumps and neural tissue-preferential expression in tail bud embryos of the Xenopus neuronal pentraxin I gene. 2016, Pubmed , Xenbase
Hawryluk, An in vivo characterization of trophic factor production following neural precursor cell or bone marrow stromal cell transplantation for spinal cord injury. 2012, Pubmed
Heo, Tumor necrosis factor-α-activated human adipose tissue-derived mesenchymal stem cells accelerate cutaneous wound healing through paracrine mechanisms. 2011, Pubmed
Houenou, Pigment epithelium-derived factor promotes the survival and differentiation of developing spinal motor neurons. 1999, Pubmed
Hsieh, Mesenchymal stem cells from human umbilical cord express preferentially secreted factors related to neuroprotection, neurogenesis, and angiogenesis. 2013, Pubmed
Jeffery, Clinical canine spinal cord injury provides an opportunity to examine the issues in translating laboratory techniques into practical therapy. 2006, Pubmed
Kalinina, Characterization of secretomes provides evidence for adipose-derived mesenchymal stromal cells subtypes. 2015, Pubmed
Kilroy, Cytokine profile of human adipose-derived stem cells: expression of angiogenic, hematopoietic, and pro-inflammatory factors. 2007, Pubmed
Kong, JAK2/STAT3 signaling mediates IL-6-inhibited neurogenesis of neural stem cells through DNA demethylation/methylation. 2019, Pubmed
König, The plexin C1 receptor promotes acute inflammation. 2014, Pubmed
Lau, Astrocytes produce and release interleukin-1, interleukin-6, tumor necrosis factor alpha and interferon-gamma following traumatic and metabolic injury. 2001, Pubmed
Lawrence, The functional organization of the motor system in the monkey. II. The effects of lesions of the descending brain-stem pathways. 1968, Pubmed
Lee, Proteomic analysis of tumor necrosis factor-alpha-induced secretome of human adipose tissue-derived mesenchymal stem cells. 2010, Pubmed
Lee, Neurotransmitters and microglial-mediated neuroinflammation. 2013, Pubmed
Lee, Endogenous expression of interleukin-4 regulates macrophage activation and confines cavity formation after traumatic spinal cord injury. 2010, Pubmed
Li, The cellular and molecular mechanisms of tissue repair and regeneration as revealed by studies in Xenopus. 2016, Pubmed , Xenbase
Liao, Transformation from a neuroprotective to a neurotoxic microglial phenotype in a mouse model of ALS. 2012, Pubmed
Lima, Systemic Interleukin-4 Administration after Spinal Cord Injury Modulates Inflammation and Promotes Neuroprotection. 2017, Pubmed
Lu, Neural stem cells constitutively secrete neurotrophic factors and promote extensive host axonal growth after spinal cord injury. 2003, Pubmed
Lu, Adipose-derived mesenchymal stem cells protect PC12 cells from glutamate excitotoxicity-induced apoptosis by upregulation of XIAP through PI3-K/Akt activation. 2011, Pubmed
Markandaya, Acute Treatment Options for Spinal Cord Injury. 2012, Pubmed
Martinez, Alternative activation of macrophages: an immunologic functional perspective. 2009, Pubmed
Masai, N-cadherin mediates retinal lamination, maintenance of forebrain compartments and patterning of retinal neurites. 2003, Pubmed
McKee, Advances and challenges in stem cell culture. 2017, Pubmed
Meyerrose, Mesenchymal stem cells for the sustained in vivo delivery of bioactive factors. 2010, Pubmed
Minor, Tissue plasminogen activator promotes axonal outgrowth on CNS myelin after conditioned injury. 2009, Pubmed
Minor, Decorin promotes robust axon growth on inhibitory CSPGs and myelin via a direct effect on neurons. 2008, Pubmed
Miyamoto, N-cadherin-based adherens junction regulates the maintenance, proliferation, and differentiation of neural progenitor cells during development. 2015, Pubmed
Mothe, Advances in stem cell therapy for spinal cord injury. 2012, Pubmed
Mukaino, Anti-IL-6-receptor antibody promotes repair of spinal cord injury by inducing microglia-dominant inflammation. 2010, Pubmed
Munoz, Human stem/progenitor cells from bone marrow promote neurogenesis of endogenous neural stem cells in the hippocampus of mice. 2005, Pubmed
Muñoz, Regeneration of Xenopus laevis spinal cord requires Sox2/3 expressing cells. 2015, Pubmed , Xenbase
Nakajima, Transplantation of mesenchymal stem cells promotes an alternative pathway of macrophage activation and functional recovery after spinal cord injury. 2012, Pubmed
Namavari, Semaphorin 7a links nerve regeneration and inflammation in the cornea. 2012, Pubmed
Neugebauer, Vitronectin and thrombospondin promote retinal neurite outgrowth: developmental regulation and role of integrins. 1991, Pubmed
Neumann, Regeneration of dorsal column fibers into and beyond the lesion site following adult spinal cord injury. 1999, Pubmed
Neumann, Neurotrophins inhibit major histocompatibility class II inducibility of microglia: involvement of the p75 neurotrophin receptor. 1998, Pubmed
Novotna, IT delivery of ChABC modulates NG2 and promotes GAP-43 axonal regrowth after spinal cord injury. 2011, Pubmed
NULL, Xenopus laevis: practical uses in cell and molecular biology. 1991, Pubmed , Xenbase
Oh, Mesenchymal Stem Cells Increase Hippocampal Neurogenesis and Neuronal Differentiation by Enhancing the Wnt Signaling Pathway in an Alzheimer's Disease Model. 2015, Pubmed
Oyinbo, Secondary injury mechanisms in traumatic spinal cord injury: a nugget of this multiply cascade. 2011, Pubmed
Pasterkamp, Semaphorin 7A promotes axon outgrowth through integrins and MAPKs. 2003, Pubmed
Paul, The secretome of mesenchymal stem cells: potential implications for neuroregeneration. 2013, Pubmed
Pereira, IL-10 regulates adult neurogenesis by modulating ERK and STAT3 activity. 2015, Pubmed
Piazza, Timing of Surgery After Spinal Cord Injury. 2017, Pubmed
Pinho, Immunomodulatory and regenerative effects of the full and fractioned adipose tissue derived stem cells secretome in spinal cord injury. 2022, Pubmed
Pires, Unveiling the Differences of Secretome of Human Bone Marrow Mesenchymal Stem Cells, Adipose Tissue-Derived Stem Cells, and Human Umbilical Cord Perivascular Cells: A Proteomic Analysis. 2016, Pubmed
Popovich, Cellular inflammatory response after spinal cord injury in Sprague-Dawley and Lewis rats. 1997, Pubmed
Popovich, Depletion of hematogenous macrophages promotes partial hindlimb recovery and neuroanatomical repair after experimental spinal cord injury. 1999, Pubmed
Salgado, Mesenchymal stem cells secretome as a modulator of the neurogenic niche: basic insights and therapeutic opportunities. 2015, Pubmed
Sasaki, Protection of corticospinal tract neurons after dorsal spinal cord transection and engraftment of olfactory ensheathing cells. 2006, Pubmed
Sato, Interleukin-1 participates in the classical and alternative activation of microglia/macrophages after spinal cord injury. 2012, Pubmed
Scott, Semaphorin 7a promotes spreading and dendricity in human melanocytes through beta1-integrins. 2008, Pubmed
Shen, Overexpression of beta-1,4-galactosyltransferase I in rat Schwann cells promotes the growth of co-cultured dorsal root ganglia. 2003, Pubmed
Slack, Cellular and molecular mechanisms of regeneration in Xenopus. 2004, Pubmed , Xenbase
Sousa, Pre-Clinical Assessment of Roflumilast Therapy in a Thoracic Model of Spinal Cord Injury. 2023, Pubmed
Sroga, Rats and mice exhibit distinct inflammatory reactions after spinal cord injury. 2003, Pubmed
Steele, Pigment epithelium-derived factor: neurotrophic activity and identification as a member of the serine protease inhibitor gene family. 1993, Pubmed
Su, Immediate expression of Cdh2 is essential for efficient neural differentiation of mouse induced pluripotent stem cells. 2013, Pubmed
Sumi, The expression of tissue and urokinase-type plasminogen activators in neural development suggests different modes of proteolytic involvement in neuronal growth. 1992, Pubmed
Talac, Animal models of spinal cord injury for evaluation of tissue engineering treatment strategies. 2004, Pubmed
Tan, N-cadherin-dependent neuron-neuron interaction is required for the maintenance of activity-induced dendrite growth. 2010, Pubmed
Tan, AMP-activated kinase mediates adipose stem cell-stimulated neuritogenesis of PC12 cells. 2011, Pubmed
Tang, Differential Roles of M1 and M2 Microglia in Neurodegenerative Diseases. 2016, Pubmed
Tanimoto, Pigment epithelium-derived factor promotes neurite outgrowth of retinal cells. 2006, Pubmed
Taylor, Activation of group II metabotropic glutamate receptors underlies microglial reactivity and neurotoxicity following stimulation with chromogranin A, a peptide up-regulated in Alzheimer's disease. 2002, Pubmed
Taylor, Activation of microglial group III metabotropic glutamate receptors protects neurons against microglial neurotoxicity. 2003, Pubmed
Tran, Stem cells as drug delivery methods: application of stem cell secretome for regeneration. 2015, Pubmed
Tseng, Apoptosis is required during early stages of tail regeneration in Xenopus laevis. 2007, Pubmed , Xenbase
Tsuda, IFN-gamma receptor signaling mediates spinal microglia activation driving neuropathic pain. 2009, Pubmed
Vizoso, Mesenchymal Stem Cell Secretome: Toward Cell-Free Therapeutic Strategies in Regenerative Medicine. 2017, Pubmed
Wang, Human progenitor cells from bone marrow or adipose tissue produce VEGF, HGF, and IGF-I in response to TNF by a p38 MAPK-dependent mechanism. 2006, Pubmed
Yang, Severity-dependent expression of pro-inflammatory cytokines in traumatic spinal cord injury in the rat. 2005, Pubmed
Yang, Resistance of interleukin-6 to the extracellular inhibitory environment promotes axonal regeneration and functional recovery following spinal cord injury. 2017, Pubmed
Yang, Transplantation of human umbilical mesenchymal stem cells from Wharton's jelly after complete transection of the rat spinal cord. 2008, Pubmed
Zhang, Decorin is a pivotal effector in the extracellular matrix and tumour microenvironment. 2018, Pubmed