The secretome of Mesenchymal Stem Cells as a cell-free based therapy for Spinal Cord Injury

Universidade do Minho Escola de Medicina Rita Catarina Assunção Ribeiro Silva dezembro de 2019 The secretome of Mesenchymal Stem Cells as a cell-free based therapy for Spinal Cord Injury Rita Catarina Assunção Ribeiro Silva The secretome of Mesenchymal Stem Cells as a cell-free based therapy for Spinal Cord Injury UMinho|2019 Rita Catarina Assunção Ribeiro Silva dezembro de 2019 The secretome of Mesenchymal Stem Cells as a cell-free based therapy for Spinal Cord Injury Trabalho efetuado sob a orientação do Doutor António Salgado e da Doutora Luísa Pinto Tese de Doutoramento Doutoramento em Ciências da Saúde Universidade do Minho Escola de Medicina ii DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS Este é um trabalho académico que pode ser utilizado por terceiros desde que respeitadas as regras e boas práticas internacionalmente aceites, no que concerne aos direitos de autor e direitos conexos. Assim, o presente trabalho pode ser utilizado nos termos previstos na licença abaixo indicada. Caso o utilizador necessite de permissão para poder fazer um uso do trabalho em condições não previstas no licenciamento indicado, deverá contactar o autor, através do RepositóriUM da Universidade do Minho. Atribuição-NãoComercial-SemDerivações CC BY-NC-ND https://creativecommons.org/licenses/by-nc-nd/4.0/ iii AGRADECIMENTOS/ACKNOWLEDGEMENTS Findo uma das etapas mais desafiantes da minha vida profissional, é hora de agradecer a todos os que partilham esta conquista comigo. Ao António Salgado, orientador e mentor, e amigo, pelo apoio incondicional, presença constante, confiança e dedicação a este trabalho que exige tanto de nós. Tó, estou-te eternamente grata. À Luísa, pela orientação, apoio, sugestões e discussões científicas. Aos professores Nuno Sousa, Jorge Pedrosa e João Bessa pelo apoio institucional da Escola de Medicina, do ICVS e dos NERDs. À Tó Team, pelo companheirismo e partilha de ideias e ideais científicos, que enriqueceram o meu trabalho. Em especial aos meus companheiros Eduardo e Rui, por todo o apoio e momentos de descontração, e à “mice team” – Andreia, Inês, Jorge e Susana – que mobilizaram todos os seus esforços para me ajudar na fase final deste trabalho. A todos os outros colegas de equipa e do domínio, que de certa forma colaboraram neste trabalho. To Abhay Pandit, for the opportunity to work in a reputed institute such as Cúram, and to all the mentoring and relevant scientific discussion on the work I did there. To Aniket for all the help in the lab. To Ana, Shubha and Niranjan, Portuguese and Indian friends in Ireland to which I am sincerely grateful for all the experiences you presented me with. Aos meus amigos Bárbara, Eduardo, Cláudia, Patrícia, Joana S., Rita, Joana C., Ana e Luís, que tornaram Braga a minha casa e viveram comigo as minhas alegrias, tristezas, certezas e incertezas. Tenho-vos num espacinho especial no meu coração. Por fim, um agradecimento especial àqueles que me relembram todos os dias de onde venho e do que sou feita: ao meu eterno “namorido”, Pedro, o meu porto de abrigo e companheiro de vida; ao meu Pai e à minha Irmã – Nemum curat quam familae . Aos meus sogros, à Ana e ao Bruno, aos meus avós, tios e primos. E às minhas duas Belinhas... A inspiração, força e alegria do meu passado, presente e futuro. Esta tese é tão minha quanto vossa! The work presented in this thesis was performed at the Life and Health Sciences Research Institute (ICVS) and at Behavioral & Molecular Lab (BnML), at the School of Medicine, University of Minho. Part of this work was also performed at the Center for Research in Medical Devices (Cúram), through financial support from Science Foundation Ireland (SFI) and the European Regional Development Fund (Grant Number 13/RC/2073). Financial support was provided from Prémios Santa Casa Neurociências - Prize Melo e Castro for Spinal Cord Injury Research (MC-04/17); Portuguese Foundation of Science and Technology [Doctoral Fellowship to R. C. Assunção-Silva (PDE/BDE/113596/2015)]; funded by FEDER, through the Competitiveness Internationalization Operational Programme (POCI), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the projects POCI-01-0145FEDER-007038; TUBITAK/0007/2014; PTDC/DTP-FTO/5109/2014; POCI-01-0145-FEDER-029206; POCI-01-0145-FEDER-031392; PTDC/MED-NEU/31417/2017 and NORTE-01-0145-FEDER-029968. This work has also been developed under the scope of the project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). iv STATEMENT OF INTEGRITY I hereby declare having conducted this academic work with integrity. I confirm that I have not used plagiarism or any form of undue use of information or falsification of results along the process leading to its elaboration. I further declare that I have fully acknowledged the Code of Ethical Conduct of the University of Minho. v TÍTULO: O SECRETOMA DE CÉLULAS ESTAMINAIS MESENQUIMATOSAS COMO UMA TERAPIA PARA LESÕES VERTEBROMEDULARES RESUMO As lesões vertebro-medulares (LVM) resultam de um trauma na espinal medula, seguido da perda parcial ou completa da função motora e sensorial abaixo do nível de lesão. A transplantação celular tem estado na vanguarda de muitas estratégias, mas apresentam uma taxa de implantação e sobrevivência baixas perante o ambiente de lesão nocivo. Como alternativa, estratégias baseadas no uso do secretoma de células estaminais têm sido muito exploradas. O secretoma de células estaminais mesenquimatosas (MSCs) têm mostrado efeitos benéficos através de citoquinas anti-inflamatórias e fatores de crescimento e regenerativos. Uma expressão diferencial destas moléculas por MSCs derivadas do tecido adiposo (ASCs), medula óssea (BMSCs) e do cordão umbilical (HUCPVCs) sugerem um impacto terapêutico distinto. Esta hipótese foi confirmada por experiências in vitro , onde o secretoma de ASCs promoveu níveis de crescimento axonal superiores aos das outras populações celulares. De seguida, o potencial terapêutico do secretoma de ASCs foi avaliado num modelo de LVM em Xenopus Laevis . A sua administração na espinal medula dos Xenopus após transeção completa aumentou a regeneração axonal e crescimento neuronal no local de lesão. Os animais tratados apresentaram formação de uma ponte axonal na zona lesionada, assim como uma melhoria da função motora. Finalmente, o potencial terapêutico do secretoma de ASCs foi avaliado num modelo de LVM em ratinho. O secretoma foi administrado por via intravenosa após transeção completa torácica da espinal medula. Os animais tratados demonstraram melhorias das funções motoras e sensoriais, acompanhado por uma redução evidente do número de células inflamatórias no local de lesão, o que sugere uma ação anti-inflamatória do secretoma das ASCs. Foi também observado crescimento e regeneração axonal após tratamento, assim como uma diminuição da cavidade de lesão. Em resumo, os resultados aqui apresentados providenciam evidências do potencial terapêutico do secretoma de ASCs após LVM, considerando os seus efeitos positivos ao nível da inflamação neuronal e crescimento e regeneração axonal, observado nos modelos in vitro e in vivo aqui estudados, e que estão associados à recuperação motora dos Xenopus Laevis e do ratinho. Palavras-chave: Células estaminais mesenquimatosas, crescimento axonal, lesões vertebro-medulares, neuroinflamação, secretoma. vi TITLE: THE SECRETOME OF MESENCHYMAL STEM CELLS AS A CELL-FREE BASED THERAPY FOR SPINAL CORD INJURY ABSTRACT Spinal cord injury (SCI) results from a mechanical trauma to the spinal cord, followed by partial or complete loss of motor and sensory function below the level of injury. Cell transplantation has been in the forefront of regenerative medicine strategies, but often presents low engraftment and survival rate within the aggressive environment of SCI. Alternatively, cell-free based strategies using the secretome of stem cells has been highly explored. Mesenchymal stem cells (MSCs) secretome has been showing beneficial effects through anti-inflammatory cytokines and regeneratingand growth-permissive factors. A differential expression of these molecules by adipose tissue (ASCs)-, bone-marrow (BMSCs)- and umbilical cord (HUCPVCs)-derived MSCs suggested a distinct therapeutic outcome. Indeed, this was confirmed in in vitro experiments, where ASCs secretome promoted significantly higher levels of axonal growth, when compared to the other cell populations. Following this, the therapeutic potential of ASC secretome was evaluated in a Xenopus laevis model of SCI. ASC secretome delivery into a transected Xenopus spinal cord increased axonal regeneration and neuronal regrowth at the lesion site. Treated animals showed ablation gap closure and axonal bridge formation between the two spinal cord stumps, as well as an improved motor function. Finally, the therapeutic potential of ASC secretome was evaluated in a mice model of SCI. ASC secretome was intravenously administered into mice spinal cord after complete thoracic transection. Treated animals showed improved motor and sensorial function, accompanied by a marked reduction on the number of inflammatory cells at the lesion site, suggesting an anti-inflammatory action of ASC secretome. Axonal outgrowth and regeneration through the injury was also observed upon ASC secretome treatment, as well as decreased lesion cavities. Altogether, these results provide evidences of the therapeutic potential of ASC secretome after SCI, supported by indications on the positive effects exerted on neuroinflammation, and axonal outgrowth and regeneration, observed for in vitro and in vivo models herein studied, and that were associated to locomotor recovery to both Xenopus laevis and mice. Keywords: Axonal Outgrowth; Mesenchymal Stem Cell Secretome; Neuroinflammation; Regeneration; Spinal Cord injury. vii TABLE OF CONTENTS AGRADECIMENTOS/ACKNOWLEDGEMENTS III STATEMENT OF INTEGRITY IV RESUMO V ABSTRACT VI ABBREVIATIONS LIST VIII FIGURES & TABLES LIST XIII THESIS AIMS AND LAYOUT XV CHAPTER I – INTRODUCTION 1 1. Spinal Cord injury 2 1.1 Pathophysiology 2 1.2 Clinical Management 2 1.3 Research on SCI: fundamental studies in animal models 4 1.3.1 Basic experimental models of SCI 5 1.3.2 Mammal models of SCI 15 2. Cell transplantation: A relevant regenerative approach for SCI treatment 22 2.1 Adult Myelinating Cells 23 2.2 Stem cells 25 3. MSC secretome: A cell-free based therapy for SCI regeneration 30 3.1 MSC Paracrine Activity: Secretome vs Transplantation 30 3.2 MSC secretory profile from a mechanistic point of view 31 4. Concluding Remarks 38 CHAPTER II – EXPLOITNG THE IMPACT OF SECRETOME OF MCSS ISOLATED FROM DIFFERENT TISSUE SOURCES ON NEURONAL DIFFERENTIATION AND AXONAL GROWTH 64 CHAPTER III – XENOPUS LAEVIS – BEYOND A BASIC MODEL OF REGENERATION 88 CHAPTER IV – ADIPOSE-DERIVED MESENCHYMAL STEM CELL THERAPEUTIC EFFECT IN A MICE MODEL OF SPINAL CORD INJURY 106 CHAPTER V – GENERAL DISCUSSION AND FUTURE PERSPECTIVES 131 xiv CHAPTER V. GENERAL DISCUSSION AND FUTURE PERSPECTIVES Figure 1. The regenerative potential of MSC secretome for SCI treatment. xv THESIS AIMS AND LAYOUT In this thesis, we pursued to explore the potential of the secretome of mesenchymal stem cells (MSCs) as a cell-free based therapy for Spinal Cord Injury (SCI). For this purpose, the impact of MSC secretome on neuronal differentiation and axonal outgrowth, was initially evaluated in vitro . Furthermore, we explored how the secretome of MSCs derived from adipose tissue (ASCs) would impact the regeneration of both Xenopus Laevis and mice models of SCI after spinal cord complete transection. The thesis will present the following layout: Chapter 1 presents a general overview covering fundamental aspects of SCI pathophysiology and the clinical management strategies currently accessible for SCI patients. Furthermore, a detailed description of the animal models used for fundamental studies on SCI research is provided, along with the main cellular-based therapeutic strategies available for SCI treatment in both preand clinical setting. Chapter 2 comprises research work focused on disclosing the impact of the secretome of MSCs derived from the adipose tissue, bone marrow and umbilical cord on neuronal differentiation and axonal growth, in vitro . This chapter is presented as an original paper published in Biochimie, in 2018 (Assunção-Silva et al., 2018). Chapter 3 addresses the use of a naturally regenerating animal model – the Xenopus laevis – to study the regenerative role of ASC secretome after a complete thoracic transection of Xenopus ’ spinal cord. Behavioral and histological assessments are presented. Chapter 4 presents the work in which a spinal cord thoracic complete transection mice model was used to further explore the potential of ASC secretome in promoting functional and sensorial recovery in mammals. Moreover, the impact of ASC secretome on neuroinflammation, and axonal regeneration and outgrowth after SCI was also studied. Chapter 5 encompasses a general discussion incorporating all the findings of the work performed on the scope of this thesis. Moreover, the limitations of this work, along with future perspectives are also debated. 1 CHAPTER I INTRODUCTION 2 1. Spinal Cord injury 1.1 Pathophysiology Spinal cord injury (SCI) is commonly initiated by a mechanical trauma of the tissues surrounding the spinal column, after which the spinal cord suffers an extensive disruption. This initial mechanical event sets the primary injury at which several post-injury events are immediately triggered within the lesion site (Fig. 1). These include massive cell death, bone, muscle and vasculature damage, as well as the formation of an edema [1, 2]. Several other biomechanical and biological mechanisms follow and contribute to the progression to a secondary injury (Fig. 1), a concept first introduced by Allen and colleagues in 1991 [3]. The secondary injury comprises three phases: acute, sub-acute and chronic, as described by Alizadeh et al. [2]. From days to weeks or months, spinal cord parenchyma is invaded by inflammatory cells, and a local release of chondroitin sulfate proteoglycans (CSPGs), glutamate, and other axonal growth inhibitory molecules establishes an excitotoxicity environment [2, 4, 5]. Focal demyelination of both injured and surviving axons is also of great magnitude [4, 6]. Finally, a fluid-filled cyst is formed at the lesion site, surrounded by a glial scar mainly composed of reactive astrocytes and fibroblasts, which contributes to a progressive axonal dead and constitutes a physical barrier to axonal outgrowth and regeneration through the lesion [5, 7]. This axonal growth-restrictive character of the SCI sets a chronic injury. The location and severity of the injury determines the overall neurological deficits, and usually lead to chronic pain syndromes and hypersensitivity of the patients [2]. 1.2 Clinical Management The clinical management of traumatic SCI encompasses a rapid clinical assessment and classification of the injury, according to the American Spinal Injury Association (ASIA) grade [2], and further stabilization of the patients to minimize the primary injury and prevent the secondary injury [1]. Along with the neurological impairments, neurogenic and spinal shock are also other SCI-associated conditions, that together with respiratory, circulatory, cardiovascular, urinary and bowel complications are the main causes of morbidity and mortality of the SCI patients [8-10]. Therefore, it is extremely important that patients’ vital signs are stabilized and eventual hemorrhages are controlled in the acute phase of injury upon their entrance in the healthcare unit [9, 11]. Respiratory support, namely tracheostomy and mechanical ventilation, should be immediately provided to the patients in cases of potential airway and breathing obstruction [12], especially for cervical and thoracic SCI , that affects the spinal nerves innervating the respiratory muscles [13, 14]. Further surgical decompression and/or stabilization of the spinal cord is a standard procedure [9, 15] to restore spinal alignment and stability. Actually, surgical 3 Figure 1. Pathophysiology of SCI. Within an injured spinal cord, both healthy (upper right) and injured (bottom right) environments can be found. The pathophysiology of SCI comprises different events, namely the infiltration of peripheral T and B cells (1) and macrophages (2) releasing inflammatory cytokines and axonal growth-inhibitory molecules, activation of resident microglia and astrocytes (3), oligodendrocyte damage and consequent axonal demyelination (4), axonal damage and retraction (5), and formation of a glial scar composed of activated astrocytes surrounding the lesion site (6). The astrocytic scar surrounds a fluid-filled cystic cavity and constitutes a physical barrier for axons to cross the lesion site, thus impairing the re-establishment of the neural circuitry and the regeneration and repair of the spinal cord. [1, 2, 4] intervention is the most crucial step in the management of SCI patients, after the control over the lifethreatening complications. Early spinal decompression was correlated to improved clinical and neurological outcomes following injury and reduced health care costs [15, 16]. However, it is more likely that surgical decompression cannot completely cease the secondary injury [17]. For that purpose, pharmacological treatment using steroids is the current treatment option [18, 19]. Within these, the most common is Methylprednisolone (MP). The effect of MP in the neurological function of SCI patients was shown for the first time in 1984, and later in 1990 and 1997 by the American National Acute Spinal Cord 4 Injury Study – phases I-III [20-23]. The findings of these studies provided a standard protocol for the administration of high dosage of MP within 8h post-injury, which showed significant sensory and motor improvements of SCI patients one year following SCI. MP is believed to act upon specific secondary injuryrelated mechanisms such as inflammation and ischemia [24]. However, the first studies were severely criticized regarding the methodology used, as well as its scientific and statistical reliability. For example, a very small size of sample population showing beneficial effects were considered, and statistical differences could only be found upon stratification of the data. Moreover, the clinical relevance of the statistical functional improvements was not well defined [25-28]. In addition, severe side effects were presented by the patients upon MP treatment, such as respiratory complications that resulted in death, pneumonia and wound infections [22, 23]. In a scenario where the positive outcomes of MP treatment may not compensate the hazardous effects, new Guidelines for the Management of Acute Cervical Spine and Spinal Cord Injury were released, in which the use of MP treatment for acute SCI was not recommended [29]. The controversy around MP treatment for SCI still exists nowadays, and currently there is no pharmacological alternative. Nonetheless, several pre-clinical and clinical tests are currently running with other neuroprotective drugs, such as riluzole, minocycline, and glutamate antagonists [21, 30]. The therapeutic window for intervention in the primary phase of injury is usually difficult to tackle, and the exacerbating secondary injury hamper specialized intervention [4]. The above-mentioned clinical complications that follow the mechanical trauma contributes for this delay in the management of SCI. Therefore, deeper knowledge over the secondary mechanisms is needed to tailor post-traumatic therapeutic strategies that counteract the current clinical failure in the treatment SCI. 1.3 Research on SCI: fundamental studies in animal models Conceptually, animal models are in vivo systems that provide investigational methods that are used to assess specific events under controlled conditions. They are extremely valuable to investigate a given human disease or condition. However, to be reliable and reproductive enough to further translate these investigations to clinics, in vivo animal studies must mirror all aspects of the disease or condition, not only in terms of the cause but also concerning its physiological and pathological repercussions. Given the complexity of the pathophysiology of SCI, finding a strategy that completely promotes the regeneration of the spinal cord after lesion is inevitably dependent on innumerous factors, and the efforts in the clinics are yet not enough. The wide variability in human SCI accounts for this. The inhibitory nature of the biological mechanisms following SCI to axonal regeneration in mammals contribute to the failure 5 of human SCI to regenerate [2, 5, 7]. But there are other factors to consider. For example, the anatomical level of the trauma can determine the severity of the injury [10]. The time-window and dosage response at which a therapy is given to the patient is also crucial to avoid the progression of the damage [15, 31]. Moreover, some biomechanical factors associated to the injury, such as the mechanism, velocity, and compression level may also affect the treatment outcomes [5]. Therefore, an effective therapy should ideally tackle all these SCI-related aspects, which is probably the most puzzling issue faced by researchers and clinicians in the field. So, before that, determining what features should be addressed in such therapies and how to overcome them is crucial to tailor the most suitable approach that could hold its efficacy at the different stages of one lesion. 1.3.1 Basic experimental models of SCI Under the premise that robust and fully axonal regrowth must be achieved to promote complete functional regeneration after injury, the common aim of any strategy is to represent the pathophysiology of human SCI as close as possible. As no in vitro system is able to mimic the complex environment after injury, different animal models, with varying degrees of complexity have been established throughout the years (Fig. 2). Amphibians Amphibians are a group of animals that include anurans, such as frogs, and urodeles, such as salamanders. This group of animals have been used to study embryonic development since the 1900s. After Spallanzani demonstrated that frogs tadpoles and salamanders regenerate their tails, limbs and eye lens [32], a new window of research upon the natural regenerative ability of these animals was opened. The two classes of animals differ in their regenerative process throughout life, although they share closely related biology [33]. The main difference between them relies on the fact that salamanders can regenerate throughout all stages of life, while regeneration in frogs is restricted to early stages of development. Salamander Urodeles, such as the Salamanders possess the ability to regenerate tail, limbs, and the lens of the eye, throughout any stage of development. Life cycle of salamanders is very similar to the Xenopus laevis , but unlike these, salamanders rarely goes through metamorphosis. In fact, they are neotenous, thus never complete metamorphosis. Nevertheless, salamanders that undergo this process, either spontaneously or hormonally induced, did not present any effect on their regenerative capacity [34, 35]. Only subtle 6 changes were observed in the immune system upon induced metamorphosis [36], in comparison to the extreme changes observed in Xenopus laevis [33], highlighting the impact that an unbalanced immune system might have in the regeneration. Salamanders are the only amphibians able to regenerate the spinal cord in any stage of development, including embryonic, larval, juvenile and adult. However, using urodeles as a model of SCI and regeneration is not as simple as it might seem. There are different patterns of regeneration among the different developmental stages, with embryonic and larvae animals showing higher numbers of new and qualitatively different neurons than adults [37-39], as well as differences on the time course of regeneration, with adult urodeles taking longer to regenerate than larval and juvenile after trunk transection [40]. The injury models used in salamanders may also influence the overall regenerative processes. The two major models of SCI in salamanders are spinal cord transection and tail amputation. Both share common features, and spinal cord regeneration is easy to follow, where the interactions between growing axons with ependymal cells is very clear [41]. Crush injuries had also been used in salamanders since it is the most studied in mammals, however this model presents an extensive cell death and debris that do not allow a clean view over the regenerative process [42]. It has been established that the regeneration process of urodele’s spinal cord after injury is divided in three main parts: ependymal outgrowth, formation of an ependymal tube and channels that facilitate tissue regeneration, and axonal regrowth [43]. Following SCI, a controlled reorganization of ependymal radial glia cells occurs to form an ependymoglial tube in the continuity of the spinal cord’s central canal [44]. An interaction of epithelial and mesenchyme occurs to bridge the gap between the two stumps of the spinal cord [43]. Simultaneously, GFAP-expressing ependymoglial cells along with infiltrating macrophages migrate to the lesion gap and clear cell debris and extracellular matrix (ECM) [45, 46]. During salamander spinal cord regeneration, neuronal repopulation of the lesion gap is extremely important to obtain a fully functional spinal cord. Cell tracking studies suggested the ependymoglial cells that are mobilized into the injury site as progenitor cells that generate several cell types, including neurons and glia [47, 48]. How these progenitor cells were committed to differentiate into neural cells was later reported by Schnapp et al. [49] to be due to signaling of healthy portions of the spinal cord through the expression of specific transcription factors such as Msx1, PAX7 and PAX6. Other studies also suggests that at least some embryonic patterning and differentiative processes are required to fully regenerate the spinal cord and restore its function [50, 51]. This was in fact confirmed by Clarke et al. [52], showing that these cells retained the expression of a transcriptional factor code from the embryonic development, which may be related to the continued neurogenesis observed in the salamanders spinal cord throughout 7 life. Later on, the spinal cord resident Sox2+ progenitor cells were found to establish a gene expression program where downregulation of pro-neural genes and upregulation of proliferation-promoting genes was observed [53]. Additionally, they switched from a neurogenic to a proliferative state, supporting spinal cord outgrowth [54]. This goes in accordance to previous reports showing that spinal cord cells did not proliferate after tail amputation in Sox2+ progenitor cells knockdown models. This resulted in the total absence of spinal cord in the regenerating tail [55], therefore confirming the role of Sox2-dependent progenitor cells to reconstitute different spinal cord cell types in salamanders after SCI [56]. Others have also shown that the regenerating spinal cord cells activate the expression of some genes associated with Wnt and PCP signaling pathways [53, 57], and that also express factors related to tight junctions proteins and to cell-cycle such as MARCKS protein [58]. Successful spinal cord regeneration in salamanders involves not only stem cell growth and neural replacement but also axonal regrowth into the spinal cord lesion. The features of salamander’s neural-ependymal cells contribute to a directed outgrowth of the newly formed neurons.!In addition, descending axons from the healthy spinal cord were found to extend into the lesion site, also contributing to the initial formation of newly spinal cord [59]. This pro-regenerative response of the healthy axons in salamanders might be explained by a miRNA array-based analysis performed by Diaz Quiroz et al. [60]. In this analysis, miR-125b was found to be highly expressed in the spinal cord of these animals after transection, and associated to the downregulation of the axon-repulsive gene semaphoring (SEM)-4D. Additionally, increased levels of miR-125 were correlated with better functional outcome of the transected animals [60]. In summary, salamanders’ ependymal cells with radial glia phenotype seem to be the key cell type for a successful regeneration. But whether this is enough to identify and counteract to what is missing in the mammals and go towards their injury environment is still to be clarified. Xenopus laevis Frog’s ability to regenerate after injury started to be reported around 1915, when Hooker and colleagues showed a physiological and anatomical regeneration of frog larvae following spinal cord transection [61]. Interestingly, Piatt and Piatt later revealed that the same was not observed for adult frogs after spinal cord transection [62]. In light of these observations, the recovery and regeneration of spinal cord after transection in frogs in all stages of development became a matter of study. It is now well recognized that the regenerative capacity of anuran amphibians is highly dependent on metamorphosis [63]. A good example of this class of amphibians is the Xenopus laevis frog. The development of this specie comprises 65 anatomically identifiable stages, according to Nieuwkoop and Faber’s table [64]. In fact, Xenopus 14 15 Figure 2. Vertebrate and Mammal models of research on SCI. Fundamental studies on SCI can be performed in basic models such as vertebrates and more complex models like mammals. These animal models provide important insights on the signature of SCI, and allow to identify potential targets for new therapies. In addition, new treatment methodologies can be tested in these models within a variety of environmental challenges aiming to represent the pathophysiology of SCI as close as possible. [33, 40, 74, 94, 110, 124-129] 1.3.2 Mammal models of SCI Non-mammalian regenerating models can give us insights on the biomolecular and neuromorphological signature of SCI events, as well as on the environmental and neuron-intrinsic and -extrinsic factors involved in the regenerative process. By doing so, potential targets for new therapies can be identified and encourage the development of new treatment approaches. However, it is worth to note that these smaller animal models do not allow to address the heterogeneity of SCI pathology. Therefore, it is still of the utmost importance to keep SCI studies in non-human mammals that share many similarities with human spinal cord. Several animal models are available, including rodents, dogs/cats, pigs and non-human primates. Moreover, different environmental challenges can be addressed by using different patterns of injury (spinal cord contusion, compression, transection, hemissection, dislocation and chemical) [124, 125]. The contusion or compression injuries are preferred [125, 126], because most of human SCI happen due to a blunt trauma to the spinal cord. Contusion, for example, is the underlying SCI cause in 49% of paraplegic patients [127]. While contusion allows to better investigate the pathophysiology of SCI [3, 128] and may also be more appropriate for acute management strategies [129], compression models enables to study post-traumatic spinal cord decompression techniques or neuroprotective treatment [130]. Transection is also a preferred pattern of injury in SCI models. These models provide a clean injury environment, where both ascending and descending spinal tracks are totally interrupted and there is no issue of axon sparing. However, they are rarely encountered in clinics. Nevertheless, transection models are useful in studying processes of axon degeneration and regeneration [131], and neuroplasticity [132134], and are the most suitable for the implementation of a tissue engineering related strategies, in which scaffolds can be easily implanted within the injury site [129]. All of these models can also be used in less or more severe context, by performing partial (incomplete) or complete lesions, respectively [134]. Different levels of trauma (cervical, thoracic, lumbar) [126], and treatment’s time-window, route and frequency of administration, can also be other variants differently 16 tested among the current SCI models. It is also important to establish the time period between the injury itself and the intervention or treatment to be performed. For that, it is necessary to distinguish the acute phase of injury [135, 136] – within hours or days post-injury – from the chronic phase [137, 138]. Rodents Rodents SCI models include both rat and mice. Similar histopathological, behavioral and neurophysiological outcomes after SCI can be found between both species [129, 139], besides sharing a similar genome to human. Nevertheless, rat models are still the most widely used [125, 126], due to their similarity to human SCI pathophysiology, such as the development of large cystic cavities at the lesion site [6, 140]. Rats are, however, quadrupeds and their corticospinal tract is mainly dorsal [141], unlike the biped’s of humans. On the other hand, mice do not exhibit a cystic cavity in the lesion site, but instead a densely packed cellular mass that gradually decreases in size [142, 143]. Moreover, these animals offer the possibility to easily manipulate their genome and thus create transgenic models than can be useful to study cellular and molecular aspects of SCI [140]. The differences between rat and mice regarding the pathophysiology of SCI goes beyond the formation of the cystic cavity in the lesion site. In fact, main differences have been correlated to species-specific variances in the distribution, magnitude and composition of the inflammatory response after injury [144]. After spinal contusion in rats and mice, microglia/macrophages reactions were similar between the two species, but lymphocytes and leucocytes infiltration was different [145]. In addition, mice presented a unique fibroblast-like cells in the injured spinal cord, which was previously thought to be involved in the wound repair by activating T-cells and release neuroprotective cytokines to the injured milieu [145]. Interestingly, reactive astrocytes were also found to be important players in the protection of spinal cord tissue and motor function preservation in transgenic mice with reactive astrocytes ablation after crush injury [146]. The absence of these cells aggravated tissue disruption, cellular degeneration, wound spread and severe motor deficits in the injured animals [146, 147]. This goes in contrast to the reported implication of the reactive astrocytes in the formation of the glial scar in rats after SCI, which has long thought to be detrimental to axonal regeneration and functional improvement [148, 149]. However, this is still a matter of debate, as several evidences attribute a rather tissue-protective effects of this cell population in SCI response [150]. Infiltrating macrophages were also reported to play an anti-inflammatory role in the recovery of SCI in mice, which was correlated with motor improvements of the animals [151]. So far, not only the inflammatory response after SCI in rodents has been a matter of study. Functional 17 analysis techniques as indicator of recovery are also well-established [152], such as behavioral and electrophysiological assessments [124, 153-155]. Evaluation of other biomolecular and cellular events have also been extensively reviewed. Cell necrosis and apoptosis in response to SCI is a good example of that. Crowe et al. [156] and Shuman et al. [157] showed that post-traumatic neural necrosis and oligodendrocyte apoptosis mediated secondary axonal degeneration and chronic demyelination, as a consequence of microglial activation. Other dimensions such as depression and/or anxiety, and pain recently came into light in what regards SCI animal models [158]. It is worth to note that the development of depression and chronic pain are very common among human SCI patients [159, 160], thus requiring appropriate attention to be given to this particular behavior in animal models of SCI. Therefore, the establishment of tools to assess depressive/anxiety-like phenotypes, as well as pain behaviors in both rat and mice has been increasing [158, 161]. In light of the above reported findings, rodents are probably the most suitable animal models for SCI research. The more evident difference between rodents and humans are the size of the spinal cord. The heterogeneity of injury, and the less complex functional organization of rodent nervous system should also be considered [162]. Therefore, the direct translation from rodents to clinics remains under debate. While some believe in the accuracy of rodent models by itself, others keep requiring further investigation in upper, larger animal models, as well as in non-human primates [163-165]. Larger animals and non-human primates Large animal models such as dogs/cats and pigs, and non-human primates are the closest to human researchers can get in pre-clinical setting. However, unlike rodents, that are easy of handle, with a relatively low cost handling [126], the use of larger animals usually involves practical and ethical concerns, so that they are rarely used [133]. Canine/Feline Canine SCI model has been mostly considered for SCI research due its similarities in the mechanisms of injury, classification and functional monitoring to those of human patients. In fact, dogs usually suffer from contusive-compressive injuries as result of road traffic accidents or disc degeneration, at both cervical and thoracolumbar levels [166-168]. Whenever naturally injured dogs are not used, balloon compression injuries are the most preferred injury pattern to use due to it reproducibility [169, 170]. The pathophysiology of canine SCI is also very similar to rodents and humans [171]. In addition, scales to 18 determine canine SCI severity has been established and are very similar to the ASIA scale used for human SCI patients [172]. Finally, functional assessment of dogs has also been established. In this regard, electrophysiological studies have been performed in both acute and chronic SCI dogs [170, 173]. Following severe SCI, dogs usually loose pelvic limb sensation but regain their ambulation, resulting in a phenomenon known as “spinal walking”. Recent electrophysiological recordings tried to correlate the incidence of this phenomenon and pelvic limb sensation with changes in somatosensory and motor evoked potentials, but no apparent association between them was found, suggesting that other factors may be leading to the motor recovery of these animals [173]. Cats also present this phenomenon of spinal walking, even after complete SC transection at the cervical level, if they receive appropriate physical training [174, 175]. The existence of central pattern generators in the spinal cord has been linked to the occurrence of this phenomenon. Contrarily to this, non-human primates and humans are unable to walk after spinal cord complete transection [176, 177] . Alike dogs, cats also frequently suffer from SCI as result of road traffic accident or falls. Most of them occurs at the thoracolumbar and sacrococcygeal level and are usually accompanied by concomitant injuries [178, 179]. Cats rarely die from SCI, and the primary clinical care encompasses spinal and systemic stabilization [180], to avoid the progression to secondary injury, following the procedures that are taken with human SCI patients. Neurological assessment follows with the assessment of multiple spinal lesions and the presence of nociception, to determine the prognosis of the animal [181, 182]. From a more histological point of view, cellular response to cat SCI has been explored since the early 60’s. Bunge et al. [183] was the first to show remyelination events on the adult mammalian central nervous system. However, the author used an experimental injury based on the manipulation of cerebrospinal fluid by withdrawal and reinjection in cat spinal cord, so a limited demyelinating lesion could be produced. Following injury, the demyelinated axons remained intact, and the first evidences of newly formed myelin appeared 19 days post-injury, with all axons showing myelination by 64 days post-injury. This follows the surrounding of axons by glial cells, resembling the mechanism of myelinogenesis [183]. However, these data do not go in agreement with the present evidences on the mechanisms of demyelination and remyelination of mammalian systems. Additionally, the experimental injury model used by this author has nothing to do with the SCI that is usually used in a pre-clinical setting with SCI animal models and observed in clinics. In fact, later in 1983, Blight et al. [184] showed that a contusion injury at mid-thoracic spinal cord of female cats provoked severe neuronal death in the damaged spinal cord at a chronic stage, accompanied by marked and sustained axonal demyelination and selective elimination 19 of large fibers. Most of the animals did not recover their locomotion. For those who did, the recovery of effective locomotion was associated with the maintenance of 5-10% of neuronal population in the lesion site. No spontaneous axonal remyelination was, however, observed. Porcine Adult pig SCI models have been developed over the last years, aiming to provide an intermediate valid model between rodents and humans. Recently, the corticospinal tract in pigs was found to be anatomically similar to human, suggesting the importance of the porcine as a pre-clinical model. One of the first studies on pigs subjected to SCI was reported in 1991 by Blight et al. [185]. In this study, guinea pigs were moderately injured by compression of the spinal cord at the thoracic level, and comparable to contusion injuries in cats regarding the type and distribution of axonal damage. At the chronic stage of injury, some degree of functional recovery was obtained, which correlated with the number of surviving axons in the lesion. Following the time course of the injury, a delayed secondary response to injury was observed, which was suggested by the authors to be due to a slower phagocytic activity of macrophages during the inflammatory response at the injury site [185]. More recently, a sacrocaudal SCI porcine model was developed using minipigs [186]. After sacrocaudal spinal cord transection, marked neurological deficits restricted to animals’ tail was observed. The aim of this study was to use this model for cell transplantation in an acute SCI phase, which at the end proved valid for that effect [186]. In a more severe injury context, Zahra et al. [187] demonstrated that a complete cervical SCI in piglets provoked acute hemodynamic alterations and withdrawal of sympathetic tone. The establishment of more chronic injuries in porcine models has also been described. For example, a chronic complete paraplegia porcine model was wellestablished by Zurita and colleagues [162]. Several procedures of postoperative care in both acute and chronic phase of injury were validated, as well as the treatment of possible complications. Neurological evaluations of the animals were also optimized and established and enabled to assess the development of neuropathic pain and autonomic dysreflexia. Importantly, a new scale for hindlimb motor function assessment was developed based on a modification of the previously reported Tarlov scale [188]. Physical rehabilitation of the hindlimbs and other locomotion-based training were also applied, such as kinesitherapy and daily gait training in a treadmill [189]. Electrophysiological studies were also performed in these animals to assess the integrity of ascending and descending spinal cord tracts, which in the future can be used to confirm the potential of the tested therapeutic approaches in this model [162]. Eletrophysiological and electromyographical studies in several porcine models of SCI had been already 20 described by others [190]. More recently, a new reliable Porcine Thoracic Injury Behavior Scale was developed in order to assess locomotor recovery of minipigs following thoracic SCI contusion [191]. In this study, varied degrees of injury severity were induced, and the locomotor recovery of the animals differed accordingly, and correlated with the extent of SC white and gray matter sparing, as well as neurofilament expression at the epicenter of the lesion [191]. Non-human Primates The need to demonstrate a therapy’s efficacy in non-human primate SCI models before moving with a clinical trial is still under intense debate [163]. Nevertheless, non-human primates are undoubtedly the closest to human that researchers can get in the pre-clinical setting. The anatomical organization of their motor system and the contribution of the descending cortical and subcortical spinal cord pathways in controlling their movement in an injury context corresponds to that found in the other species, such as cats, and humans [192]. This may explain the remarkably spontaneous neuroplasticity of the corticospinal projections in the primate spinal cord after cervical hemissection SCI, which was found to reconstitute 60% of the pre-lesion axon density in the injury site [193]. Motor function recovery of muscle recruitment, hand function and locomotion was correlated with this improved anatomical recovery. The motor recovery in these animals was evaluated by electromyography (EMG) recordings and kinematics during the performance of fine motor tasks and treadmill training [193]. The use of EMG as a measure of impairment and recovery after traumatic injury had also been used by others [194], which demonstrated to be a feasible and safe method to use. The reported improvement on motor recovery of mammals has been associated with mechanisms of spontaneous recovery, either by the use of the spared systems after injury [195], and axonal sprouting or remyelination [132]. In primates, endogenous neurogenesis was also a mechanism suggested to contribute to this, by replacing oligodendrocytes and astrocytes within the lesion area [196]. The newly formed olygodendrocytes were seen to ensheath and remyelinate the host axons. This goes in accordance to other studies, also highlighting the importance of neurogenesis as a compensatory mechanism after injury in mammals through cell replacement [196]. The main goal of SCI research in animal models is, in fact, to extensively characterize all the available SCI animal models and standardize procedures so several interventions and assessments can be comparable among studies, as well as their correlations with molecular, biochemical and histological 21 evidences. Only then the insights given by this understanding over a pathology will enable to move towards the development of strategies that can be further used to control or revert the consequences of that pathology in the clinics. The proximity with humans makes non-human primates excellent models to study the effectivity of certain therapies and its reliability for the clinics. Moreover, the differences between rodents and larger animals such as dogs and cats, and monkeys are significant. As well reviewed by Courtine et al. [197], major differences concern the evolution of the neuroanatomy throughout the evolution of the species, namely the motor cortex and its corticospinal tract (CST) projections. While CST lesions had almost no effect on stepping in rodents, suggesting a poor motor cortex influence in sustaining locomotion in rat and mice [198], monkeys showed marked deficits in stepping after CST damage [199], and humans were unable to walk independently after that [200]. Likewise, different inflammatory and immune responses after SCI were found between rodents and monkeys [201], which could largely influence the cellular and molecular events characteristic of the SCI pathophysiology. These and other differences among species may result in divergent anatomical and functional outcomes. Nonetheless, there are some constrains regarding the use of larger animals, namely monkeys. For instance, proper housing and post-operative care of these animals are under extreme regulation control. Plus, it is not always easy to apply and maintain urinary catheters or incontinence pads on these animals to control the incontinency consequent of the injury [162]. Regarding monkeys, the difficulty in maintaining them in their upper limbs limits the rehabilitation care to passive techniques, specially in cases of complete paraplegia, instead of using active rehabilitation and physiotherapy such as that applied to SCI human patients [202]. On the other hand, the relatively large size of monkeys is obviously more comparable to human than to lower animals, which is not only reflected in the neuroanatomy and physiology of the monkeys, but in other dimensions such as cognition and behavior [197]. In conclusion, all animal models have advantages as well as caveats. The choice on the best model to use highly depends on the specific aims of the study and which outcomes are expected from there, which normally goes from determining the effect of a therapeutic approach in terms of spinal axonal growth, neuronal cell recovery, remyelination and regeneration, and the inflammatory responses after injury and/or treatment, but also to assessment of functional recovery. Fundamental studies on rodents and larger animals should be continued, especially if never-tested therapies are being explored. After that, and before moving to human clinical trials, non-human primates are most likely the unique models that provide ways to truly understand the safety and efficacy of that therapy, and examine their effects on 22 multiple variables. Figure 3. Injury models of SCI. Contusion-, compression-, and transection-based models are the main injury models available to study the pathophysiology of SCI and to test new therapeutic approaches [131, 132]. 2. Cell transplantation: A relevant regenerative approach for SCI treatment The above referred established animal models have been of great importance not only to understand the basic pathophysiology of SCI, but also for the possible development of therapeutic strategies. Indeed, the establishment of therapies for SCI patients that lead to functional improvements is still a priority for researchers and clinicians. The challenge relies in obtaining robust pre-clinical evidence from animal model research before moving to clinics. At the moment, various cellular, molecular and tissue engineering strategies are being studied under different conceptual rationales. Cell therapies have been in the forefront of most SCI regenerative strategies, due to their neuroprotective and/or neuroregenerative potential [203]. In the context of SCI, cells can have the ability to replace the lost ones, promote and guide axonal growth and myelinate newly and resident neurons [203]. Going beyond these functions, many cells secrete an array of trophic factors which give support to the resident cells and modulate the local inflammatory response as well as potentiate plasticity in the spared spinal cord [204-207]. With this plethora of potential mechanisms, a 23 variety of cells from several tissue sources have been investigated for SCI, namely pluripotent and adult stem/progenitor cells, and adult myelinating cells. The vast majority of studies have been conducted in rodent models, but also some in larger mammals or primates. Along this sub-chapter, the most recent and promising pre-clinical studies using cell transplantation on animal SCI models will be addressed. As some of these strategies have reached clinical trials, their translation to human SCI will also be tackled. 2.1 Adult Myelinating Cells Schwann Cells SCs are the myelinating cells of the peripheral nervous system. As so, they can be valuable to use in CNS regenerative strategies. In fact, these cells have been extensively used in SCI animal models, with the first studies concerning SC transplantation being presented by Duncan and colleagues, who showed the ability of these cells to remyelinate demyelinated host axons in both the brain and spinal cord models of demyelination [208, 209]. Since then, several animal models have been used for SC transplantation studies. In a rat SCI model of contusion, the transplantation of SC suspensions supported the extension of sensory and spinal axons into SC grafts, with some of them showing evidence of remyelination, accompanied by improved motor recovery after transplantation [210]. Similar results had also been previously shown by Pinzon et al. [211] in a thoracic transection SCI in rats, in which the transplantation of matrigel channels-containing SCs increased axonal myelination and regeneration through SC grafts, with improved measurable electrophysiological conduction. More recently, SC grafts were shown to support axonal growth, both descending and ascending fibers, in rats with chronic thoracic SCI, correlated with improved motor function of the animals [212]. Interestingly, the genetic modification of SCs to express neurotrophins, namely Brain derived neurotrophic factor (BDNF) and Neurotrophin (NT)-3 and further transplantation into thoracic transected rat spinal cord, was shown to increase axonal regrowth and regeneration [213, 214]. Due to the positive results obtained in animal models, some of this work has been translated to the clinics. For instance, Saberi and colleagues used autologous transplantation of SCs in patients with chronic thoracic SCI. However, there were no beneficial neither adverse effects following treatment [215]. Later in 2016, the Miami Project to Cure Paralysis initiated a phase I clinical trial to evaluate the safety of autologous human SC transplantation in patients with subacute complete SCI (NCT01739023) [216]. No adverse events were found upon SC usage up to one year post-transplantation, proving the safety of both the administration procedure and treatment. No additional spinal cord damage or formation of mass 30 3. MSC secretome: A cell-free based therapy for SCI regeneration 3.1 MSC Paracrine Activity: Secretome vs Transplantation Over the years, MSCs contribution to the repair and regeneration of damaged tissues following SCI has been confirmed in several studies, by supporting host axonal remyelination and outgrowth [304-306] and contributing to overall tissue protection and preservation [307, 308]. Furthermore, these cells present anti-inflammatory, anti-scarring, anti-apoptotic and pro-angiogenic properties in response to SCI [309312]. However, the percentage of surviving MSCs upon transplantation is surprisingly low [276, 313, 314], so long-term engraftment and survival of these cells is very unlikely. This strongly suggests that their neuroprotective and neuroregenerative potential relies mostly on a trophic activity of these cells. In fact, MSCs secrete a broad spectrum of bioactive molecules and/or microvesicles, known as secretome, which is believed to mediate their regenerative properties [315-317]. Therefore, a detailed discrimination of which factors MSCs secrete, and under which conditions, is the key to understand the innate capacity of MSCs to contribute to the injury response. Protein analysis performed on the BM-MSC secretome revealed the presence of IGF-1, hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and TGF-b1, which supported hippocampal neuronal survival and axonal outgrowth in culture [318]. Our group found similar results using the secretome of ASCs and UC perivascular cells (HUCPVCs), which were related to the presence of NGF, VEGF, HGF and stem cell factor (SCF) in their secretome [319]. Likewise, the viability, proliferation and densities of cortical and cerebellar neuronal cultures were found to be increased by the secretome of HUCPVCs [320]. In another study, the secretome of ASCs was reported to protect cortical neurons from inflammation in vitro [321], by increasing TGF-β1 and interleukin (IL)-10 while decreasing TNF-alfa (α), nitric oxide or prostaglandin E2 in culture. Many in vivo studies, although not applying the secretome itself, have explored gene and protein expression in the transplanted cells following SCI. For instance, in a rat SCI model, the expression of human neutrophil-activating protein2 (NAP-2), NT-3, bFGF, glucocorticoid induced tumor necrosis factor receptor (GITR) and VEGF-receptor 3 (VEGFR-3) by Wharton’s Jelly UC-MSCs was related to an overall improvement of injured animals [322]. Another evidence was shown after the transplantation of HUC-MSCs in rats after SCI, where the neurotrophic factors GDNF and VEGF were detected in animals’ spinal cord, possibly explaining the improvements observed in neurological functions [323]. NAP-2, NT-3, bFGF, GITR and VEGF were also detected in the spinal cord of completely transected rats following Wharton’s Jelly UC-MSCs, accompanied by axonal growth and regeneration, and locomotor improvements of the treated animals [324]. 31 Altogether, these data indicate that MSCs might engage in a paracrine profile, providing a favorable environment for neuroregeneration after SCI. In recent years, the progress in discriminating the composition of MSC secretome triggered the idea of using the secretome of MSCs therapeutically, in alternative to cellular transplantation. In line with this, pre-clinical studies concerning the use of MSCs secretome were first shown by Cantineaux et al. [325] with the intrathecal delivery of BM-MSCs secretome into the spinal cord of rats after SCI contusion. The secretome presented a pro-angiogenic character, promoting a reduction in cystic cavities and leading to functional recovery. No effect on axonal regrowth was observed though, neither on modulation of the inflammatory response after injury. These results were confirmed by exploring the properties of BMSC secretome in vitro , performed simultaneously by the authors [325]. Similar results were later reported by others [326, 327]. Kanekiyo et al. [326] and Cizkova et al. [327] have shown that repeated intrathecal injections of BMSC secretome in rats after SCI contusion induced interesting levels of functional recovery. This was correlated with increased regeneration of the damaged tissues, indicated by higher axonal densities throughout the injury site, spared spinal cord tissue, axonal remyelination and enhanced GAP-43 expression [326, 327]. In contrary to Cantinieaux et al. [325], Cizkova and colleagues reported attenuated inflammation following BMSC secretome treatment, further confirmed by decreased levels of pro-inflammatory markers, namely IL-6 and TNF-a [327]. All these promising data suggest that the secretome of MSCs by itself is able to promote neurological improvements after SCI and provide means for spinal cord repair after injury. Therefore, the therapeutic application of MSC secretome seems to provide a safe and viable alternative to cell transplantation for SCI treatment. Nevertheless, the extensive characterization of MSC secretome pointed to significant distinct secretory profiles of MSCs [328, 329], which seems to vary according to the experimental conditions being used [330-332], as well as to the intrinsic properties of the cells, and to the different available donor and tissue-sources [333, 334]. These variances may have impact on their efficacy, therefore should be carefully considered. 3.2 MSC secretory profile from a mechanistic point of view Differences among MSC populations go beyond donor and experimental disparity [333], and include tissue-source variations in respect to cell-surface markers expression, yield and expandability, differentiation patterns, and epitope profile [292, 335, 336]. Thus, it would be expected that different tissue-source MSCs have distinguishable secretory patterns as well as different therapeutic profiles. This has been in fact confirmed over the years. Hsiao and colleagues provided a comparative analysis on the 32 expression of several factors in human BM-MSCs, ASCs and Dermal tissue-derived MSCs [329]. VEGF-A, angiogenin, bFGF, and NGF were expressed by all the MSC populations at comparable levels, whereas IGF-1, VEGF-D, and IL-8 were preferentially expressed by ASCs, which contributed to a better performance of this cell population on angiogenesis [329]. More recently, we have also found different patterns and composition on the secretome of BM-MSCs, ASCs, and HUCPVCs [337], which might explain differential levels of axonal growth on DRGs observed among the three cell populations [338]. These data were consistent with previous work from our group showing that different sets of growth factors were being secreted by HUCPVCs and ASCs under the same culture conditions, which acted differently on neuronal cell density and metabolic activity [339]. Interestingly, when triggered by inflammatory stimuli, UCB-MSCs showed higher anti-inflammatory effect than ASCs and BM-MSCs. Besides variations in the secretory profile among MSC populations, the same population can also alter the production of factors when exposed to different agents. For instance, ASCs were found to increase the levels of VEGF, HGF and IGF1 in response to an inflammatory stimulus [340]. Hypoxia also triggered prominent production of VEGF when compared to normoxia conditions, providing a more angiogenic and anti-apoptotic activity of MSC secretome in a mice model of ischemia [341]. The differences among MSC populations should not be considered a problem upon the use of MSC secretome for SCI therapeutic applications. On the contrary, the progress made in the determining the exact content of MSC secretome enables to discriminate the corresponding therapeutic impact along with the putative mechanisms being activated or controlled by it. These are important insights, as it might give the opportunity to achieve the most suitable cocktail of paracrine factors for a specific context of injury in future studies. Plus, it should be emphasized that the response of MSCs to the environment offer ways to manipulate and control the features of the secretome, an advantage not provided by cellular transplantation strategies. 33 Table 1. Studies of stem cell-based regenerative strategies for SCI repair and regeneration. SCI model Injury Strategy Administration route Administration Frequency & Timing Main achievements Ref Schwann Cells Rat Contusion - T9 Transplantation of SCs suspensions Intraspinal injection - epicenter 2x106 cells; once, 7 dpi Sensory and spinal axons outgrowth and remyelination; Motor function improvement [342] Transection - T8 Implantation of SCs-containing matrigel channels in SCI site Local application within a guidance channel N/A; once, immediately post-injury Axonal myelination and regeneration; improved axonal electrophysiological conduction [211] Chronic contusion - T9 SCs transplantation Intraspinal injection - epicenter 2x106 cells; once, 8 wpi Axonal growth of descending and ascending fibers; motor function improvement [212] Transection - T8 Transplantation of genetically modified SCs to express BDNF and NT-3 Local application and intraspinal caudal injection 1x106 cells; once, immediately post-injury Axonal regrowth and regeneration [343] Patients Chronic midthoracic injury Autologous SCs transplantation Intraspinal injections - syrinx 3 to 4.5x106 cells; once, > 6 mopi No beneficial nor adverse effects [215] Subacute complete Autologous SCs transplantation - Phase I clinical trial for safety studies (MIAMI Project) Intraspinal injection - epicenter 5 to 15x106 cells; once, 4-7 wpi No adverse effects up to 1-year posttransplantation; No additional spinal cord trauma; No formation mass lesions. Safe administration procedure and treatment [216] Chronic thoracic and cervical Autologous SCs transplantation - Phase I clinical trial for safety studies N/A N/A Ongoing N/A Olfactory Ensheathing Cells Rat Hemissection - C1/C2 OECs transplantation Intraspinal injection - epicenter N/A; once, immediately post-injury CST fibers regeneration; motor function improvement [221] Complete transection - T9 OECs transplantation + treadmill training Intraspinal injection – rostral/caudal to lesion 4x105 cells; once, immediately post-injury Motor improvement; no regeneration of serotonergic fibers bellow SCI site [223] Transection - T9/T10 Primate-derived OECs transplantation Intraspinal injection – rostral/caudal to lesion 4x105 cells; once, immediately post-injury Modest regeneration of 5-HT fibers; no CST regeneration [224] Dorsal hemissection - T11/T12 OECs transplantation Local application and intraspinal rostral/caudal injections 4x105 cells; once, 4 wpi No rubrospinal nor CST axonal regeneration [225] 34 Patients Chronic complete injury - T4-T7 Autologous OECs transplantation - Phase I/IIa clinical trial Intraspinal injections Once, 18-32 mopi No adverse effects up to 3 years posttransplantation; No motor improvements [228, 344] Chronic complete transection/compr ession – T3-T11 Autologous OECs transplantation in three patients + neurorehabilitation Intraspinal microinjection – rostral/caudal to lesion; around lesion site 3x104-20x104 cells; 120-212 injections; 1.3-5 ypi Functional improvement in 2 out of 3 patients - upgraded ASIA scale from A to B and C; Motor and sensory amelioration in 1 of 3 patients with no upgraded ASIA scale. [229] Embryonic Stem Cells Rat Contusion – T8/T9 Transplantation of neuronand glial-restricted precursor cells derived from ESCs + single dose of MP (10 min postinjury; Immunosuppression with cyclosporine A (3dpi and up to 2wpi) Intraspinal injections – rostral/epicenter/caudal 1x105 cells; once; 9dpi Motor improvement; Bladder function improvement; descending axonal outgrowth; ESCs differentiation into neuronand glial-like cells [234] Contusion – T9T10 ESCs-derived NPCs transplantation Intraspinal injections 1x106 cells; once; 9dpi ESCs-derived NPCs survival and differentiation into neural lineages; Motor function improvement [345] Induced demyelination – T10 ESCs-derived OPCs transplantation Intraspinal injections – dorsal column white matter 1,25x105 cells; once; 3dpi Axonal remyelination; motor function improvement [346] Patients Subacute complete thoracic ESC-derived OPCs transplantation - safety studies (Geron Corporation) N/A 2x106 cells; once; 7-14dpi No observations of positive effects; discontinuity of the program [237] Subacute cervical ESC-derived OPCs transplantation - Phase I/Iia clinical trial for safety studies (Asterias Biotherapeuthics) N/A N/A Results to be provided N/A Induced Pluripotent Stem Cells Mice Compression – T6 Transplantation of iPSCsderived NSPCs; Immunosuppression with cyclosporine A Intraspinal injections – rostral/caudal to lesion 5x104 cells; once; 7dpi Axonal remyelination; motor function improvement [252] Rat Mild-contusion – T10 Acute transplantation of iPSCsderived OPCs Intraspinal injections - epicenter 5x105 cells; once; 9dpi Axonal remyelination; serotonergic innervations; synaptic contacts [253] 35 establishment; cavity size and glial scar reduction; motor and electrophysiological improvement Nonhuman primate Contusion – C5 Transplantation of murine and human iPSCs-derived NPCs; Intraspinal injections - epicenter 1x106 cells; once; 9dpi Grafted cells survival and differentiation into neural cells; Axonal outgrowth; angiogenesis; Motor function improvement [254] Neural Stem/Progenitor Cells Mice Dysmyelination Adult NSPCs transplantation into transgenic mice + delivery of bFGF/EGF/PDGF-AA Intraspinal injections for cell transplant – epicenter; Intrathecal injections for growth factors – lesion site 5x104 cells; twice; immediately post-injury NPCs differentiation into oligodendrocyte-like cells; robust axonal ensheathment and myelination; Improved axonal conduction [261] Rat Demyelination and dysmyelination – T8/T9 Transplantation of Spinal cordderived NSCs Intraspinal injections – lesion site 1x105cells; once; 3dpi NSCs differentiation into myelinating OECand SC-like cells [262] Compression – T7-T8 Adult spinal cord-derived NPCs transplantation + Immunosuppression with cyclosporine A (daily) Intraspinal Injections – midline of spinal cord/rostral/epicenter/caudal 2x105cells; once; 1 wpi Modest change in trophin expression in the spinal cord of NGF, LIF, IGF-1, TGFb1; Co-infusion of trophins led to increased levels of trophic factors: BDNF, bFGF, NGF, CNTF, IGF-1, LIF [264] Adult brain-derived NPCs + delivery of EGF/bFGF/PDGF-A + Immunosuppression with cyclosporine A (daily) + minocycline (10 days) Intraspinal injections for NPCs – 2 midlines/rostral/epicenter/caudal; intrathecal injections for growth factors 4x105 cells; once; 2 wpi Nonhuman primate Contusion – C5 NPCs transplantation into the spinal cord + immunosuppression with cyclosporine (daily, up to 8wpi) Intraspinal injection - epicenter 1x106cells; once; 9dpi NPCs survival and differentiation into neural cells; injury cavities decrease; motor function improvement [263] Patients Chronic incomplete and complete cervical and thoracic NSCs transplantation - Two Phase II clinical trial for safety and preliminary efficacy (Stem Cells Inc.); Patients temporary immunosuppression Intramedullary perilesional injections Thoracic cohort: Up to 4x106 cells; once Cervical cohort: 2x106 cells; once Positive sensory improvement; Benefits of cell transplantation disappeared upon immunosuppression withdrawal [347] Chronic cervical and thoracic Human spinal cord-derived NSCs (Neural Stem Inc.) N/A N/A Recruiting; Results expected by 2020 N/A 36 Mesenchymal Stem Cells Rat Hemicompression – T8/T9 Acute and sub-acute human UC-MSCs transplantation + immunosuppression with cyclosporine (daily) Intravenous injection 1x106 cells; once; 1 dpi or 5 dpi UC-MSCs migrated to the lesion site; motor function improvement in the acute phase [287] Compression ASCs transplantation + Immunosuppression with cyclosporine A (24h priorinjury and daily post-injury) Intraspinal injections – rostral central caudal part of lesion site 5x105 cells; once; 7dpi Motor function improvement; apoptosis and astrogliosis decrease [290] Contusion – T10 ASCs transplantation Intravenous injection 2.5x106 cells; once; 8dpi Motor function improvement; migration of ASCs to injury site (homing); reduced lesion cavity; CINC-1 secreted by ASCs contributed to cell survival and functional recovery. [291] Hemissection – C3/C4 ASCs transplantation (ASC incubation with bFGF prior transplantation) + Immunosuppression with cyclosporine A (daily) Intraspinal injection – rostral/caudal to lesion 5x104 cells; once; immediately post-injury Expression of BDNF, VEGF and FGF-2; 5HT axonal outgrowth and sprouting throughout the spinal cord; apoptosis and astrogliosis decrease [348] Complete transection Transplantation of UC-MSCs from wharton's jelly Intraspinal injection – rostral/caudal to lesion 5x105 cells; once; immediately post-injury Axonal growth and regeneration and motor improvements mediated by NAP2, NT-3, bFGF, GITR and VEGFR-3; [322] Contusion – T9 Transplantation of BM-MSCs overexpressing BDNF Intraspinal injection - epicenter 3x105 cells; once; 7dpi Reduced lesion cavity; increased spared white matter; axonal outgrowth; Increased levels of trophic factors: BDNF and GDNF [323] Contusion – T8T10 BMSCs Secretome administration Intrathecal injection BMSC secretome: 10µl; once; immediately post-injury Angiogenesis; cystic cavities reduction; functional recovery; no axonal regrowth [325] BMSC secretome: 10µl/h for 2 weeks Axonal densities increase; astrocytedevoid lesion site; Motor function improvements [326] BMSC secretome: 30µl at 1, 5, 9 and 13 dpi. pared spinal cord tissue; enhanced GAP-43 expression; attenuated inflammation (decreased IL-2 and -6 and TNF-a, and increased CNTF and VEGF); Motor function improvements [327] Pig Compression – T12-T13 Autologous BM-MSCs transplantation + rehabilitation (daily) Intraspinal injections – lesion site and adjacent subarachnoid space 15x106; twice; 3mopi Axonal outgrowth in neo-formed tissue; axonal conduction recovery; motor function improvement [188] 37 Dog Compression – L4 Transplantation of canine ASCs transplantation + MPSS Intravenous injections of ASCs and MP ASCs: 10x106 cells; Daily for 3 days; 6hpi MPSS: 5.4mg/kg/hour; 47h; 6hdpi Migration of ASCs to the lung, spleen and injured spinal cord; Motor function improvement; Antioxidative and antiinflammatory molecules increased; Several side effects of MPP; [294] Compression - L4 Transplantation of canine ASCs overexpressing Heme oxygenase-1 Intraspinal injections – rostral/epicenter/caudal 10x106 cells; once; 1wpi Decreased fibrosis and microglial cell infiltration; motor function improvement [295] Compression – L4 Transplantation of canine ASCs and chondroitinase ABC (chABC) Intraspinal injections – rostral/epicenter/caudal ASCs: 10x106 cells; once; 3wpi chABC: 5U/mL Increased expression of digested CSPGs, bIII-tubulin and neurofilament; Increased levels of COX2 and TNF-a; Significant motor function improvement [296] Thoracolumbar ASCs transplantation + Electrotherapy ASC injection by lumbar puncture; Subcutaneous electrical stimulation ASCs: 1x107 cells; once; 624 mopi. Electrotherapy: voltage 30mV, frequencies 5/10/15/20Hz; 5min/Frequency Motor function improvement [298] Patients Subacute and chronic BM-MSCs transplantation - Clinical trial for safety studies; Physical therapy Intraspinal – in and around lesion epicenter; directly into spinal canal; intravenous injections 4x108 cells; once; minimum 1ypi AIS grades, Barthel index and bladder function improvements; decreased spasticity; no tumor formation and infection; no increased pain. Multiple routes of administration feasible and safe [299] Chronic cervical BM-MSCs transplantation - Phase II/III clinical trial for safety and efficacy studies (Pharmicell Co.) intramedullary and intrathecal 1.6x107 cells (intramedullary route); 3.2x107 cells (intrathecal route); once; immediately post-injury Results expected in 2020 N/A Thoracolumbar – T10-L1 Transplantation of human UCMSCs from wharton's jelly + rehabilitation - clinical application Intraspinal injections through lumbar puncture 4x107 cells; once; 21mopi Motor and activity improvement; tactile sensation and algesia amelioration; bladder and bowel function improvement; no adverse effects [300] [301] Subacute, and early and late chronic Human UC-MSCs transplantation - phase I/II clinical trials for safety and efficacy studies Intrathecal Monthly, for 4 months Results expected in 2021 N/A chronic ASCs transplantation - Phase I clinical trial for safety studies (CELLTOP study, Mayo Clinic) Intrathecal in cerebrospinal fluid Single dose Results expected in 2023 N/A 38 4. Concluding Remarks The management of SCI and it devastating effects on motor and sensorial control in patients concerns a huge challenge in neuroscience research. Preclinical and clinical investigation has been trying to address this, but there is still no effective treatment showing functional recovery of patients. This is mostly due to the complexity of the central nervous system, which contains the spinal cord, regarding their anatomy and physiology, cell population content and neural circuitry. Therefore, injury to the spinal cord of humans has acute outcomes, followed by secondary pathophysiological events that lead to the progression of the lesion until complete and permanent loss of sensation and motor function that occurs bellow the injury level, establishing an irreversible chronic lesion. It is widely known that the spinal cord of humans lacks the capacity to regenerate after injury, in contrast to that of vertebrates, such as the amphibians and fish, which presents an extraordinary ability to fully regenerate. Thus, the investigation on the regenerative capacity of those vertebrates allow us to identify and understand the mechanisms and features of spinal cord regeneration that lack in human and that may be translatable to clinics. On the other hand, mammalian models that share the inability to regenerate with humans, such as rodents, pigs and nonhuman primates, are of the utmost importance to study the regeneration of the spinal cord along with testing the therapeutic efficacy of a given therapy in a loss-of-function paradigm. While the insights given by the more basic models may be directed to the understanding of the pathophysiology of SCI and thus lead to more preventive strategies, larger models provide means to follow the cure of SCI in more applied and translatable systems. Ideally, the key to develop new therapies for SCI patients with functional improvements would be to focus in combining both resources. However, robust pre-clinical evidences should first be obtained so that it can be translated to clinics, which remains a challenge. Nevertheless, the application of stem cells to SCI have been shown promising in many studies. Their therapeutic role has been mostly attributed to a neuroprotective and neuroregenerative activity towards the repair of damaged tissues. Among the variety of cell populations available, MSCs have been shown interesting. These cells have an innate capacity to respond to injury by stimulating the endogenous repair of spinal cord. 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McDonald, J.W., et al., Transplanted embryonic stem cells survive, differentiate and promote recovery in injured rat spinal cord. Nat Med, 1999. 5 (12): p. 1410-2. 346. Liu, S., et al., Embryonic stem cells differentiate into oligodendrocytes and myelinate in culture and after spinal cord transplantation. Proceedings of the National Academy of Sciences U S A, 2000. 97 (11): p. 6126-6131. 347. Levi, A.D., et al., Emerging Safety of Intramedullary Transplantation of Human Neural Stem Cells in Chronic Cervical and Thoracic Spinal Cord Injury. Neurosurgery, 2018. 82 (4): p. 562-575. 348. K., K.M., et al., The Therapeutic Effects of Human Adipose-Derived Stem Cells in a Rat Cervical Spinal Cord Injury Model. Stem Cells and Development, 2014. 23 (14): p. 1659-1674. 64 CHAPTER II EXPLOITING THE IMPACT OF THE SECRETOME OF MSCS ISOLATED FROM DIFFERENT TISSUE SOURCES ON NEURONAL DIFFERENTIATION AND AXONAL GROWTH Rita C. Assunção-Silva, B. Mendes-Pinheiro, P. Patrício, L. Behie, F.G. Teixeira, L. Pinto, A.J. Salgado Biochimie DOI: 10.1016/j.biochi.2018.07.026 2018 65 EXPLOITING THE IMPACT OF THE SECRETOME OF MSCS ISOLATED FROM DIFFERENT TISSUE SOURCES ON NEURONAL DIFFERENTIATION AND AXONAL GROWTH Rita C. Assunção-Silva1,2,3, B. Mendes-Pinheiro1,2, P. Patrício1,2,3, L. Behie4 F.G. Teixeira1,2, L. Pinto1,2,3, A.J. Salgado1,2 1Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus de Gualtar, 4701-057 Braga, Portugal 2ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal 3BnML, Behavioral and Molecular Lab, Braga, Portugal 4Pharmaceutical Production Research Facility, Schulich School of Engineering, University of Calgary, 2500 University Drive NW, Calgary, Alberta, Canada R C Assunção-Silva: [email protected] B Pinheiro: [email protected] P Patrício: [email protected] F G Teixeira: [email protected] L Pinto: [email protected] A J Salgado: [email protected] 66 ABSTRACT Cell transplantation using Mesenchymal stem cell (MSC) secretome have recently been presented as a possible free-based therapy for CNS related disorders. MSC secretome is rich in several bio-factors that act synergically towards the repair of damaged tissues, thus making it an ideal candidate for regenerative applications. Great effort is currently being made to map the molecules that compose the MSC secretome. Previous proteomic characterization of the secretome (in the form of conditioned media - CM) of MSCs derived from adipose tissue (ASC), bone-marrow (BMSC) and umbilical cord (HUCPVC) was performed by our group, where proteins relevant for neuroprotection, neurogenic, neurodifferentiation, axon guidance and growth functions were identified. Moreover, we have found significant differences among the expression of several molecules, which may indicate that their therapeutic outcome might be distinct. Having this in mind, in the present study, the neuroregulatory potential of ASC, BMSC and HUCPVC CM in promoting neurodifferentiation and axonal outgrowth was tested in vitro , using human telencephalon neuroprogenitor cells and dorsal root ganglion explants, respectively. The CM from the three MSC populations induced neuronal differentiation from human neural progenitor cells, as well as neurite outgrowth from dorsal root ganglion explants. Moreover, all the MSC populations promoted the same extent of neurodifferentiation, while ASC CM demonstrated higher potential in promoting axonal growth. Keywords Mesenchymal Stem Cells, Secretome, Neuroregulatory Factors, Neurodifferentiation, Axonal outgrowth, Cell-free based CNS therapy 67 1. INTRODUCTION An extensive body of literature suggests that Mesenchymal stem cell (MSC)-mediated paracrine activity plays a role in promoting tissue repair. MSCs were firstly identified by Friedenstein as multipotent stem cells characterized by the capacity to self-renew, to adhere to plastic and colonize, and to differentiate into three mesodermal cell lineages [1]. However, the real interest around MSCs is their contribution towards regeneration of tissues upon injury. In fact MSC-based therapies have been used in the context of several neurodegenerative diseases, where neuronal survival has been reported in animal models of stroke [2] and traumatic brain injury (TBI) [3]. Additionally, the recovery of motor function of induced models of Parkinson’s disease (PD) [4] and SCI, with an observed remyelination and reconnection of the neural circuitry [5, 6], has also been observed upon MSCs administration. Direct evidences attribute the regenerative potential of MSCs to their ability to secrete several biomolecules and trophic factors, namely neurotrophic growth factors, chemokines, cytokines, and extracellular matrix proteins, as well as extracellular vesicles, that might be relevant in a clinical setting [7-10]. In the context of neuroregeneration, pre-clinical and clinical findings show that these molecules can directly stimulate the recruitment, proliferation and differentiation of the endogenous cells [11, 12]. Additionally, they can regulate local mechanisms such as apoptosis, scarring and revascularization, as well as modulate immune and inflammatory responses [9, 13], thus contributing to reduce tissue damage. Recent insights on the effective therapeutic role of MSC-secreted bio-factors, especially given the fact that MSCs have a limited engraftment and survival rate when delivered into a damaged tissue [14], opens the possibility of using MSC secretome as a cell-transplantation free based regenerative therapy. In fact, the therapeutic application of the secreted molecules in replacement of stem cells presents enormous advantages as it should minimize stem cell-related ethical and immune-compatibility issues [15], as well as allow a precise dosing and localized delivery to the damaged tissues in a minimally invasive manner [16]. The perspective of safer and more effective strategies motivated further investigation towards the identification of the molecules composing the cell secretome, rather than only looking to its overall regenerative effects. In this line, proteomic profiling of MSC conditioned media (CM) became intensively explored in recent years [17-21]. While valuable tools for MSC secretome characterization were being used, several studies highlighted significant differences between different tissue-sources of MSCs. In fact, the heterogeneity of MSCs residing in different tissues has been reported some time ago [10]. So far, the best characterized and the most studied sources of adult MSCs are obtained from the bone-marrow (BMSCs), adipose tissue (ASCs) 68 and umbilical cord. Several studies provided us with distinct characteristics of each population, that goes from different expression of cell surface markers [22], to specific differentiation processes [23] and immunomodulatory functions [24]. A relevant point of interest that arises from a collective analysis on this heterogeneity studies is the fact that different tissue sources of MSCs are likely to have different secretion profiles [19, 25]. For instance, we have previously shown that the exposure of primary cultures of hippocampal neurons to the CM of ASCs and Human Umbilical Cord Perivascular Cells (HUCPVCs) had different effects on cell proliferation and metabolic activity [26]. Hsieh and colleagues also found that MSCs isolated from Wharton’s jelly secreted more factors related to angiogenesis and neurogenesis than BMSCs, which improved neural differentiation and migration and decreased cell apoptosis in an in vitro model of acute ischemic stroke [27]. Considering the existence of such differences on the secretome composition of MSCs obtained from different tissue-sources, the choice of the best MSC population for a particular application must be determined according to their characteristics and secretory profile. Thereafter, our group found that it is crucial to perform a detailed mapping of the CM obtained from BMSCs, ASCs and HUCPVCs. In a recently published proteomic analysis, we have shown that all these populations were able to secret important factors known to be involved in processes of several CNS disorders/injuries [28]. Moreover, the pattern and composition of ASCs, BMSCs and HUCPVCs CM differed, a fact that could indicate a certain degree of specificity towards different CNS related conditions. After this, it remains unclear if these different secretion profiles could activate distinct mechanisms by which the repair and regeneration of tissues from the nervous system may be regulated. Based on these results, the present study proposes to further explore the previously analyzed MSC CM potential in promoting the differentiation and axonal growth of neural populations in vitro . 2. MATERIALS AND METHODS 2.1 Cell Culture 2.1.1 Human bone marrow mesenchymal stem cells (BMSCs), adipose tissue derived stem cells (ASCs), and Human umbilical cord perivascular cells (HUCPVCs) BMSCs (Stem Cell Technologies, Grenoble France) were thaw and expanded according to protocol established in our lab [29]; ASCs were kindly provided by Professor Gimble (Pennington Biomedical Research Center/Tulane University, USA); and HUCPVCs were kindly provided by Professor Davies (University of Toronto, Canada). ASCs and HUCPVCs were isolated as previously described [30, 31]. Cells were cultured and maintained in α-MEM medium (Invitrogen, USA) supplemented with sodium bicarbonate (NaHCO3; Merck, USA), 10% of fetal bovine serum (FBS; Biochrom, Germany) and 1% 69 Penicilin-Streptomycin antibiotic (P/S; Invitrogen, USA). Upon confluence, cells were enzymatically dissociated with 0.05% trypsin/EDTA (Invitrogen, USA), re-plated at a density of 4000 cells/cm2 and maintained at 37ºC, 5% humidified CO2, 95% air and 90% relative humidity. 2.1.2 Human Telencephalon Neural Progenitor Cells (htNPCs) htNPCs were isolated from 10-week human fetus telencephalon region, as previously described [31]. Ethical consent was approved by the Conjoint Health Research Ethics Board (CHREB), University of Calgary (ID: E-18786). htNPCs were thaw and plated in Nunc T-25 flask containing 5 mL of a serum-free medium PPRF-h2, described in detail by Baghbaderani et al. [32]. Cells were maintained in culture for 48h, during which aggregated into neurospheres. After this time, htNPCs were mechanically dissociated and re-plated into fresh medium. Every 4 days, 40% of the medium was replaced by fresh. 2.2 Conditioned media (CM) collection The CM of ASCs, HUCPVCs and BMSCs was collected from cells in passage 5. For that, cells were plated at a density of 4000 cells/cm2, and allowed to grow for 72 hours. After this, cells were washed 5 times with PBS without Ca2+ and Mg2+ (Invitrogen, USA), and once with the conditioning medium. For neurodifferentiation experiments, Neurobasal A medium (Invitrogen, USA) supplemented with 1% Kanamycin (Invitrogen, USA) was added to the cells. For axonal growth assays, Neurobasal medium (Invitrogen, USA) supplemented with 1% Pen-Strep was used. After 24h of conditioning period, the CM was collected and frozen at -80ºC until used. 2.3 htNPCs culture with MSC CM For neurodifferentiation experiments, htNPCs were enzymatically dissociated with 0.05% trypsin-EDTA, and plated onto glass coverslips pre-coated with poly-D-lysine hydrobromide (100 μg/mL; Sigma) and laminin (10 μg/mL; Sigma) at a density of 5.5 x 104 cells. Cells were maintained in culture for 5 days with the CM collected from the three MSC populations, at 37°C, 5% CO2, 95% air and 90% relative humidity. htNPCs culture with Neurobasal-A medium supplemented with 1% of kanamycin was used as control. 2.4 Isolation and culture of dorsal root ganglion (DRG) explants with MSC CM Dorsal root ganglion explants were used for axonal growth experiments. For that, DRGs from 5 days-old neonatal Wistar-Han rat pups were dissected as previously described [33]. Briefly, DRGs from cervical 70 and thoracic regions of the spine of neonatal rat pups (P5) were dissected and the remnants of peripheral nerve processes were cleaned. The explants were placed on top of collagen hydrogels, prepared as previously described [34] and incubated with MSC CM for 7 days. DRG cultures in collagen gels in Neurobasal Medium supplemented with B27, L-glutamine, glucose and 1% of P/S was used as control. 2.5 Immunostaining htNPCs. htNPCs were fixed, washed, and blocked as previously described [4]. The following primary antibodies were then used: rabbit anti-doublecortin (DCX; 1:500, Abcam, Cambridge, MA, USA) to detect immature neurons, and mouse anti-rat microtubule associated protein-2 (MAP-2; 1:500, Sigma) to detect the mature ones, for 1h at 37ºC. After washing, samples were incubated with the secondary antibodies Alexa Fluor 488 goat anti-rabbit immunoglobulin G (IgG, Life Technologies) and Alexa Fluor 594 goat antimouse immunoglobulin G (IgG, Life Technologies) for 1h at 37°C. Further incubation with 4-6-diamidino2-ph enylindole-dihydrochloride (DAPI; Life Technologies) was performed for 10 min at Room Temperature (RT). Samples were then observed under an Olympus BX-61 Fluorescence Microscope (Olympus, Hamburg, Germany). DRGs. For the immunocytochemistry (ICC) of DRGs, the following antibodies were used: Mouse monoclonal anti-human neurofilament 200 kDa (Millipore) as the primary antibody and Alexa fluor 488 goat antimouse IgG (Invitrogen) as the secondary antibody. DRGs were fixed with 4% paraformaldehyde (PFA) in PBS for 45 min at RT and washed with PBS. A further incubation 0.3% Triton X-100 (Sigma, USA) for 10 min at RT was used for cell permeabilization, and washing with PBS. Samples were then incubated with a blocking buffer solution [PBS containing 10% fetal bovine serum (FBS)] for 90 min at RT, after which they were incubated with the primary antibody (diluted 1:200 in PBS solution with 10% FBS) for 48 h at 4 °C. After washed with a PBS solution containing 0.5% FBS, samples were incubated with the secondary antibody (diluted 1:1000 in PBS/0.5%FBS solution) overnight at 4°C. After PBS washing, DAPI (1 μg ml−1; Invitrogen) was added to the samples for 10 min to stain cell nuclei. Samples were finally washed and maintained hydrated in PBS for fluorescence microscopy analysis (Olympus BX-61 Fluorescence Microscope, Olympus, Hamburg, Germany). 2.6 Neurodifferentiation Assessment Neurodifferentiation of htNPCs was inferred by qRT-PCR for NeuroD1 (ND1) and bIII-Tubulin (bIII-Tub), which protocol is described in the next sub-section, and by the number of Doublecortin (DCX) and 71 Microtubule associated protein (MAP-2) expressing cells. For this purpose, three coverslips and ten representative fields per condition were chosen and imaged using a fluorescence microscope as referred above. To normalize the data between the different experiments, the results are presented in percentage (%) of cells. This was calculated by counting the number of cells with positive staining for DCX and MAP2 markers, and dividing this value by the total number of cells/field (DAPI-positive cells; n=3). 2.7 Protein Association Network analysis and quantitative Real time PCR For neurodifferentiation experiments, the mRNA expression levels of selected genes of interest (Table 1) were measured by quantitative real time polymerase chain reaction (qRT-PCR), after htNPCs incubation with MSC CM. These genes were selected based on protein-protein interaction networks from the previously identified biomolecules in the MSC CM [28] using STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) bioinformatics tool. The neurodifferentiation markers NeuroD1 and Tubulin beta 3 Class III (TUBB3) were also used to assess the neurodifferentiation stage after incubation of htNPCs with MSC CM. The oligonucleotide primers for the target genes were designed using PrimerBLAST software (NCBI). The real time reactions were performed in an Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems, LLC, CA, USA) using PerfeCTa SYBR Green SuperMix, Low ROX (Quanta Biosciences). Target gene expression levels were normalized against the housekeeping gene Beta2-microglobulin (B2M), and presented as fold-change of mRNA levels compared to the Control group. The 2-ΔΔCT method was used to calculate fold-change levels. Table 1. Forward and reverse sequences of oligonucleotide primers used in the qRT-PCR, and the respective gene symbol, name and product size. Gene symbol Gene name Primer sequence 5’–3’ forward=reverse Product Size (bp) TUBB3 Tubulin beta 3 class III Fw GGC CTC TTC TCA CAA GTA CG Rv CCA CTC TGA CCA AAG ATG AAA 317 NeuroD1 Neuronal differentiation 1 Fw CCG TCC GCC GAG TTT G Rv GCG GTG CCT GAG AAG ATT G 173 FLT1 Fms-related tyrosine kinase-1 Fw CTG GGC AGC AGA CAA ATC CT Rv AAA AGT CAC ACC TTG CTT CGG 113 NRP1 Neuropilin 1 Fw CGC AAG GCG AAG TCT TTT GA Rv TGT GAG CTG GAA GTC ATC ACC 265 78 of proteins with neuroprotection, neurogenic, neurodifferentiation, and axon guidance and growth functions was identified. The three MSC populations differed in their secretion profile, posing the question of whether their neuroregulatory action may differ accordingly. Therefore, the study herein presented aimed at evaluating the role of MSC secretome in mediating neurodifferentiation and axonal growth as a function of tissue source. For that purpose, htNPCs and DRGs were incubated with the CM of ASCs, BMSCs and HUCPVCs, for neurodifferentiation and axonal growth assessment, respectively. htNPCs are normally expanded as neurospheres in the presence of a serum-free medium PPRF-h2, as demonstrated by Teixeira et al [4]. Upon removal of the expansion medium, these cells are described to spontaneously differentiate into neural phenotypes. For that reason, this cell population was used in this study to test the neurodifferentiation potential of MSC CM. The incubation of htNPCs with the MSC CM induced significantly higher levels of differentiation into neuronal phenotypes in comparison to control conditions, as shown by the expression of DCX (immature neurons) and MAP-2 (mature neurons) markers. On the other hand, no differences were observed in BIII-tubulin levels, another marker of fully mature neurons, as assessed by gene expression (Fig.1 and 2). Moreover, no differences were observed between the MSC populations, suggesting that the different CM had the same differentiation potential. Regarding the effects of MSC CM on axonal growth, the well described DRG-based in vitro model of neurite outgrowth [35] was used. After incubation with MSC CM, we observed that neurite extension from DRGs was promoted by the CM of all populations, in comparison to controls (Fig 4). Moreover, we verified that ASC CM induced more neurite extension from the explants, with significant differences regarding BMSC and HUCPVC CM, and control (Fig.5). The results herein presented showing the neuroregulatory potential of MSC secretome in neural cultures goes in accordance to some previous studies. For example, our group has shown that the CM of both ASCs and HUCPVCs promoted both proliferation and metabolic activity of hippocampal neurons [26]. We have also observed that BMSC CM improved both neuronal and glial cell survival. In that study, different CM collection times were tested – 24h and 96h. While the former increased the survival of neurons, the later was more prone to improve glial cell survival [36]. High in vitro neuronal differentiation [4, 37] and in vivo cell proliferation in the dentate gyrus (DG) of adult rat hippocampus [4] was also found using HUCPVC CM. Interestingly, proteomic analysis on these CM revealed differences in proteins related with neural cell viability, proliferation and differentiation, namely 14-3-3, Ubiquitin C-Terminal Hydrolase L1 (UCHL1), Heat shock protein (hsp) 70 and Peroxiredoxin-6 (PRDX6), which may explain the above-referred results [36]. Others observed the neurotrophic factors Brain-derived neurotrophic factor (BDNF) and Betanerve growth factor (β-NGF) to be correlated with the ability of undifferentiated MSCs to induce the survival 79 and neurite outgrowth of neuroblastoma cells and DRGs, respectively [7]. Some other factors not related to MSC so far but that were recently shown to have regenerative and neurotrophic functions are the ASCsecreted Macrophage-colony stimulating factor (MCSF), Matrix metalloproteases (MMPS), Follistatin (FST)-like 1, Mesencephalic astrocyte-derived neurotrophic factor (MANF), and Neuron derived neurotrophic factor (NDNF) [38]. Others like Semaphorins (SEM), Galectins (Gal), Platelet-derived growth factor (PDGF) and Transforming growth factor-beta (TGF-β) were found in the secretome of BMSCs by Cizkova et al. [39]. Altogether, these results suggest the existence of tissue-source based differences, as recently evidenced by Pires et al [28]. In this proteomic data, the expression of some factors related to neuronal differentiation, namely of PEDF, SEM7A, CDH2 and IL-6 varies in the CM of ASC, BMSCs and HUCPVCs, which would indicate that neurodifferentiation and axonal growth processes mediated by the MSC CM would be distinct. Still, the role of these factors in the CNS is robustly proved. PEDF, for instance, was shown to induce a neuronal phenotype in cultured human retinoblastoma cells in vitro [40], as well as to contribute to the survival and differentiation of embryonic chick spinal cord motor neurons [41]. CDH2 was also found to be essential for the neural differentiation of mouse induced pluripotent stem cells [42], and to regulate the pattern of neurodifferentiation in P19 carcinoma cells [43, 44]. Similarly, IL-6 was recently demonstrated to promote neural differentiation of pluripotent stem cells upon treatment with an immunosuppressive drug [45]. However, the presence of these factors in the CM of MSCs did not translate into a fully maturation into neurons at least using this period of CM exposure. Nonetheless, we have shown that htNPCs express some of the receptors that have been implicated in the promotion of neurogenesis, neurodifferentiation and neuronal migration, namely FLT-1 [46] and TGFBR1 [47], suggesting they might be responsive to the factors present in the MSC CM and that longer culturing periods may potentiate further their differentiation into fully mature neurons (Fig.3). The putative downstream signaling molecules involved in neuronal differentiation signaling cascades upon activation of these receptors, such as NRP1 [48], SMAD2 [49] and STAT3 [50, 51], were not differentially expressed in the cells from the different experimental groups (Fig3). In fact, signaling molecules display a very quick turnover, which may have accounted for the lack of significant differences in their expression between groups. Facing these observations, the absence of differences of htNPCs neurodifferentiation herein observed can therefore suggest that the protein levels presented may not be within the optimal dose to fully exert their effects, opening up the possibility to explore new approaches that may promote the secretion of higher doses of these molecules by MSC. Yet, the existence of innumerous distinct protocols among the different studies evaluating the differentiation of neural cultures may also account for different outcomes. Another possible explanation to be considered is the existence of multidirectional function of 80 most of neuroregulatory factors. For example, the upregulation of PEDF in ASC CM does not necessarily mean it should promote higher levels of neurodifferentiation of htNPCs. On the contrary, this factor is also related to axonal growth [52], which supports the pronounced effects of ASC CM in the levels of neurite outgrowth from DRG explants, depicted in figure 5. So, in this case, PEDF appears to be mediating axonal growth over neurodifferentiation. A similar phenomenon might be happening for IL-6. Indeed, this factor was shown to have a role in both neurodifferentiation and axonal growth [53]. But there is a clear upregulation of this factor in HUCPVCs CM in the proteomic analysis [28]. However, the effect of MSC secretome on axonal growth is clearly provided by ASC population (Fig 5), and not by HUCPVCs, suggesting a poor contribution of IL-6 in that effect. This is supported by the upregulation levels of SEM7A and Glial-derived nexin (GDN) in the ASC CM, which suggests that it is more likely that these two factors are responsible for mediating axonal growth, rather than IL-6. The potential of SEM7A and GDN in CNS has been in fact shown. The SEM7A-mediated axonal guidance has been proven to be required for proper axon tract formation during embryonic development [54], and to promote spreading and dendricity in human melanocytes [55]. On the other hand, the action of GDN has been also reported several years ago to improve chick sympathetic neurons [56] and hippocampal pyramidal cell [57] neurite extension in vitro . Based on these results, we believe that ASC-mediated neurite outgrowth of DRG explants is mostly based on the combined action of PEDF, SEM7A and GDN. Adding to this, Beta-1,4-galactosyltransferase 1 (β4Gal-T1) protein was found only in the ASC CM [28]. β4Gal-T1 was suggested to regulate the neurite outgrowth on PC12 cells [58] and DRG when co-cultured with Schwann Cells [59]. Moreover, the overexpression of this protein in the lesion site after sciatic nerve crush suggest it involvement in the regeneration of the injured tissue [60]. Therefore, the exclusive presence of this molecule on ASC CM may have indeed improved their impact in the in vitro DRG model of neurite outgrowth herein used. Another multi-functional protein relevant for axonal growth guidance is the extracellular chaperone Clusterin (CLUS), found highly expressed by ASC CM in comparison to BMSC and HUCPVC CM [28]. It is mainly involved in modulating toxic protein deposition in CNS disorders such as Alzheimer’s disease [61]. However, studies have also found this protein to contribute to neurite outgrowth of PC12 cells [62] and to induce sensory nerve outgrowth after sciatic nerve transection [63]. Finally, Decorin (DCN), a leucine proteoglycan protein, was also found to be upregulated in ASC CM in our proteomic analysis [28]. This anti-scarring molecule was elsewhere reported to promote robust neurite outgrowth across SCI lesion sites, by reducing the expression of chondroitin sulfate proteoglycan (CPSGs) [64, 65]. 81 5. CONCLUSIONS This study revealed that the incubation of ASC, BMSC and HUCPVC CM in htNPCs induced their differentiation towards the neuronal phenotype although with no significant differences among them. Moreover, all MSC CM improved neurite/axonal outgrowth in an in vitro model of axonal regeneration based on DRG explants. However, ASC CM provided higher extent of axonal growth when compared to BMSC and HUCPVC. The presence of important neuroregulatory factors in the secretome of MSCs, namely PEDF, CADH2, IL-6, SEM7A and GDN, may explain the observed results. Specifically, the upregulation of PEDF, SEM7A and GDN on ASC CM may be responsible for the higher levels of axonal growth observed with this population. Therefore, these results suggest two mechanisms underlying MSC secretome therapeutic action, namely neurodifferentiation and axonal growth. However, its potential is not limited to these two mechanisms. The modulation of other events such as excitotoxicity, apoptosis, inflammation, should be elucidated to understand their relevance in recovering the normal function of the CNS. The molecular and cellular pathways implicated on them may also be clarified to envisage the future application of MSC secretome in a clinical setting. ACKNOWLEDGMENTS The authors acknowledge the financial support by Prémios Santa Casa Neurociências - Prize Melo e Castro for Spinal Cord Injury Research (MC-17-2013 and MC-04-2017); Portuguese Foundation for Science and Technology (Doctoral fellowships PDE/BDE/113596/2015 and SFRH/BD/120124/2016 to R.C Assunção Silva and B. Mendes-Pinheiro, respectively; Post-doctoral fellowhip to F.G. Teixeira - SFRH/BPD/118408/2016; IF Starting Grant to L. Pinto and IF Development Grant to A. J. Salgado); Canada Research Chair in Biomedical Engineering (LAB). This work is funded by national funds through FCT under the scope of grante reference TUBITAK/0007/2014. This article has been developed under the scope of the projects NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). This work has been funded by FEDER funds, through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the project POCI-01-0145-FEDER007038. HUCPVCs and ASCs were kindly provided by Prof. John E. Davies (University of Toronto, Canada) and Prof. Jeff Gimble (LaCell Inc, USA). 82 CONFLIT OF INTERESTS The authors declare no conflict of interests associated with this publication. REFERENCES [1] A.J. Friedenstein, R.K. Chailakhjan, K.S. Lalykina, The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells, Cell and tissue kinetics, 3 (1970) 393-403. [2] L.R. Zhao, W.M. 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Davies, Decorin promotes robust axon growth on inhibitory CSPGs and myelin via a direct effect on neurons, Neurobiology of disease, 32 (2008) 88-95. 94 2.3 Spinal Cord Injury and post-operative care Tadpoles in the stages 45-47 (refractory period) and 50-54 (regenerative period) were used in this work. For that, animals were closely check daily for their developmental stages. When the desired stage was reached [29], animals were carefully collected and used for the experiments. A complete transection of the spinal cord of Xenopus laevis was the injury model used in this work, for both stage 45 and 50 tadpoles. To inflict the transection into the animal’s spinal cord, the protocol described by Edwards-Faret et al. [30] was used as reference. Briefly, tadpoles were anesthetized with 2% (w/v) of freshly prepared tricaine methanesulfonate (MS222) by immersing the animals in the solution for 1-2 minutes. Animals were then carefully immobilized on their abdominal area using forceps under a dissecting microscope. A small incision on the skin and dorsal muscles perpendicular to the body’s axis were made at the mid-thoracic level of the animals, at the central level of the gut. The meningeal layer was then removed using forceps, to completely expose the spinal cord [30]. At this stage, animals were grouped according to the procedure/treatment to receive: 1) non-injured tadpoles, injected with saline (SH group; n=12); 2) tadpoles subjected to SCI, injected with Neurobasal-A medium (NB group, n=12); and 2) tadpoles subjected to SCI, injected with ASC secretome (CM group, n=12). To fully transect the spinal cord of the tadpoles, a tip of a 30-gauge needle was used to make a clean cut at the thoracic level, perpendicular to the spinal cord. A successful spinal cord transection was confirmed by checking the presence of a dark line between rostral and caudal stumps of the spinal cord [30]. After surgery, all animals were transferred to small contents containing 1x MBS and antibiotics [penicillin (5,000 U/ml)- streptomycin (5 mg/ml) solution (Sigma-Aldrich, Germany) and gentamycin (1.25 mg/ml, Fisher Scientific, UK)], defined as 0,1x MBS + 3A, and kept at 20-21 ºC until they recover from anesthesia. The post-operative care of the animals with the antibiotics were maintained for 3 days, with the 0,1x MBS + 3A solution being replaced twice a day. 2.4 Secretome injection in the Xenopus laevis tadpole’s spinal cord after injury 2.4.1 FITC-labelling of the secretome. To visualize that the secretome was being correctly administered in the ependymal canal of the spinal cord of Xenopus laevis tadpoles, and that it was not being washed out by the time the animal has been placed back in the water, the secretome was labelled with Fluorescein isothiocyanate (FITC) prior to injection. Using the FluoReporter FITC Protein Labelling Kit (Invitrogen, USA), one has the possibility to bind a FITC dye to free amines of proteins larger than 30kDa in a certain 95 solution. As so, a dye-protein conjugate displaying a fluorescent signal (Em: 580nm, Ex: 488nm) was formed, providing means to label the desired proteins of the secretome herein used. The labelled secretome was imaged in real time using a fluorescent microscope (Olympus IX-53), immediately, 1 and 2 days after injection. 2.4.2 Local injection of ASC secretome. The secretome of ASCs was injected locally in the rostral stump of the spinal cord, immediately after injury. For that, a Pneumatic Pico Pump System (Narishige Group, Tokyo, Japan) was used. First, a pulled-glass capillary needle was filled with 2µl of the labelled secretome. The needle was then set in the needle holder, and the tip of the needle was carefully placed on the ependymal canal, rostral to the spinal cord, with the help of the micromanipulator under the dissection microscope. The secretome was administered at a rate of 0,05µl/s. NB group animals were injected Neurobasal-A medium. 2.5 Swimming Behavior of Xenopus Laevis tadpoles To evaluate the effect of ASC secretome on the functional recovery of the animals after injury, their free-swimming behavior was analyzed at 2, 3 and 5 days post-injury. Animal’s swimming trajectory was evaluated using a custom-made optimized vibrating six-well plate along with a video-tracking system (DanioVision, Noldus, Netherlands). For that, animals were individually placed in wells of a six-well plate containing vibrating motors attached to their walls, and their movement was tracked and recorded by a camera inside the DanioVision chamber. The parameters of the test were set using an Ethovision software. The animals were left to acclimatize the environment without disturbance for 10 minutes before testing. Once the vibration mode was set to ON, animals were subjected to cycles of 4 seconds of vibration, followed by 12 seconds of resting period (no vibration), to a total of 80 seconds of test. The recording data of the animals was acquired by the EthioVision software. 2.6 Histological preparation of the animals Tadpoles were sacrificed by deeply anesthetize by immersion into 2% (w/v) of MS222 for 15 minutes, and placed in 4% (w/v) of paraformaldehyde (PFA) solution for 1 hour. Animals were washed 3 times with 1x PBS, and placed on a solution of saccharose at 30% (w/v). After 24-48 hours, animals were carefully immersed in section medium (Neg-50, Thermo Scientific, USA), 96 frozen in liquid nitrogen, and stored at -20ºC. Later on, longitudinal cross sections of 20"m thickness were taken using Leica CM1900 cryostat and kept at -20ºC until required for immunohistochemistry. 2.7 Immunohistochemisty (IHC) Tadpole’s spinal cord sections were immunostained for axonal growth and regeneration [bIIITubulin and growth associated protein (GAP)-43, respectively]. For that, sections were first washed with 0.1% (v/v) of Triton-X 100 (Sigma) in 1x PBS (PBS-T), 3 times, for 5 minutes to remove the excess of frozen section medium. Sections were then incubated with 3% (v/v) of PBS-T for 10 minutes for permeabilization of the tissue. After that, tissue sections were blocked with a solution of 3% (w/v) bovine serum albumin in PBS-T for 1 hour at room temperature (RT) to avoid unspecific binding of the antibodies. Next, sections were incubated for 1 hour with the following antibodies: mouse monoclonal Acetylated Anti-Tubulin (1:500, Sigma) and rabbit anti-GAP-43 (1:500, Abcam). Sections were then exposed for 1 hour at RT to the respective secondary antibodies: Alexa Fluor 488 rabbit anti-mouse and Alexa Fluor 594 goat anti-rabbit (1:500; Invitrogen). Finally, all sections were counterstained with DAPI (4',6'-diamino-2-fenil-indol; 1mg/ml, Invitrogen) for 5 minutes at RT. 3 washes with 1x PBS were performed between steps. The sections were mounted in ImmuMount (Thermo Scientific, USA) and imaging of the tissue was performed using a confocal pointscanning microscope (Olympus FV1000) at 20x magnification. All images were treated and analyzed using Image J software. 2.8 Tissue Histological Analysis Quantification of bIII-Tubulin and GAP-43 positivity was performed on epicenter segment of the spinal cord, in both injured secretome-treated (CM group) and non-treated (NB group) animals, for stage 45 and 50. Using the Image J software, all acquired images were converted into monochrome 8-bit images. Fluorescent particles appear as black pixels, and background as white pixels. The region of interest (ROI) to analyze was determined using the free-hand drawing tool. The expression of immunofluorescence within the ROI was evaluated automatically by the software. Mean values within each group were calculated as the percentage of bIII-tubulinor GAP-43expressing axons. 97 2.9 Statistical Analysis The data obtained from behavioral analysis and tissue fluorescence quantification analysis were reported as Mean±SEM. Statistical differences among groups were assessed by One-way ANOVA and Tukey’s post hoc tests using GraphPad PRISM software (version 5.00). A p-value of £ 0.05 (95% confidence level) was set as criteria for statistical significance. Significant values were denoted with * for p<0.05, ** for p<0.01, and *** for p<0.001. 3. RESULTS In this study, the potential of ASC secretome in promoting Xenopus laevis spinal cord regeneration after injury was evaluated in both refractory and regenerative periods. For that, tadpoles in stage 45-47 and 50-54 were grouped in refractory and regenerative period groups, respectively, and SCI was performed by completely transecting their spinal cord. Immediately after injury, animals received a single injection of ASC secretome (CM group) through the ependymal canal, rostral to the injury site. Animals inflicted with SCI and injected with Neurobasal-A medium (NB group), or not subjected to SCI and injected with saline solution (SH group) were used as control groups. ASC secretome distributed well in the lesion site and was retained in spinal cord tissue The secretome of ASCs was labelled with FITC fluorochrome prior to injection in Xenopus laevis animals with the purpose to follow its distribution and retention within the spinal cord tissue. Immediately after injection, the labelled molecules were clearly observed surrounding the injury site (Fig. 1 – Day 0; arrow heads). 1 and 2 days after injection, some of the labelled molecules were seen in the spinal cord tissue, both at the lesion site and in the proximities (Fig. 1 – Day 1; Fig. 1 Day 2; arrow heads). These data suggest that the secretome of ASCs was correctly injected in the ependymal canal of the spinal cord, and that it was not washed away by the time that animals were placed back in the water, after the surgical procedure was finished. In addition, the retention of secretome’s molecules in the spinal cord tissue for at least 2 days after injection may indicate that some may still be exerting their effect upon the lesioned animals for some days after treatment. 98 Figure 1. FITC-labelled secretome at the injury site of Xenopus laevis tadpoles. Real time representative fluorescent microscopy of the Xenopus laevis immediately (Day 0), 1 (Day 1) and 2 (Day 2) days following injection of FITC-labelled secretome in the spinal cord after transection. The secretome was adequately injected in the ependymal canal of the spinal cord, and was well distributed and retained up to 2 days following injection. ASC secretome improves functional recovery of Xenopus laevis tadpoles after SCI Tadpoles motor recovery in response to treatment was assessed by monitoring animal’s freeswimming ability using a motion capturing software, for both the refractive period at 2, 3 and 5 days post-injury, and the regenerative period, at 3 and 5 days post-injury. Paralysis of all animals in both periods was observed during the two initial days post-treatment. On the following days, the ASC secretome-treated group in the refractive period showed a swimming pattern very similar to healthy animals (SH group), in opposition to NB-treated animals (Fig. 2A). Significant differences in the swimming distances between the ASC secretome-treated groups and the NB-treated group, 5 days post-treatment confirmed these observations (*p<0.05; Fig. 2B). Regarding the regenerative period, clear differences in the swimming pattern between the ASC secretomeand NB-treated groups were observed (Fig. 2E), with the first group showing a pattern very similar to the SH group. However, no significant improvements of animal’s motor function were found throughout time between the two treated groups (Fig. 2F). Additionally, although the secretome-treated group show similar swimming pattern to the SH group (Fig. 2E), 5 days after treatment, this was not reflected in the distance travelled during the swimming recording (Fig. 2F). 99 ASC secretome favors axonal sprouting and regeneration in Xenopus laevis tadpoles after SCI Neuronal regrowth and regeneration after treatment was assessed by performing anti-acetylated tubulin and anti-GAP-43 immunostaining, respectively, at 2, 3, and 5 days post-injury for refractive period animals, and 3 and 5 days post-injury for regenerative period. Substantial GAP-43 expressing cells in the lesion core (LC) and both rostral and caudal ends of the spinal cord was observed in the ASC secretome-treated group from the refractive period (Fig. 2C), 2 days after treatment, but few were observed for the NB-treated group (Fig. 2C, arrow heads). This was confirmed by significant differences in the mean percentage of GAP-43+ cells between the ASC secretome-treated group and the NB-treated group (**p<0.01, Fig. 2D). Considerable ablation gap closure and a robust axonal bridge formation was observed in the secretome-treated animals, 3 and 5 days after treatment, respectively (Fig. 2C). Furthermore, increased expression of bIII-tubulin in the LC, 5 days post-treatment, indicated neuronal regrowth throughout the injury site (Fig. 2E), though no statistical differences were found between groups. In the regenerative period, GAP-43 staining was mostly found rostral and caudally to the LC in the secretome-treated group (Fig. 2G), confirmed by the elevated, but not statistically significant, mean percentage of GAP-43+ cells in this group, in comparison to the NB-treated group (Fig. 2H), 3 and 5 days after treatment. Also, complete ablation gap closure with formation of bIItubulin positive axonal bridge was observed in the secretome-treated group, 3 days posttreatment (Fig. 2G). However, no statistical differences were found in the mean percentage of bIII-tubulin expression between secretomeand NB-treated groups (Fig. 2I), most likely due to the proximity of the two stumps of the spinal cord, not enabling to distinguish the bIII-tubulin expression between the ends of the spinal cord and the axonal bridge itself. 100 101 Figure 2. Therapeutic effects of ASC secretome on Xenopus laevis tadpoles after complete transection on swimming recovery, axonal growth and regeneration, in both refractory (A-E) and regenerative (F-J) stages. Swimming pattern and quantification of the distance travelled by the (AB) refractory animals at 2, 3 and 5 days post-treatment, and (F-G) regenerative animals 3 and 5 days post-treatment. ASC secretome promoted functional recovery of Xenopus laevis tadpoles after SCI from the refractory period, 5 days post-treatment, when compared to NB-treated animals. On the other hand, no differences were found in the locomotor performance between ASC secretomeand NB-treated animals in the regenerative stage. Representative confocal images of longitudinal crossections of Xenopus laevis spinal cord after immunostaining for bIII-tubulin (axonal sprouting) and GAP-43 (axonal regeneration), at ( C ) refractory and (H) regenerative stages . Quantification of the percentage of (D; I) bIII-tubulin and (E; J) GAP-43 positivity, at both refractory ( D, E ) and regenerative (I, J ) stages. In the refractory period, ASC secretome group (CM) shows a clear gap closure and the formation of a robust axonal bridge between the two stumps of the spinal cord, 5 days post-treatment. This was confirmed by elevated, but not statistical significant, expression of bIII-tubulin. GAP-43 positive regenerating cells were present in the spinal cord tissue of these animals, with significant differences observed at 2 days post-treatment. In the regenerative period, CM group presented axonal bridge formation and some GAP-43+ cells caudally to the injury site, 5 days post-treatment. However, no statistical differences were observed in the percentage of both bIII-tubulin and GAP-43 positivity in this stage. Mean±SEM; n=15 for locomotor assessment; n=5 for histological evaluation *p < 0.05; **p < 0.01; ***p<0.001. 4. DISCUSSION The secretome of ASCs was previously shown by our group to contain several neurotrophic factors with anti-apoptotic, pro-inflammatory, angiogenic, and neuromodulatory roles in the CNS [17]. When applied to in vitro systems, ASC secretome was reported to be effective for neuronal survival and differentiation, and neurite growth [16]. These results motivated further investigation on the effectiveness of ASC secretome in an in vivo context. The present study demonstrated that the treatment of Xenopus laevis tadpoles with the secretome of ASCs after a complete transection of the spinal cord could promote locomotor improvements and tissue repair after SCI. So far, these naturally-regenerating species were exclusively used for investigation of the regenerative process, from understanding the pathophysiology of SCI to disclosing the mechanisms responsible for their ability to fully regenerate their tail and spinal cord after lesion. However, 102 Xenopus laevis is the unique amphibian that present shifts on their potential to regenerate throughout their life from a regenerative to non-regenerative contexts. This particular property makes them ideal to be used in experimental paradigms dealing with the establishment of therapies for SCI regenerative medicine. However, the impact of a regenerative therapy based on a cell-free approach on a Xenopus laevis SCI model was herein investigated for the first time. After complete transection of tadpoles’ spinal cord in the refractive period (stage 45-47), NB-treated animals (NB group) showed a low regenerative ability following injury, as shown by the absence of bIII-tubulin+ axonal sprouting from both rostral and caudal ends of the spinal cord and GAP-43+ regenerating cells in the lesion cavity, from 2 to 5 days post-injury (Fig. 2C). In contrast, tadpole’s treatment with ASC secretome at this stage promoted significant increased levels of regenerating cells at 2 days post-treatment (Fig. 2D), and supported extensive axonal sprouting accompanied by the formation of a robust axonal bridge between the two stumps of the spinal cord at 5 days post-treatment (Fig. 2E). Regarding tadpoles in the regenerative stage (stage 50-54), ASC secretome treatment increased GAP-43+ regenerating cells and the formation of an axonal bridge in the lesion cavity already at 3 days post-treatment (Fig. 2G), whereas the NB group presented few regenerating cells in the lesion cavity and only discrete axonal sprouting at 5 days post-injury (Fig. 2G). According to Beck et al. [5], refractory period tadpoles never progressed to tissue regeneration after tail amputation, whereas regenerative tadpoles showed regenerated tails, including the spinal cord, within approximately 1 week after injury. In addition, complete cellular bridge formation across the lesion cavity in regenerative tadpoles was only reported at 10 days post injury by Munoz and colleagues [31]. Others have reported similar dynamics of regeneration [32, 33]. Thus, our observations suggest that ASC secretome treatment is restoring the regenerative ability of the refractive tadpoles, and anticipating the regenerative process in the regenerative tadpoles. Additionally, the recovery of the swimming ability was observed for both refractory and regenerative tadpoles after ASC secretome treatment (Fig. 2A-B and E-F, respectively), in contrary to NB-treated animals, which correlated with the observed histological improvements. Interestingly, the temporary loss of tadpoles’ regenerative ability in the refractory period has been previously attributed to a suppression of specific molecular pathways [5, 34] and to a transitory imbalanced immune response [3], as well as to a marked decrease of Sox2/3 positive cells recruitment to the lesion site following injury [31, 33], all contributing differently but possibly synergistically to the decreased regenerative capabilities of these animals. Whether ASC secretome may be acting upon these or other mechanisms was not unveiled in this study. In future, 103 studies directed to explore the mechanisms underlying the effects of ASC secretome on the regeneration of tadpoles after SCI will be of the utmost importance. CONCLUSION Following our previous findings concerning the potential of ASC secretome in promoting in vitro neuronal differentiation and axonal growth, the present study provides evidences on the impact that the secretome of these cells may have in the regeneration of Xenopus laevis tadpole’ spinal cord tissue after SCI and in the motor functional recovery of the injured animals treated with this molecular cocktail. So, these promising data support the potential of ASC secretome as a cell-free based strategy for the treatment of SCI, and encourage the use of Xenopus laevis to study the efficacy of therapeutic strategies for SCI, expanding the boundaries of their use in research, taking them beyond development studies to, also, regeneration paradigms. REFERENCES 1. Bloom, O., Non-mammalian model systems for studying neuro-immune interactions after spinal cord injury. Experimental neurology, 2014. 258 : p. 130-140. 2. Li, J., S. Zhang, and E. Amaya, The cellular and molecular mechanisms of tissue repair and regeneration as revealed by studies in Xenopus. Regeneration, 2016. 3 (4): p. 198208. 3. Fukazawa, T., et al., Suppression of the immune response potentiates tadpole tail regeneration during the refractory period. Development, 2009. 136 (14): p. 2323-2327. 4. Godwin, J.W. and N. Rosenthal, Scar-free wound healing and regeneration in amphibians: immunological influences on regenerative success. Differentiation, 2014. 87 (1-2): p. 6675. 5. Beck, C.W., B. Christen, and J.M. Slack, Molecular pathways needed for regeneration of spinal cord and muscle in a vertebrate. Developmental cell, 2003. 5 (3): p. 429-439. 6. Beck, C.W., J.C. Izpisua Belmonte, and B. Christen, Beyond early development: Xenopus as an emerging model for the study of regenerative mechanisms. Developmental Dynamics, 2009. 238 (6): p. 1226-1248. 7. Adams, D.S., A. Masi, and M. Levin, H+ pump-dependent changes in membrane voltage are an early mechanism necessary and sufficient to induce Xenopus tail regeneration. Development, 2007. 134 (7): p. 1323-1335. 8. Tseng, A.-S., et al., Apoptosis is required during early stages of tail regeneration in Xenopus laevis. Developmental biology, 2007. 301 (1): p. 62-69. 9. Hofstetter, C.P., et al., Marrow stromal cells form guiding strands in the injured spinal cord and promote recovery. Proc Natl Acad Sci U S A, 2002. 99 (4): p. 2199-204. 110 with this, the in vivo application of the secretome of bone-marrow derived MSCs (BM-MSCs) in rats’ spinal cord after injury, markedly improved animal’s locomotor function and fostered tissue repair, by stimulating axonal outgrowth and regeneration, and attenuating infiltration of astrocytes and inflammatory response, in comparison to vehicle-treated animals [30-32]. BM-MSCs are the most widely MSC population, however the isolation procedure of these cells is too painful and invasive, and is likely to cause generalized infections [33]. There are other available MSC sources, from which the adipose-tissue has emerged as a potential alternative. In contrary to BM-MSCs, Adipose-derived MSCs (ASCs) are of easy access and can be obtained through a simple and less invasive procedure. Moreover, ASC paracrine activity upon damaged tissues has also been attributed to the composition of their secretome, providing them with both neuroprotective and anti-inflammatory roles [34, 35]. In fact, the therapeutic impact of ASCs on SCI has been reported in several SCI animal models. The transplantation of ASCs into rodent and dogs following SCI was reported to support functional improvements, and to contribute to tissue repair and regeneration, mostly by decreasing apoptosis and astrogliosis after injury while supporting axonal growth and regeneration [36-41]. Considering the beneficial effects of ASCs shown in animal models, this cell population has been put forward to clinical trials, where some preliminary improvements were found in some patients, with no adverse effects associated with ASC transplantation [42, 43]. Currently, a phase I clinical trial using autologous ASCs in chronic SCI patients is ongoing at the Mayo Clinic, with expected outcomes to be reported in 2023. Recent evidences attribute the therapeutic effects of ASCs to the biomolecules presented in their secretome. In fact, previous studies from our group reported that the presence of NGF, VEGF, HGF and stem cell factor (SCF) in ASC secretome supported hippocampal neuronal survival in culture [27]. Moreover, cortical neurons were found to be protected from inflammation in vitro , due to an increase of TGF-β1 and IL-10 and decrease of Tumor necrosis factor-alfa (TNF-α), nitric oxide or prostaglandin E2 in culture by ASC secretome [29]. More recently, we have shown that ASC secretome was able to promote axonal regeneration of dorsal root ganglion explants and neurodifferentiation of neural progenitor cells [44]. Considering such promising data, ASCs seems to be a good alternative to BM-MSCs. Thus, ASC secretome is herein proposed as a cell-free based therapy for SCI to better elucidate it potential in promoting axonal growth and functional recovery in vivo . 111 2. METHODS 2.1 Adipose tissue-derived mesenchymal Stem Cells (ASCs) 2.1.1 ASC culture ASCs were kindly provided by Professor Gimble (Pennington Biomedical Research Center/Tulane University, USA). After thaw, cells were cultured in alfa-Minimum Essential Medium (α-MEM, Invitrogen, USA) supplemented with sodium bicarbonate (NaHCO3; Merck, USA), 10% (v/v) of fetal bovine serum (FBS; Biochrom, Germany) and 1% (v/v) Penicilin-Streptomycin antibiotic (P/S; Invitrogen, USA). When confluent, cells were enzymatically dissociated with 0.05% (v/v) trypsin/EDTA (Invitrogen, USA), re-plated at a density of 4000 cells/cm2 and maintained at 37ºC, 5% humidified CO2, 95% air and 90% relative humidity. 2.1.2 Conditioning and secretome collection. The secretome, denoted as conditioned media (CM), was collected from cells in passage 5, as previously described [44]. Cells were plated at a density of 4000cells/cm2 and maintained in culture for 72 hours. Cells were then washed 5 times with PBS without Ca2+ and Mg2+ (Invitrogen, USA), and 1 time with the conditioning medium – Neurobasal A Medium supplemented with 1% (v/v) Kanamycin (Invitrogen, USA). After 24 hours of conditioning period in supplemented Neurobasal-A medium, the secretome was collected and centrifuged to remove cell debris. The collected secretome was concentrated 100x using a Vivaspin 20 centrifugal concentrators (MWCO 5kDa, Sartorius™ Vivaspin™ 20, Germany) at 3000g, and frozen at -80ºC until further required. 2.2 Spinal cord injury and post-operative care Eight weeks-old female C5Bl/6 mice (Charles River, France), were used in this in vivo study. Animals were group housed - 5 per cage, on corncob bedding with access to food and water ad libitum, and holding rooms were maintained on a 12-hour light/dark cycle. A complete transection of the spinal cord was the injury model herein used. Briefly, animals were anesthetized with a mixture of 1.5x Imalgene and 1x Dorbene. When no reaction to pinch was observed, animals were considered ready for surgery. First, animals were placed under a dissecting microscope. An incision on the skin and dorsal muscles was performed from T2-T10 and the muscles retracted. A laminectomy was performed at the T8 level, and the spinal cord exposed. At this stage, animals were grouped according to the procedure and/or treatment to receive: 1) mice subjected to sham operations - laminectomy but no SCI, injected with Neurobasal-A medium (SH group, n=8); 2) mice subjected to SCI, injected with Neurobasal-A medium (NB group, n=7); 112 and 3) mice subjected to SCI, injected with ASC secretome (CM group, n=9). The spinal cord of NB and CM group animals was totally cut using a microdissection scissor. The complete separation of both ends of the spinal cord was confirmed under the microscope using forceps. Animals were finally closed with Vicryl sutures (Johnson and Johnson, USA). After the surgical procedure, anesthesia effect was reverted by a single subcutaneous administration of atipamezole (5mg/ml, Antisedan/Pfizer, USA). Post-operative care consisting in subcutaneous administration of the analgesic butorphanol (10mg/ml, Butamidor, Richter Pharma AG, Austria), the antibiotic enrofloxacin (5mg/ml, Baytril/Bayer, Germany), 0.9% (v/v) NaCl and vitamins (Dulphalyte, Pfizer) was then given to every animal. Animals were then kept under heat lamps until recover from anesthesia. Post-operative care was maintained twice a day for 1 week postinjury. Manual bladder evacuation was performed twice a day until animals recover their bladder control completely. The general health of the animals was carefully checked every day for sins of illness and weight loss of the animals, during the time of post-surgery recovery and treatment. 2.3 ASC secretome administration to mice after SCI Animals from the CM group were intravenously administrated with ASC secretome. The treatment was given systemically in the three 24 hours post-injury, the first immediately after SCI, and then weekly until 6 weeks post-injury (100"l per injection). SH and NB group animals were administered with 100"l of Neurobasal-A medium. 2.4 Motor and emotional behavior of mice after SCI 2.4.1 BMS score. Basso Mouse Scale (BMS) scoring was used to assess the locomotor recovery of secretome-treated mice after SCI [45]. The first BMS evaluation was performed 2 days after injury in order to confirm hindlimbs paraplegia in all animals. Scores of 0 were selected for the experiment. BMS scoring for locomotion evaluation was then performed weekly until the end of the experiment (6 weeks post-injury). Mice were allowed to explore an open field arena for 5 minutes, while their locomotion was being recorded by videocamera. Two blinded researchers were evaluating mice locomotion during all time of the trial. 2.4.2 Von-Frey Test and Ultrasonic Vocalizations’. The ability of secretome-treated SCI mice to respond to a mechanical stimulus at the hindlimb paws was assessed by the Von Frey test [46]. For that, mice were individually placed in a clear glass in an elevated grid and the plantar surface of the hind paws was poked with Von Frey filaments of varying forces – 2g, 113 1.4g, 1g, 0.6g, 0.4g, 0.16g, 0.07g, 0.04g, 0.02g, and 0.008g. The trial started using the middle force filament (0.16g) and went further up the higher force filaments in case of no reaction (=0), or down to the lower ones in case a reaction occurred (=X), in a total of 6 measurements [47]. If no response was obtained up to the maximal filament (2g), or if a positive response occurs down to the minimal (0.008g), the 1.4 and 0.008 values were assumed for the measurement of that animal, respectively. Positive reactions considered included paw withdrawal, licking, shaking or extension of the paw, either during application of the stimuli or immediately after. The presence of nociception or hypersensitivity on the hindpaws was indicated by an exaggerated reaction to the lower diameter filaments. The response to Von Frey filaments was deduced as the 50% response threshold, calculated using the formula 50%&'()*+(,-. = 012345.7 01111 , where X is the value corresponding to the final Von Frey filament tested (in log units); k is the tabular value concerning the pattern of positive (=X) and/or negative (=0) responses, and 8 is the mean difference between stimuli (in log units) [48]. Low threshold is indicative of hypersensitivity, while high threshold indicates normal sensitivity to the mechanical stimuli, usually found among healthy individuals [46]. Another possible indicator of animal’s sensitivity is the presence of ultrasonic vocalizations (USVs) by the animals at the time of the stimuli [49]. As so, ultrasound vocalizations were recorded during the Von Frey trial. For that, ultrasound Microphones (CM16/CMPA, Avisoft Bioacoustics) sensitive to frequencies of 10-200 KHz were used, next to the Von Frey apparatus. Vocalizations were recorded using the AvisoftRecorder (version 5.1.04), and identified by automatic data processing on the software. Vocalizations were analyzed by the DeepSqueak software [50] and the total number and duration of the vocalizations per animal were the considered parameters. Vocalizations of 22Hz, the so called “negative vocalizations”, were the only considered in this assessment, as they are related to a reaction of the animals to an unpleasant or noxious stimulus [51], as is the case of the Von Frey. According to Portfors et al. [51], 22Hz vocalizations typically present a frequency between 18 and 35KHz, therefore the data obtained was analyzed following this parameter. 2.5 Histological preparation of the animals Six weeks after SCI, mice were deeply anesthetized by intraperitoneal injection of sodium pentobarbital (200mg/ml, Eutasil, Ceva Saúde Animal, Portugal), and transcardially perfused with 0.9% NaCl followed by cold 4% (w/v) PFA. The spine and the spinal cord were dissected and incubated with PFA for 24 hours, at 4ºC. The spinal cord was then carefully dissected and placed on a solution of 30% (w/v) of saccharose for 24 hours at 4ºC. After that, 3cm length of spinal cord tissues were cut having the lesion site at the 114 middle point, carefully immersed in section medium (Neg-50, Thermo Scientific, USA), frozen in liquid nitrogen, and stored at -20ºC. Longitudinal cross sections of 20"m thickness were then taken using Leica CM1900 cryostat and kept at -20ºC until required for immunohistochemistry. 2.6 Immunohistochemisty (IHC) Mouse spinal cord sections were immunostained for axonal growth and regeneration [bIII-Tubulin and growth associated protein (GAP)-43], de/re-myelinization (Fluoromyelin) and neuroinflammation [Ionized calcium binding adaptor molecule 1 (Iba-1)]. For that, sections were permeabilized with 0.2% (v/v) of PBS-T for 10 minutes, and washed 3 times with PBS 1x. All sections were incubated with a blocking solution of 5% (v/v) fetal calf serum in 0,2% (v/v) PBS-T for 30 minutes at RT, and incubated overnight at RT with the following antibodies: rabbit anti-beta III tubulin (1:1000, Abcam), mouse anti-GAP-43 (1:1000, Abcam), rabbit anti-Iba1 (1:750, Wako). Sections were then incubated for 1 hour at RT with the following respective secondary antibodies: Alexa Fluor 488 rabbit anti-mouse, and Alexa Fluor 594 goat anti-rabbit and rabbit anti-mouse (1:1000; Invitrogen). Cell nuclei was counterstained with DAPI (4',6'-diamino-2fenil-indol) for 10 minutes. For Fluoromyelin staining, sections were incubated with the FluoroMyelin™ Green Fluorescent Myelin Stain (1:300, ThermoFisher), along with DAPI, for 10 minutes at RT. 3 washes were performed between steps. The sections were mounted in Immu-Mount (ThermoFisher Scientific, USA) and kept at 4ºC until imaged. bIII-Tubulin, GAP-43 and Fluoromyelin were imaged by fluorescence microscope (Olympus BX61), and Iba-1 immunostained sections by confocal point-scanning microscope (Olympus FV1000). 2.7 Tissue Histological Analysis bIII-tubulin, GAP-43, Fluoromyelin and Iba-1 positivity was quantified on rostral, epicenter and caudal segments of the spinal cord, for both CM and NB groups. The process of imaging analysis was performed using Image J software. Thus, mean values of immunofluorescence within the region of interest (ROI) defined were calculated as the percentage of bIII-tubulin+ or GAP-43+ axons, per group. Fluoromyelin staining was used to quantify the lesion areas of the spinal cord, for both CM and NB groups. For that, the ROI to analyze was determined using the free-hand drawing tool and the corresponding area was automatically calculated by the software. Finally, Iba-1 positivity was used to compare the area occupied by activated and deactivated inflammatory cells throughout the spinal cord tissue of the CM and NB groups. For that, ROI was determined by the free-hand drawing tool and the mean values of 115 immunofluorescence within the ROI was calculated as the percentage of Iba-1 positivity. bIII-tubulin, GAP43 and Iba-1 measures were normalized to the total segment of the spinal cord analyzed. 2.8 Statistical Analysis The data obtained from behavioral analysis and tissue fluorescence quantification analysis were reported as Mean±SEM. Statistical differences among groups were assessed by One-way ANOVA and Tukey’s post hoc tests using GraphPad PRISM software (version 5.00). A p-value of £ 0.05 (95% confidence level) was set as criteria for statistical significance. Significant values were denoted with * for p<0.05, ** for p<0.01, *** for p<0.001, and **** for p<0.0001. 3. RESULTS The impact of ASC secretome treatment of mice after spinal cord complete transection was herein evaluated. The secretome of ASCs was intravenously administered through animal’s tail vein after injury, immediately, 24, 48 and 72 hours post-injury, and then weekly for a total of 6 weeks. Control animals were administered with Neurobasal-A media after laminectomy (SH group) or SCI (NB group). ASC secretome improves motor function and sensitivity recovery of mice after SCI Animal’s functional recovery after treatment was weekly assessed using the BMS score for a total of 6weeks, as depicted in Figure 1A. Two days post-injury, all SCI animals presented complete paralysis of the hindlimbs, when compared to laminectomy animals (SH group). On the following weeks, ASC secretome-treated animals show a gradual recovery of the hindlimbs movement up to 6 weeks, while the NB-treated animals only showed limited and slight movement of one or two joints. The CM-group improvement was found to be significantly higher already at 2 weeks post-treatment in comparison to NBgroup (*** p<0.01), and persisted to increase from 3 to 6 weeks (**** p<0.001). At 6 weeks posttreatment, secretome-treated animals showed the ability to frequently or consistently perform plantar stepping, accompanied by some degree of coordination. The motor function improvements of the secretome-treated SCI mice were accompanied by a sensorial recovery of the hindlimbs. The ability of SCI mice to respond to a mechanical stimulus at the hindlimb paws was assessed by the Von Frey test at 2 and 6-weeks post-treatment (Fig. 1B). NB-treated animals presented lower magnitude of response to the Von Frey filaments than secretome-treated animals, at both time points. Healthy individuals usually present high threshold of response, indicating normal sensitivity to the mechanical stimuli, while low threshold is indicative of hypersensitivity [46]. Thus, the 116 higher level of response of the CM-group to the Von Frey filaments, suggest a recovery of the sensitivity of this group when compared to NB group. At the time of the mechanical stimulus, animal’s vocalizations were recorded, also as indicator of animal’s sensitivity. 22Hz vocalizations were assessed as indicative of a reaction to a noxious or discomfort stimulus [51], provoked by the Von Frey filaments. The number of 22Hz vocalizations of the CM group was slightly lower than for the NB group, although no statistical differences were found (Fig. 1C), indicating that the mechanical stimuli with the Von Frey filaments had provoked less noxious sensations in the secretome-treated animals. Moreover, the duration of the vocalizations (Fig. 1D) was slightly higher for the CM group when compared to NB group, again with no statistical differences found. Altogether, these results suggest an important role of ASC secretome in both motor and sensorial recovery after SCI. Figure 1. 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 significantly improved the locomotor function of the transected animals, when compared to NB treatment. 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-weeks 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, when compared to NB group. (C-D) Recordings of USVs from mice during Von Frey trial was performed at 2 and 6 weeks after ASC secretome treatment. Mice treated with ASC secretome was shown to vocalize less ( C ) but for longer 117 periods of time ( D ), when compared to NB group, although no statistical differences were obtained. Data is presented as Mean ± SEM; n=8 (SH), n=7 (NB), n=9 (CM); *p < 0.05; **p < 0.01; ***p<0.001; ****p<0.0001 ASC secretome modulated neuroinflammation in mice after SCI The inflammatory response following injury was clearly different between the CM and NB groups, as suggested by the Iba-1 staining between the two groups throughout the spinal cord tissue (Fig. 2A). Different distribution of resting and activated inflammatory cells was also shown in the two groups, with prominent round-shape reactive cell accumulation beyond the lesion site for the NB group, as outlined in Figure 2A (dashed lines). Quantification of the percentage of Iba-1 positivity confirmed the significantly higher levels of activated inflammatory cells (Fig. 2E; * p<0.05) and lower resting ones (Fig. 2E; ** p<0.01) in the NB and CM groups, respectively. ASC secretome reduces lesion cavity in mice after SCI The border of the lesion area, shown in Figure 2B (dashed line), was clearly outlined by fluoromyelin staining. Larger cavities were found for the NB group (Fig. 2B, NB), when compared to the CM group (Fig. 2B, CM), further confirmed by the quantification of the area of the lesion cavity (Fig. 2F) 6 weeks after treatment, which was significantly higher in the former group (*** p<0.001). 118 119 Figure 2. Therapeutic effects of ASC secretome in mice spinal cord 6 weeks after complete transection on neuroinflammation, lesion cavity, axonal growth and regeneration. (A-D) Representative confocal images of longitudinal crossections of mice spinal cord after immunostaining for Iba-1 (A, neuroinflammation), fluoromyelin (B, lesion cavity), bIII-tubulin (C; axonal growth) and GAP-43 (D, axonal regeneration). Quantification of the (E) percentage of Iba-1 positivity, (F) area of lesion cavity, (G) percentage of bIII-tubulin and (H) percentage of GAP-43. CM group presented decreased inflammatory response of microglial cells and significant reduced area of the lesion cavity. Moreover, axonal outgrowth and regeneration at the lesion site, as measured by a significant increase of bIII-tubulin and GAP-43 expression, respectively, were observed for CM group, in comparison to NB group. Data is presented as Mean±SEM; n=5; *p < 0.05; **p < 0.01; ***p<0.001; ****p<0.0001. ASC secretome promotes neurite regeneration and sprouting in mice spinal cord after injury Axonal sprouting and regeneration were evaluated 6 weeks after treatment by bIII-tubulin and GAP-43 positivity in mice spinal cord tissue, respectively. Immunohistochemistry analysis of the spinal cord tissue of ASC secretome treated animals showed bIII-tubulin+ axons sprouting from the stumps of the spinal cord into the lesion area (epicenter) (Fig. 2C, CM-epicenter; white arrows). In contrast, NB group presented only few bIII-tubulin+ axons in the proximities of the epicenter, but did not go through the lesion site (Fig. 2C, NB-epicenter; white arrows). These observations were further confirmed by the significant differences on the percentage of bIII-tubulin positivity in the epicenter between the two groups (Fig. 2G; ****p<0.0001). 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Considering the complexity of SCI pathophysiology, it has been suggested that an effective therapy should tackle all the SCI-related aspects and provide sensory and motor improvement to SCI patients. For that, the current aim of any therapeutic approach for SCI relies in providing neuroprotection by modulating the inflammatory response and avoid the exacerbation of the injury, and support neuroregeneration by replacing damaged neural tissues and cells and stimulating axonal outgrowth and endogenous repair. Acknowledging the current SCI treatment paradigm, cell transplantation is one of the most explored approaches for SCI, aiming to provide an adequate growth-promoting and regenerative environment to the injured spinal cord. Among the innumerous available cell populations, mesenchymal stem cells (MSCs) have been in the forefront of many of these approaches. Studies showing the beneficial effects of MSC transplantation after SCI have been proposing a paracrine action of these cells on the injured tissues, through the secretion of protective and trophic factors, rather than attributing it to the action of cells itself [1-5]. Thus, the work herein developed have specifically focused on the potential neuroregenerative effect of the secretome of MSCs as a cell-free based therapy for SCI. The main challenge of any strategy proposed for SCI treatment relies in obtaining robust pre-clinical evidences from in vitro and in vivo models, before moving to the clinics. So, the regenerative potential of MSC secretome was herein explored in two in vitro models of regeneration and in two in vivo models of SCI (Fig. 1). In vitro (chapter 2), the secretome of bone marrow mesenchymal stem cells (BM-MSCs), adipose tissuederived MSCs (ASCs) and Human Umbilical Cord Perivascular Cells (HUCPVCs) induced neuronal differentiation from human neural progenitor cells (hNPCs), and neurite outgrowth from dorsal root ganglion (DRGs), suggesting a mediation of their neuroregulatory actions through these two mechanisms of repair. The three MSC populations seems to share equal differentiation profiles, but acted differently on axonal outgrowth, with ASC secretome revealing higher induction of DRG neurite outgrowth. This indicated that distinct processes of regeneration were being mediated by the MSCs as a function of tissue source, going in accordance with previous reports [6-9]. Under this assumption, our group and others have been putting effort in mapping the molecules secreted by these cells under specific conditions, aiming to determine which factors are upregulated and disclose which mechanisms of repair underlie their action. A varied expression of factors on the secretome of ASCs, BMSCs and HUCPVCs that are 133 related to neuronal differentiation and axonal outgrowth have been in fact previously shown [3, 7, 10-13]. These evidences support our hypothesis that the neurodifferentiation and axonal growth processes mediated by the MSC secretome in this study is more likely to translate the differences in the secretome composition among the three cell populations. In the end, the absence of differences among the three MSC populations in promoting neurodifferentiation was not expected, as some of the identified proteins in their secretome, namely IL-6, PEDF, CDH2, elsewhere reported to promote neuronal differentiation [14-17], presented different levels of upregulation. In addition, the receptors necessary to respond to these factors were present in hNPCs when incubated with all MSC secretome, even though signaling molecules involved in neuronal differentiation signaling cascades upon activation of these receptors were not differentially expressed. These results may be explained by protein levels below the optimal dose to exert some effect, a quick turnover of the molecules, or even a multidirectional function of the factors identified with their action directed to axonal outgrowth over neurodifferentiation. Therefore, this study would have benefited from a clarification on this. The usage of more concentrated version of secretome, different times of cell culture, or more frequent renewal of the secretome in culture would be of interest to test. Other in vitro models of neuronal differentiation could also have been tackled to further understand the results obtained. Likewise, other neuronal populations, such as motor neurons, could have been used to further address axonal outgrowth. Nevertheless, from all the MSC populations tested, ASC secretome demonstrated higher potential in this study regarding axonal outgrowth, thus being selected for further studies in the scope of this work. Moving forward to an in vivo context, ASC secretome have demonstrated positive results in two SCI models – non-mammalian regenerating Xenopus Laevis and mammalian non-regenerating mice (Fig. 1). In the former model (chapter 3), the treatment of Xenopus laevis tadpoles with ASC secretome after spinal cord complete transection favored axonal sprouting and regeneration and improved functional recovery after injury. At first, the use of a naturally regenerating model in the scope of this work seems irrelevant, since we were proposing to study a condition that results from the inability of the spinal cord to regenerate and recover after injury in mammals. In fact, Xenopus laevis was the only non-mammalian regenerating model that could be used to test the effects of therapeutic strategy for SCI, as it exclusively presents shifts from a regenerative to non-regenerative context throughout life, providing a system where loss and gain of function experiments can be directed at understanding what exactly inhibit or stimulate regeneration. Therefore, the regenerative potential of ASC secretome was herein evaluated in Xenopus Laevis tadpoles, in both refractory (non-regenerative) and regenerative stages, after complete transection of the spinal 134 cord. In the refractory period, a single injection of ASC secretome was sufficient to restore the regenerative ability of the tadpoles, indicated by the recovery of locomotor function of the treated animals. The notable recovery could be attributed to significant increased levels of regenerating cells and extensive axonal sprouting at the lesion site. Refractory tadpoles were reported to present no signs of tissue repair after tail amputation [18], therefore indicating that the recovery of the transected animals in this study is due to the effect of ASC secretome. Regarding the regenerating period, it is more likely that the treatment of ASC secretome was able to anticipate the regeneration of the injured spinal cord, as locomotor function recovery occurred earlier than for non-treated animals, accompanied by increased regeneration and axonal sprouting. The mechanisms upon which the secretome of ASCs may be acting was not elucidated, but studies directed to explore this question would be of great importance. Nevertheless, these promising data support the potential of ASC secretome as a cell-free based strategy for SCI treatment. Moreover, the use of Xenopus laevis is strongly encouraged as a model for SCI, opening the way to be used as novel, simple and low-cost tool for SCI research field, which provides a clear regeneration environment to test the therapeutic effect of new strategies. Following the positive outcomes obtained from the Xenopus laevis SCI model, we hypothesized whether ASC secretome would have a therapeutic effect in a larger animal, such as mice, using the same type of lesion (chapter 4; Fig. 1). The application of ASC secretome to lesioned animals revealed impressive in promoting motor and sensorial recovery of the treated animals, accompanied by significant axonal elongation and regeneration in the lesion site, suggesting a neuroprotective and neuroregenerative effect of the secretome. In addition, an attenuated inflammatory response after injury was observed, which is indicative of the anti-inflammatory action of the secretome of these cells. The complete transection of the spinal cord is not the most relevant in the clinics, as contusion or compression injury models are, since it does not reflect the SCI pathophysiology in humans [19, 20]. Besides, it is a very aggressive model of injury which could have been impeditive of observing any type of therapeutic effect of the strategy herein proposed. Nonetheless, transection is advantageous in providing a clean injury environment for studying processes of regeneration after lesion, therefore having shown valuable to assess our hypothesis on whether ASC secretome could promote axonal elongation and regeneration throughout the lesion site after SCI. Still, the therapeutic effect of ASC secretome in other lesion models such as contusion could have been addressed. Although mice share many similarities with human SCI pathophysiology, there are slightly differences mostly related to the absence of cystic cavity formation and to the inflammatory response following the injury [21, 22]. Thus, going up to larger animals with this strategy, namely dogs, 135 pigs and primates, would be of great interest to take us closer to the human SCI pathology. Another important aspect of this work is the route and frequency used to administer the secretome of ASCs. The secretome has been intravenously delivered to the animals through their tail vein (IV route). A concentrated formulation of ASC secretome has been injected once a day in the first three 24 hours postinjury and then weekly for 6 weeks, under the rationale that a sufficient supply of the molecules composing the secretome should be available during both acute and chronic phases of injury to increase the chances of having a measurable therapeutic effect. The continuous supply of ASC secretome to transected animals might explain the behavioral and histological outcomes of this study, and may have accounted for the prolonged regenerative effect observed 6-weeks post-injury, which is normally due in shorter periods of time [23-25], as well as to the marked modulation of microglia and macrophages recruitment to the injury site. These evidences are promising to envisage a SCI therapeutic strategy based on the use of ASC secretome. Although the IV route has been successfully used for the administration MSCs into rats and dogs after SCI [26, 27], this kind of administration poses some constraints to the animals, that have to be injected several times in short periods of time, which may add to some pain and stress associated to the injections. Also, it requires a very precise technique and practice of the animal’s handler not only for the sake of the animals’ well-being but also to avoid other issues such as the loss of secretome associated with less precise injections. The use of other routes of administration should therefore be considered in the future, namely through intrathecal catheters, intraspinally or subcutaneously, as successfully applied by others for the administration of either MSCs or MSC secretome [28-34]. Altogether, the work of this thesis demonstrates that the secretome of ASCs is able to promote the desired effects in stimulating the repair and regeneration of the spinal cord after lesion, highlighting the benefits of using the secretome of MSCs in alternative to cell transplantation. In fact, the transplantation of cells into the injury site would have some advantages, such as the possibility to provide a continuous secretion of biomolecules, adjusted to the needs of the lesion microenvironment, especially given that these cells have the capacity to respond to the stimuli of the surroundings, migrate into the injured tissues and adjust their activity accordingly [35-37]. However, cell transplantation approaches are very time and money consuming, and it requires high number of cells [38]. Plus, the survival rate of the transplanted cells is too low [39, 40]. The use of the secretome not only avoids these limitations, but can still be provided in a continuous way, depending on the route and frequency of administration used, as above discussed. Overall, the findings of this work are indicative of the positive effects exerted by the secretome of ASCs on axonal outgrowth and regeneration, and neuroinflammation, observed for the three models herein