Functionalized hydrogels and cell based therapies: A tissue engineering approach for spinal cord injury regeneration

Dissertação de Mestrado em Ciências da SaúdeAs Lesões da Medula Espinal (LME) continuam a ser das doenças mais devastadoras do Sistema Nervoso Central (SNC), devido aos graves danos funcionais causados, bem como o fardo que representam para os pacientes. A falta de uma terapia eficaz para tratar esta condição ou pelo menos atenuar a sua extensão levou a comunidade científica a procurar novas estratégias possíveis para abordar esta doença. Desta forma, hoje em dia terapias moleculares e celulares têm sido aplicadas em modelos animais de LME com resultados promissores. Contudo, terapias singulares, normalmente falham quando aplicadas na clínica, portanto a combinação de mais do que uma técnica poderá ser a solução. Assim sendo, neste trabalho conjugamos um hidrogel (Gellan Gum-GRGDS) com células mesenquimais do tecido adiposo (ASCs) e células gliais do bolbo olfativo (OECs), com o intuito de promover a recuperação de ratos com LME. Em primeiro lugar, as interações in vitro entre os dois tipos de células foram avaliadas através de co-culturas de contacto direto e através do secretoma de cada tipo celular. Depois, foi determinado o crescimento de ASCs e OECs quando encapsuladas no hidrogel GGGRGDS. Também foi verificado o potencial regenerativo do secretoma de ambas as células em estudo, com recurso a um modelo in vitro de regeneração axonal, baseado em Gânglios da Raiz Dorsal (DRGs). Finalmente, o valor terapêutico da nossa abordagem foi determinado usando um modelo in vivo de LME. Os ratos foram sujeitos a uma hemissecção do lado esquerdo da medula espinal (T10-T11) e divididos em diferentes grupos de acordo com o respetivo tratamento: animais não tratados (grupo HS); animais tratados com células (Cells); com GG-GRGDS (GG); e com células encapsuladas em GG-GRGDS (GG+cells). Foi também incluído um grupo de animais sem lesão, apenas sujeito a uma laminectomia (Sham). Os resultados demonstraram que as ASCs e OECs foram capazes de crescer em co-culturas de contacto direto sem alterações significativas da sua morfologia e do seu número total; ambas as células apresentaram uma taxa de crescimento normal quando encapsuladas em GG-GRGDS, o que demonstra a capacidade deste último como veículo de transporte celular. Relativamente aos explantes de DRGs, os fatores secretados pelas ASCs e OECs parecem potenciar a regeneração axonal. Finalmente, os animais tratados com a conjugação de hidrogel com ASCs+OECs apresentaram melhorias significativas das suas capacidades motoras e ao mesmo tempo menor astrogliose. Em suma, estes resultados indicam que ao conjugar GG-GRGDS com ASCs e OECs, poderá ser possível desenvolver terapias alternativas direcionadas para a recuperação de LME.Spinal Cord Injury (SCI) remains one of the most devastating diseases of the Central Nervous System (CNS) due to the drastic functional impairments caused, as well as the burden it represents for patients. The lack of an effective therapy to treat this condition or at least minimize the injury extent, led the scientific community to search for new possible strategies to address this question. Consequently, nowadays molecular and cell based therapies are often applied on SCI animal models with promising results. However single therapies usually fail when translated to the clinics, so the combination of more than one technique appears to be the best solution. Having this in mind, in the present work, we aimed at conjugating a hydrogel based scaffold (gellan gum-GRGDS) with Adipose tissue derived Mesenchymal Stem Cells (ASCs) and Olfactory Ensheathing Cells (OECs) in order to promote the recovery of SCI rats. Firstly, the in vitro interactions between both cell types were assessed through direct co-cultures and using the secretome of each cell. Then, the growth of ASCs and OECs while encapsulated within the GGGRGDS hydrogel was evaluated. We also verified the regenerative potential of both cells’ secretome, using an in vitro model of axonal regeneration based on Dorsal Root Ganglia explants (DRGs). Finally, the therapeutic value of our proposed strategy was determined in an in vivo model of SCI. Rats were subjected to an hemisection injury on the left side of the spinal cord (T10-T11) and divided in different groups according to the respective treatment: no treated animals (HS group); cells treated (Cells); GG-GRGDS-treated (GG); and GG-GRGDS with cellstreated animals (GG+cells). An additional group of animals without injury, only subjected to a laminectomy was also included (Sham). The results showed that ASCs and OECs were able grow together on direct co-cultures without significant alterations in cell morphology and cell numbers; both cells also exhibited a standard growth profile while inside the GG-GRGDS hydrogel, which proves the suitability of this hydrogel as a vehicle for cell transplantation. Regarding DRGs explants, ASCs and OECs secreted paracrine factors seemed to potentiate axonal regeneration. Finally, the animals treated with the conjugation of GG-GRGDS hydrogel with ASCs+OECs presented significant improved motor skills, while simultaneously disclosing an evident decrease in astrogliosis. All together, these results indicate that by using GG-GRGDS conjugated with ASCs and OECs, it may be possible to develop alternative therapeutic routes for SCI repair/regenerationÀs entidades que financiaram este trabalho, nomeadamente: 1) Fundação para a Ciência e a Tecnologia (FCT) (PTDC/SAU-BMA/114059/2009); 2) BIOHYBRID - Templates for Peripheral Nerve Regeneration (EU-FP7-Health-2011-collaborative project 278612); 3) Programa Operacional Regional do Norte (ON.2 – O Novo Norte), ao abrigo do Quadro de Referência Estratégico Nacional (QREN), através do Fundo Europeu do Desenvolvimento Regional (FEDER)

Combinatorial therapies for spinal cord injury: strategies to induce regeneration

The authors want to acknowledge the financial support from Prémios Santa Casa Neurociências ‐ Prize Melo e Castro for Spinal Cord Injury Research (MC-04/17); from Portuguese Foundation for Science and Technology [Doctoral fellowship (SFRH/BD/103075/2014) to EDG; Post‐ Doctoral fellowship (SFRH/BPD/97701/2013) to NAS; IF Development Grant to AJS]. This work is funded by national funds through FCT under the scope of grant reference TUBITAK/0007/2014 and 3599-PPCDT Project: PTDC/DTP-FTO/5109/2014. 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-FEDER-007038.info:eu-repo/semantics/publishedVersio

Hydrogels and cell based therapies in spinal cord injury regeneration

Spinal cord injury (SCI) is a central nervous system- (CNS-) related disorder for which there is yet no successful treatment. Within the past several years, cell-based therapies have been explored for SCI repair, including the use of pluripotent human stem cells, and a number of adult-derived stem and mature cells such as mesenchymal stem cells, olfactory ensheathing cells, and Schwann cells. Although promising, cell transplantation is often overturned by the poor cell survival in the treatment of spinal cord injuries. Alternatively, the therapeutic role of different cells has been used in tissue engineering approaches by engrafting cells with biomaterials. The latter have the advantages of physically mimicking the CNS tissue, while promoting a more permissive environment for cell survival, growth, and differentiation. The roles of both cell- and biomaterial-based therapies as single therapeutic approaches for SCI repair will be discussed in this review. Moreover, as the multifactorial inhibitory environment of a SCI suggests that combinatorial approaches would be more effective, the importance of using biomaterials as cell carriers will be herein highlighted, as well as the recent advances and achievements of these promising tools for neural tissue regeneration.The authors would like to acknowledge the Portuguese Foundation for Science and Technology (Grant no. PTDC/SAU-BMA/114059/2009; IF Development Grant to António J. Salgado); Prémios Santa Casa Neurociências for funds attributed to António J. Salgado under the scope of the Prize Melo e Castro for Spinal Cord Injury Research; cofunded by Programa Operacional Regional do Norte (ON.2—O Novo Norte), ao abrigo do Quadro de Referência Estratégico Nacional (QREN), através do Fundo Europeu de Desenvolvimento Regional (FEDER)

Neuroinflammation and Parkinson’s disease - from neurodegeneration to therapeutic opportunities

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disorder worldwide. Clinically, it is characterized by a progressive degeneration of dopaminergic neurons (DAn), resulting in severe motor complications. Preclinical and clinical studies have indicated that neuroinflammation can play a role in PD pathophysiology, being associated with its onset and progression. Nevertheless, several key points concerning the neuroinflammatory process in PD remain to be answered. Bearing this in mind, in the present review, we cover the impact of neuroinflammation on PD by exploring the role of inflammatory cells (i.e., microglia and astrocytes) and the interconnections between the brain and the peripheral system. Furthermore, we discuss both the innate and adaptive immune responses regarding PD pathology and explore the gut–brain axis communication and its influence on the progression of the disease.The present work was supported by Prémios Santa Casa Neurociências— Prize Mantero Belard for Neurodegenerative Diseases Research (MB-28-2019), the Portuguese Foundation for Science and Technology (FCT) to S.M. (CEECIND/01902/2017) and F.G.T. (2021.00643.CEECIND), the European Regional Development Fund (FEDER), the Competitiveness Internationalization Operational Programme (POCI), national funds through the Foundation for Science and Technology (FCT), under the scope of projects UIDB/50026/2020, UIDP/50026/2020, POCI-01-0145-FEDER-029751, and EXPL/MED-PAT/0931/2021, project NORTE-01-0145-FEDER-000023, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), and under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER)

Citalopram administration does not promote function or histological recovery after spinal cord injury

Citalopram is a selective serotonin reuptake inhibitor, and although widely used as an antidepressant, this drug has also demonstrated interesting repairing properties leading to motor recovery and pathology amelioration in animal models of stroke and degeneration. Here, we tested the efficacy of both 7-day and 8-week citalopram treatment in a contusive spinal cord injury (SCI) rat model. A combination of behavioral tests, histological and serum cytokine analysis was used to assess overall recovery. Despite promoting a mild reduction of inflammatory cells as well as an early, but transient increase of specific serum cytokines, citalopram administration showed no overall beneficial effects on motor performance or lesion extension. Our results do not support citalopram treatment as a therapeutic strategy for SCI.This research was funded by Prémios Santa Casa Neurociências—Prize Melo e Castro for SpinalCord Injury Research, Grant Number MC-04/17 and the Portuguese Foundation for Science and Technology(FCT) through the Scientific Employment Stimulus to N. Silva and S. Monteiro (CEECIND/04794/2017 and CEECIND/01902/2017) and fellowships to RL (PD/BDE/127836/2016); EDG (SFRH/BD/103075/2014);NLV (SFRH/BD/136952/2018) and R.AS (PDE/BDE/113596/2015).This work was also funded by FEDER,through the Competitiveness Internalization Operational Program (POCI) and by National funds, throughthe Foundation for Sciences and Technology (FCT), under the scope of the projects POCI-01-0145-FEDER-007038,POCI-01-0145-FEDER-029206, POCI-01-0145-FEDER-029751 and PTDC/BTM-MAT/29968/2017. This work hasbeen funded by ICVS Scientific Microscopy Platform, member of the national infrastructure PPBI - PortuguesePlatform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122; by National funds, through the Foundationfor Science and Technology (FCT) - project UIDB/50026/2020 and UIDP/50026/2020; and by the projectsNORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Program (NORTE2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF)

Influence of different ECM-like hydrogels on neurite outgrowth induced by adipose tissue-derived stem cells

Mesenchymal stem cells (MSCs) have been proposed for spinal cord injury (SCI) applications due to their capacity to secrete growth factors and vesicles-secretome-that impacts important phenomena in SCI regeneration. To improve MSC survival into SCI sites, hydrogels have been used as transplantation vehicles. Herein, we hypothesized if different hydrogels could interact differently with adipose tissue-derived MSCs (ASCs). The efficacy of three natural hydrogels, gellan gum (functionalized with a fibronectin peptide), collagen, and a hydrogel rich in laminin epitopes (NVR-gel) in promoting neuritogenesis (alone and cocultured with ASCs), was evaluated in the present study. Their impact on ASC survival, metabolic activity, and gene expression was also evaluated. Our results indicated that all hydrogels supported ASC survival and viability, being this more evident for the functionalized GG hydrogels. Moreover, the presence of different ECM-derived biological cues within the hydrogels appears to differently affect the mRNA levels of growth factors involved in neuronal survival, differentiation, and axonal outgrowth. All the hydrogel-based systems supported axonal growth mediated by ASCs, but this effect was more robust in functionalized GG. The data herein presented highlights the importance of biological cues within hydrogel-based biomaterials as possible modulators of ASC secretome and its effects for SCI applications.This study is funded by Prémios Santa Casa Neurociências—Prize Melo e Castro for Spinal Cord Injury Research. This is also partially funded by EU-FP7-Health-2011-Collaborative Project 278612, Biohybrid—Templates for peripheral nerve regeneration, and Portuguese Foundation for Science and Technology (IF Development Grant to A. J. Salgado; postdoctoral fellowship to N. A. Silva—SFRH/BPD/97701/2013; PhD fellowships of R. C. Assunção-Silva and E. D. Gomes—PDE/BDE/113596/2015 and SFRH/BD/103075/2014, resp.). This article is a result of the project (NORTE-01-0145-FEDER-000013) supported by the Norte Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF); Cofinanciado pelo ProgramaOperacional Regional do Norte(ON.2 SR&TD Integrated Program—NORTE-07-0124-FEDER-000021), ao abrigo do Quadro de Referência Estratégico Nacional (QREN), através do Fundo Europeude Desenvolvimento Regional (FEDER); Projeto Estratégico—LA 26–2011-2012 and Projeto Estratégico—LA 26–2013-2014 cofinanciado por fundos nacionais, através da Fundação para a Ciência e a Tecnologia (PEst-C/SAU/LA0026/2011; PEst-C/SAU/LA0026/2013), e pelo Fundo Europeu de Desenvolvimento Regional (FEDER), através do COMPETE (FCOMP-01-0124-FEDER-022724; FCOMP-01-0124-FEDER-037298). The authors would like to thank Professor Jeffrey Gimble at the Tulane University Center for Stem Cell Research and Regenerative Medicine and LaCell LLC (New Orleans, Louisiana, USA) for kindly providing the ASCs used in this study.info:eu-repo/semantics/publishedVersio

Impact of mesenchymal stem cells' secretome on glioblastoma pathophysiology

Background: Glioblastoma (GBM) is a highly aggressive primary brain cancer, for which curative therapies are not available. An emerging therapeutic approach suggested to have potential to target malignant gliomas has been based on the use of multipotent mesenchymal stem cells (MSCs), either unmodified or engineered to deliver anticancer therapeutic agents, as these cells present an intrinsic capacity to migrate towards malignant tumors. Nevertheless, it is still controversial whether this innate tropism of MSCs towards the tumor area is associated with cancer promotion or suppression. Considering that one of the major mechanisms by which MSCs interact with and modulate tumor cells is via secreted factors, we studied how the secretome of MSCs modulates critical hallmark features of GBM cells. Methods: The effect of conditioned media (CM) from human umbilical cord perivascular cells (HUCPVCs, a MSC population present in the Wharton's jelly of the umbilical cord) on GBM cell viability, migration, proliferation and sensitivity to temozolomide treatment of U251 and SNB-19 GBM cells was evaluated. The in vivo chicken chorioallantoic membrane (CAM) assay was used to evaluate the effect of HUCPVCs CM on tumor growth and angiogenesis. The secretome of HUCPVCs was characterized by proteomic analyses. Results: We found that both tested GBM cell lines exposed to HUCPVCs CM presented significantly higher cellular viability, proliferation and migration. In contrast, resistance of GBM cells to temozolomide chemotherapy was not significantly affected by HUCPVCs CM. In the in vivo CAM assay, CM from HUCPVCs promoted U251 and SNB-19 tumor cells growth. Proteomic analysis to characterize the secretome of HUCPVCs identified several proteins involved in promotion of cell survival, proliferation and migration, revealing novel putative molecular mediators for the effects observed in GBM cells exposed to HUCPVCs CM. Conclusions: These findings provide novel insights to better understand the interplay between GBM cells and MSCs, raising awareness to potential safety issues regarding the use of MSCs as stem-cell based therapies for GBM.The authors would like to acknowledge the funding agencies that supported this work: Fundacao para a Ciencia e Tecnologia (FCT), Portugal, projects reference: PTDC/SAU-GMG/113795/2009 (BMC); SFRH/BD/88121/2012 (JVdC); SFRH/BD/103075/2014 (EDG); IF/00601/2012 (BMC); IF/00111/2013 (AJS); SFRH/BD/81495/2011 (SIA); PTDC/NEU-NMC/0205/2012, PTDC/NEUSCC/ 7051/2014, PEst-C/SAU/LA0001/2013-2014 and UID/NEU/04539/2013 (BM); Fundacao Calouste Gulbenkian (BMC); Liga Portuguesa Contra o Cancro (BMC); " COMPETE Programa Operacional Factores de Competitividade, QREN, the European Union (FEDER-Fundo Europeu de Desenvolvimento Regional) and by The National Mass Spectrometry Network (RNEM) under the contract REDE/1506/REM/2005; 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-FEDER-007038; and 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). The funding body did not have a role in the design of the study, in collection, analysis or interpretation of data, or in writing the manuscript

Induction of neurite outgrowth in 3D hydrogel-based environments

The ability of peripheral nervous system (PNS) axons to regenerate and re-innervate their targets after an injury has been widely recognized. However, despite the considerable advances made in microsurgical techniques, complete functional recovery is rarely achieved, especially for severe peripheral nerve injuries (PNIs). Therefore, alternative therapies that can successfully repair peripheral nerves are still essential. In recent years the use of biodegradable hydrogels enriched with growth-supporting and guidance cues, cell transplantation, and biomolecular therapies have been explored for the treatment of PNIs. Bearing this in mind, the aim of this study was to assess whether Gly-Arg-Gly-Asp-Ser synthetic peptide (GRGDS)-modified gellan gum (GG) based hydrogels could foster an amenable environment for neurite/axonal growth. Additionally, strategies to further improve the rate of neurite outgrowth were also tested, namely the use of adipose tissue derived stem cells (ASCs), as well as the glial derived neurotrophic factor (GDNF). In order to increase its stability and enhance its bioactivity, the GDNF was conjugated covalently to iron oxide nanoparticles (IONPs). The impact of hydrogel modification as well as the effect of the GDNF-IONPs on ASC behavior was also screened. The results revealed that the GRGDS-GG hydrogel was able to support dorsal root ganglia (DRG)-based neurite outgrowth, which was not observed for non-modified hydrogels. Moreover, the modified hydrogels were also able to support ASCs attachment. In contrast, the presence of the GDNF-IONPs had no positive or negative impact on ASC behavior. Further experiments revealed that the presence of ASCs in the hydrogel improved axonal growth. On the other hand, GDNF-IONPs alone or combined with ASCs significantly increased neurite outgrowth from DRGs, suggesting a beneficial role of the proposed strategy for future applications in PNI regenerative medicineEU-FP7-Health-2011-collaborative project 278612, Biohybrid—Templates for peripheral nerve regeneration; Prémios Santa Casa Neurociências—Prize Melo e Castro for Spinal Cord Injury Research; Portuguese Foundation for Science and Technology (IF Development Grant to A J Salgado; Post-Doctoral fellowship to N A Silva — SFRH/BPD/97701/2013); co-funded by Programa Operacional Regional do Norte (ON.2—O Novo Norte), ao abrigo do Quadro de Referência Estratégico Nacional (QREN), através do Fundo Europeu de Desenvolvimento Regional (FEDER); Professor Jeffrey Gimble at the Tulane University Center for Stem Cell Research and Regenerative Medicine and LaCell LLC (New Orleans, Louisiana, USA) for kindly providing the ASCs used in this studyinfo:eu-repo/semantics/publishedVersio

Modulation of bone marrow mesenchymal stem cell secretome by ECM-like hydrogels

It has been demonstrated that bone marrow mesenchymal stem cell (BM-MSCs) transplantation has beneficial effects on several central nervous system (CNS) debilitating conditions. Growing evidence indicate that trophic factors secreted by these cells are the key mechanism by which they are acting. These cells are frequently used in combination with 3D artificial matrices, for instance hydrogels, in tissue engineering-based approaches. However, so far, no study has been reported on the influence of such matrices, namely the presence or absence of extracellular matrix motifs, on BM-MSCs secretome and its effects in neuronal cell populations. In this sense, we herein studied the impact of a hydrogel, gellan gum, on the behavior and secretome of BM-MSCs, both in its commercial available form (commonly used in tissue engineering) and in a fibronectin peptide-modified form. The results showed that in the presence of a peptide in the gellan gum hydrogel, BM-MSCs presented higher proliferation and metabolic activity than in the regular hydrogel. Moreover, the typical spindle shape morphology of BM-MSCs was only observed in the modified hydrogel. The effects of the secretome of BM-MSCs were also affected by the chemical nature of the extracellular matrix. BM-MSCs cultured in the modified hydrogel were able to secrete factors that induced higher metabolic viabilities and neuronal cell densities, when compared to those of the unmodified hydrogel. Thus adding a peptide sequence to the gellan gum had a significant effect on the morphology, activity, proliferation and secretome of BM-MSCs. These results highlight the importance of mimicking the extracellular matrix when BM-MSCs are cultured in hydrogels for CNS applications.We would like to acknowledge the funds attributed by the Portuguese Foundation for Science and Technology (FCT) (Grant No PTDC/SAU-BMA/114059/2009; pre-doctoral fellowships to N.A. Silva, SFRH/BD/40684/2007; Ciencia 2007 Program to A.J. Salgado; PEst-C/SAU/LA0001/2013-2014 and RNEM-REDE/1506/REM/2005). This work was partially funded by EU-FP7-Health-2011-Collaborative Project 278612, Biohybrid Templates for Peripheral Nerve Regeneration, and co-funded by Programa Operacional Regional do Norte (ON.2 - O Novo Norte), ao abrigo do Quadro de Referencia Estrategico Nacional (QREN), atraves do Fundo Europeu de Desenvolvimento Regional (FEDER)

Bioengineered cell culture systems of central nervous system injury and disease

Cell culture systems, either 2D or explant based, have been pivotal to better understand the pathophysiology of several central nervous system (CNS) disorders. Recently, bioengineered cell culture systems have been proposed as an alternative to the traditional setups. These innovative systems often combine different cell populations in 3D environments that more closely recapitulate the different niches that exist within the developing or adult CNS. Given the importance of such systems for the future of CNS-related research, we discuss here the most recent advances in the field, particularly those dealing with neurodegeneration, neurodevelopmental disorders, and trauma.Financial support is acknowledged from Prémios Santa Casa Neurociências – Prize Melo e Castro for Spinal Cord Injury Research; Portuguese Foundation for Science and Technology [Doctoral fellowship (SFRH/BD/103075/2014) to E.D.G.; IF Development Grant to A.J.S.; Starting Grant to F. Marques; PostDoctoral fellowship SFRH/BPD/97701/2013 to N.A.S.]; this work was co-funded by Programa Operacional Regional do Norte (ON.2 – O Novo Norte), ao abrigo do Quadro de Referência Estratégico Nacional (QREN), através do Fundo Europeu de Desenvolvimento Regional (FEDER).info:eu-repo/semantics/publishedVersio