Mesenchymal stem cells secretome in Parkinson’s disease regenerative medicine

Dissertação de mestrado em Ciências da SaúdeParkinson’s disease (PD) represents the second most common neurodegenerative brain disorder, which is clinically characterized by the progressive degeneration of dopaminergic neurons (DAergic neurons), mainly in the nigrostriatal pathway, leading to the appearance of characteristic motor and non-motor symptoms. Currently, pharmacological and surgical treatments are the most common approaches for the treatment of PD. However, so far, all of these treatments are focused on reducing the symptoms. In fact, they do not slow down or reverse the degenerative process, imposing the need for innovative therapeutical approaches. The use of adult stem cells cell-based strategy has emerged as a potential alternative therapy for PD, in which, among a number of promising stem cell sources, human mesenchymal stem cells (hMSCs) and neural progenitors cells (hNPCs) have stand out as a valid therapeutic option. Indeed, over the last years, a substantial effort has been performed in order to address the impact of hMSCs and hNPCs in central nervous system repair. Recently, and from an application point of view, several studies have claimed that the therapeutical effects of stem cells is mainly mediated by their trophic action namely, through their capacity of secreting a wide panel of neuroregulatory molecules (e.g. neurotrophic factors, cytokines, vesicles), which is defined as secretome. Thus, based in all these concepts, in this thesis we aimed to: 1) Characterize the secretome of hMSCs and hNPCs through proteomic-based approaches; 2) Determine the role of hMSCs and hNPCs secretome as a modulator of neuronal differentiation and 3) Investigate the effects of the hMSCs and hNPCs secretome in a rat model of PD, in comparison with cell transplantation. In vitro, experiments revealed that the secretome of hMSCs induced a more robust neuronal differentiation when compared to the one obtained from hNPCs. Additionally, it was also possible to observe that the injection of the secretome of both hMSCs and hNPCs in a 6-hydroxydopamine (6-OHDA)-rat model of PD potentiated the recovery of DAergic neurons (estimated by neuronal densities in substantia nigra and striatum) when compared to the untreated group 6-OHDA, and those transplanted with cells (hMSCs and hNPCs). Similar outcomes were observed in the motor performance of these animals as assessed by the rotarod and staircase tests. Finally, proteomic characterization of hMSCs and hNPCs secretome revealed that these cells were able to secrete important molecules with neuroregulatory actions such as, Galectin-1, 14-3-3 proteins, PEDF, DJ-1, whereby may support the effects observed both in vitro and in vivo. Overall, we concluded that the use of secretome per se was able to partially revert the motor phenotype and the neuronal structure of PD animals, indicating that the secretome of stem cells could represent a novel therapeutic tool for the treatment of PD.A doença de Parkinson (DP) é clinicamente caracterizada pela degeneração progressiva dos neurónios dopaminérgicos (ND), principalmente na via nigroestriatal, levando ao aparecimento dos sintomas motores e não motores da doença. Atualmente, os tratamentos farmacológicos e cirúrgicos representam a abordagem mais comum no tratamento da DP. Contudo, estes estão apenas focados na redução sintomática da doença, não retardando ou revertendo o processo degenerativo, sendo assim necessária a criação de abordagens terapêuticas inovadoras. O uso de células estaminais adultas tem emergido como uma potencial terapia alternativa para a DP. Dentro destas, as células humanas estaminais mesenquimatosas (hMSCs) e as células progenitoras neurais (hNPCs) têm emergido como uma válida opção terapêutica. Do ponto de vista de aplicação destas duas populações de células estaminais na DP, diversos estudos demonstraram que o seu efeito terapêutico é essencialmente mediado pela sua ação trófica, isto é, através da sua capacidade de segregar um vasto painel de moléculas neuroreguladoras (p.ex. fatores neurotróficos, citoquinas e vesículas), definido como secretoma. Assim, a presente tese teve como principais objetivos: 1) Caraterizar o secretoma de hMSCs e hNPCs através de análises de proteómica; 2) Determinar o efeito do secretoma de hMSCs e hNPCs como um modulador da diferenciação neuronal e 3) Investigar os efeitos do secretoma de hMSCs e hNPCs num modelo de rato da DP (6-OHDA), em comparação com a transplantação de células. In vitro, verificou–se uma maior diferenciação neuronal promovida pelo secretoma de hMSCs quando comparado com o das hNPCs. In vivo, observou-se que a injeção do secretoma quer de hMSCs quer de hNPCs num modelo de DP em ratos (6-OHDA) potenciou a recuperação dos ND (avaliado por densidades neuronais na substância negra e estriado) quando comparado com o grupo não tratado, e com os grupos transplantados com células (hMSCs e hNPCs). Resultados semelhantes foram observados no desempenho motor destes animais, avaliado pelos testes rotarod e staircase. Por último, a caracterização proteómica do secretoma de hMSCs e hNPCs revelou que estas células são capazes de segregar moléculas com importantes ações neuroreguladoras tais como, Galactina-1, proteínas 14-3-3, PEDF, DJ-1, suportando desta forma os efeitos observados tanto in vitro como in vivo. Em suma, podemos concluir que a utilização de secretoma por si só foi capaz de reverter parcialmente o fenótipo motor e a estrutura neuronal de animais parkinsonianos, indicando que o secretoma das células estaminais pode representar uma nova abordagem terapêutica para o tratamento da DP

Old and new challenges in Parkinson's disease therapeutics

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the degeneration of dopaminergic neurons and/or loss od neuronal projections, in several dopaminergic networks. Current treatments for idiopathic PD rely mainly on the use of pharmacologic agents to improve motor symptomatology of PD patients. Nevertheless, so far PD remains an incurable disease. Therefore, it is of utmost importance to establish new therapeutic strategies for PD treatment. Over the last 20 years, several molecular, gene and cell/stem-cell therapeutic approaches have been developed with the aim of counteracting or retarding PD progression. The scope of this review is to provide an overview of PD related therapies and major breakthroughs achieved within this field. In order to do so, this review will start by focusing on PD characterization and current treatment options covering thereafter molecular, gene and cell/stem cell-based therapies that are currently being studied in animal models of PD or have recently been tested in clinical trials. Among stem cell-based therapies, those using MSCs as possible disease modifying agents for PD therapy and, specifically, the MSCs secretome contribution to meet the clinical challenge of counteracting or retarding PD progression, will be more deeply explored.Portuguese Foundation for Science and Technology (FCT) for the PhD fellowship attributed to A.O. Pires (Reference: SFRH/BD/33900/2009) and the IF development grant to A.J. Salgado (Reference: IF/00111/2013). 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). 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-007038info:eu-repo/semantics/publishedVersio

Cell secretome based approaches in Parkinson's disease regenerative medicine

The available therapeutic strategies for Parkinson's disease (PD) rely only on the amelioration of the symptomatology of the disease, lacking neuroprotection or neuroregeneration capacities. Therefore, the development of disease modifying strategies is extremely important for the management of PD in the long term. Areas covered: In this review, the authors provide an overview of the current therapeutic approaches for PD and the emerging use of stem cell transplantation as an alternative. Particularly, the use of the secretome from mesenchymal stem cells (MSCs), as well as some methodologies used for the modulation of their paracrine signaling, will be discussed. Indeed, there is a growing body of literature highlighting the use of paracrine factors and vesicles secreted from different cell populations, for this purpose. Expert opinion: Secretome from MSCs has shown its potential as a therapy for PD. Nevertheless, in the coming years, research should focus in several key aspects to enable the translation of this strategy from the bench to the bedside.Portuguese Foundation for Science and Technology (FCT): Ciência 2007 Program and IF Development Grant [IF/00111/2013] to AJ Salgado, PhD scholarships attributed to C.R. Marques [PD/BDE/127833/2016], A. Marote [PDE/BDE/113598/2015] and B. Mendes-Pinheiro [SFRH/BD/120124/2016] and Post-Doctoral Fellowship to F.G. Teixeira [SFRH/BPD/118408/2016]. This article has been developed under the scope of the project NORTE-01-0145-FEDER-000023, 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 FCT, under the scope of the project [POCI-01-0145-FEDER-007038]info:eu-repo/semantics/publishedVersio

Exploiting the impact of the secretome of MSCs isolated from different tissue sources on neuronal differentiation and axonal growth

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.The authors acknowledge the financial support by Premios Santa Casa Neurociencias - 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 Assunçao Silva ~ and B. Mendes-Pinheiro, respectively; Post-doctoral fellowhip to F.G. Teixeira and Patrícia Patrício - SFRH/BPD/118408/2016 and SFRH/BPD/116249/2016; 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 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). 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, under the scope of the project POCI-01-0145-FEDER-007038. HUCPVCs and ASCs were kindly provided by Prof. John E. Davies (University of Toronto, Canada) and Prof. Jeff Gimble (LaCell Inc, USA).info:eu-repo/semantics/publishedVersio

Unveiling the Differences of Secretome of Human Bone Marrow Mesenchymal Stem Cells, Adipose Tissue-Derived Stem Cells, and Human Umbilical Cord Perivascular Cells: A Proteomic Analysis

The use of human mesenchymal stem cells (hMSCs) has emerged as a possible therapeutic strategy for CNS-related conditions. Research in the last decade strongly suggests that MSC-mediated benefits are closely related with their secretome. Studies published in recent years have shown that the secretome of hMSCs isolated from different tissue sources may present significant variation. With this in mind, the present work performed a comparative proteomic-based analysis through mass spectrometry on the secretome of hMSCs derived from bone marrow (BMSCs), adipose tissue (ASCs), and human umbilical cord perivascular cells (HUCPVCs). The results revealed that BMSCs, ASCs, and HUCPVCs differed in their secretion of neurotrophic, neurogenic, axon guidance, axon growth, and neurodifferentiative proteins, as well as proteins with neuroprotective actions against oxidative stress, apoptosis, and excitotoxicity, which have been shown to be involved in several CNS disorder/injury processes. Although important changes were observed within the secretome of the cell populations that were analyzed, all cell populations shared the capability of secreting important neuroregulatory molecules. The difference in their secretion pattern may indicate that their secretome is specific to a condition of the CNS. Nevertheless, the confirmation that the secretome of MSCs isolated from different tissue sources is rich in neuroregulatory molecules represents an important asset not only for the development of future neuroregenerative strategies but also for their use as a therapeutic option for human clinical trials.Foundation Calouste de Gulbenkian for the funds attributed to A.J.S.; Portuguese Foundation for Science and Technology (FCT) PhD fel- lowships attributed to A.O.P. (SFRH/BD/33900/2009) and S.I.A. (SFRH/BD/81495/2011) and Ciência 2007, IF Development Grant attributed to A.J.S., and projects PTDC/ NEU-NMC/0205/2012, UID/NEU/04539/2013; cofinanced by COMPETE Programa Operacional Factores de Compe- titividade; and by The National Mass Spectrometry Network (RNEM) (REDE/1506/REM/2005); Prémios Santa Casa Neurociências—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), and através do Fundo Europeu de Desenvolvimento Regional (FEDER). The authors also would like to thank Professor J.E.D. (University of Toronto, Canada) and Professor J.M.G. (Tulane University) for kindly providing HUCPVCs and ASCs, respectivelyinfo:eu-repo/semantics/publishedVersio

Constitutive deficiency of the neurogenic hippocampal modulator AP2γ promotes anxiety-like behavior and cumulative memory deficits in mice from juvenile to adult periods

The transcription factor activating protein two gamma (AP2γ) is an important regulator of neurogenesis both during embryonic development as well as in the postnatal brain, but its role for neurophysiology and behavior at distinct postnatal periods is still unclear. In this work, we explored the neurogenic, behavioral, and functional impact of a constitutive and heterozygous AP2γ deletion in mice from early postnatal development until adulthood. AP2γ deficiency promotes downregulation of hippocampal glutamatergic neurogenesis, altering the ontogeny of emotional and memory behaviors associated with hippocampus formation. The impairments induced by AP2γ constitutive deletion since early development leads to an anxious-like phenotype and memory impairments as early as the juvenile phase. These behavioral impairments either persist from the juvenile phase to adulthood or emerge in adult mice with deficits in behavioral flexibility and object location recognition. Collectively, we observed a progressive and cumulative impact of constitutive AP2γ deficiency on the hippocampal glutamatergic neurogenic process, as well as alterations on limbic-cortical connectivity, together with functional behavioral impairments. The results herein presented demonstrate the modulatory role exerted by the AP2γ transcription factor and the relevance of hippocampal neurogenesis in the development of emotional states and memory processes.H2020 -“la Caixa” Foundation(101003187

Preclinical assessment of mesenchymal-stem-cell-based therapies in spinocerebellar ataxia type 3

The low regeneration potential of the central nervous system (CNS) represents a challenge for the development of new therapeutic strategies for neurodegenerative diseases, including spinocerebellar ataxias. Spinocerebellar ataxia type 3 (SCA3)—or Machado–Joseph disease (MJD)—is the most common dominant ataxia, being mainly characterized by motor deficits; however, SCA3/MJD has a complex and heterogeneous pathophysiology, involving many CNS brain regions, contributing to the lack of effective therapies. Mesenchymal stem cells (MSCs) have been proposed as a potential therapeutic tool for CNS disorders. Beyond their differentiation potential, MSCs secrete a broad range of neuroregulatory factors that can promote relevant neuroprotective and immunomodulatory actions in different pathophysiological contexts. The objective of this work was to study the effects of (1) human MSC transplantation and (2) human MSC secretome (CM) administration on disease progression in vivo, using the CMVMJD135 mouse model of SCA3/MJD. Our results showed that a single CM administration was more beneficial than MSC transplantation—particularly in the cerebellum and basal ganglia—while no motor improvement was observed when these cell-based therapeutic approaches were applied in the spinal cord. However, the effects observed were mild and transient, suggesting that continuous or repeated administration would be needed, which should be further tested.This research was funded by the National Ataxia Foundation (NAF) and by Portuguese national funds, through the Foundation for Science and Technology (FCT)—projects UIDB/50026/2020, UIDP/50026/2020, POCI-01-0145-FEDER-029206, and through the Santa Casa Neuroscience Awards (Santa Casa da Misericórdia Lisboa)—project MC-04/17. Additionally, this project was funded by the ICVS Scientific Microscopy Platform, a member of the national infrastructure PPBI—Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122). S.C.S. received an individual fellowship within the project TUBITAK/0007/2014. The FCT funded individual fellowships to J.S C., A.N.-C., B.M.- P., F.G.T., R.L., S.M., N.A.S., C.S.-C., and S.D.-S. (SFRH/BD/140624/2018, SFRH/BPD/118779/2016, SFRH/BD/120124/2016, SFRH/BPD/118408/2016, PD/BDE/127836/2016, CEECIND/01902/2017, CEECIND/04794/2017, CEECIND/03887/2017, and CEECIND/00685/2020)

Estudo do papel neuroprotetor do secretoma de células estaminais mesenquimatosas derivadas da medula óssea usando modelos in vitro e in vivo da doença de Parkinson

Tese de doutoramento em Ciências da SaúdeA doença de Parkinson (DP) é a doença motora mais comum, afetando cerca de 6.1 milhões de pessoas mundialmente. Sintomas motores como bradicinesia, tremor em repouso e rigidez muscular são as principais características clínicas da DP. No entanto, sintomas não motores são também determinantes na qualidade de vida e da incapacidade geral do doente. Perda neuronal na substância nigra pars compacta (SNpc) que causa uma deficiência de dopamina (DA) ao nível do estriado, bem como inclusões intracelulares que contêm agregados de α-sinucleína, são as principais características neuropatológicas da doença. As abordagens terapêuticas atualmente disponíveis estão focadas na substituição dos níveis de DA, mas não são capazes de retardar, interromper ou reverter a neurodegeneração subjacente à DP. As células estaminais mesenquimatosas (MSCs) são uma das fontes celulares mais amplamente estudadas para aplicações em medicina regenerativa, particularmente devido à libertação de fatores solúveis e vesículas, coletivamente conhecido como secretoma. O principal objetivo desta tese foi estudar o potencial terapêutico do secretoma de MSCs derivadas da medula óssea (BM-MSCs) usando diferentes modelos da doença. Numa primeira fase, usamos um sistema de organóides humanos específicos do mesencéfalo para modelar a DP in vitro com recurso a dois modelos diferentes, nomeadamente, um modelo induzido pela neurotoxina 6-hidroxidopamina (6-OHDA), e um modelo genético da DP ligado a uma mutação na proteína DJ-1. O secretoma das BM-MSCs foi capaz de proteger a perda neuronal dopaminérgica, bem como a fragmentação das neurites dopaminérgicas, sendo estes efeitos mais evidentes no modelo genético. Em seguida, otimizamos os parâmetros de cultura das BM-MSCs através da introdução do lisado plaquetário humano (hPL) como suplemento para a sua expansão em cultura. In vivo, o nosso objetivo passou por realizar uma extensa caracterização do modelo 6-OHDA unilateral e intra-estriatal de ratinho, para mais tarde avaliar os efeitos do secretoma das BM-MSCs, comparando duas vias diferentes de administração de secretoma: injeções intracerebrais (administração única) com administração sistémica repetida. O secretoma das BM-MSCs levou à recuperação da função motora e proteção dos neurónios dopaminérgicos, no entanto, as múltiplas administrações sistémicas mostraram um efeito terapêutico de maior magnitude, sendo este resultado da extrema relevância do ponto de vista da aplicação clínica. Além disso, a análise proteómica revelou a identificação de várias proteínas neuroreguladoras no secretoma das BM-MSCs expandidas em hPL, reforçando o uso deste suplemento como uma alternativa viável face às abordagens mais convencionais. Concluindo, o nosso estudo produziu contribuições importantes para projetar novas abordagens clínicas livres de células para a DP com base na administração de secretoma.Parkinson’s disease (PD) is the most common movement disorder that affects 6.1 million individuals worldwide. Motor symptoms such as bradykinesia, rest tremor, and rigidity, are the core PD clinical features. Nonetheless, non-motor symptoms have been recognized as determinants of the patient’s quality of life and overall disability. Neuronal loss in the substantia nigra pars compacta (SNpc), which causes striatal dopamine (DA) deficiency, and intracellular inclusions containing aggregates of α-synuclein are the neuropathological hallmarks of PD. Currently available therapeutical approaches are focused on replacing DA levels, which are not able to slow, halt or reverse PD underlying neurodegeneration. Mesenchymal stem cells (MSCs) are one of the most extensively studied cell sources for regenerative medicine applications, particularly due to the release of soluble factors and vesicles, known as secretome. The main goal of this thesis was to address the therapeutic potential of the secretome collected from bone marrow-derived MSCs (BM-MSCs) using different models of the disease. Firstly, we took advantage of an optimized human midbrain-specific organoid system to model PD in vitro using two different models namely, a neurotoxin induced model through 6-hydroxydopamine (6-OHDA) exposure, and a genetic PD model linked to a mutation in the DJ-1 protein. The secretome of BM-MSCs was able to protect from the dopaminergic neuronal loss, as well as from induced fragmentation of dopaminergic neurites, being these effects more evident in the genetic model. Following on this we further optimize the culturing parameters of BM-MSCs, through the introduction of human platelet lysate (hPL) as a supplement for their expansion in culture. In vivo, we aimed to perform an extensive characterization of a unilateral intrastriatal 6-OHDA PD mouse model, to further evaluate the effects of BM-MSC secretome comparing two different routes of secretome administration: intracerebral injections (single administration) with multiple systemic administration. The BM-MSC secretome led to motor function recovery and dopaminergic loss protection, however, multiple systemic administrations showed a higher magnitude therapeutical effect being this result of the extreme relevance from the clinical application point of view. Moreover, proteomic analysis revealed the identification of several important neuroregulatory proteins in the secretome of hPL expanded BM-MSCs, reinforcing the use of this supplement as a feasible alternative over more conventional approaches. Overall, our study made important contributions to designing new cell-free clinical approaches for PD based on secretome administration.Financial support was provided by grants from the Foundation for Science and Technology (FCT/SFRH/BD/120124/2016 and COVID/BD/151719/2021 to Bárbara Pinheiro). ICVS Scientific Microscopy Platform, member of the national infrastructure PPBI-Portuguese Platform of Bioimaging PPBI-POCI-01-0145- FEDER-022122; National funds, through the FCT-project UIDB/50026/2020 and UIDP/50026/2020; the projects NORTE 01-0145-FEDER-000013, NORTE-01-0145-FEDER-000023, and NORTE-01-0145-FEDER-000039, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF); Competitiveness Internationalization Operational Programme (POCI), and by National funds, through the FCT, under the scope of the project POCI-01-0145-FEDER-032619. This work was also partially funded by Iniciativa Ibérica de Investigación y Innovación Biomedica and Fundación “a Caixa”, under the scope of the “la Caixa” Foundation Health Research 2020 Call (HR20-00249). Part of the work was also developed at Luxembourg Centre for Systems and Biomedicine (LCSB) at Luxembourg University and financially supported by the Fonds National de la Recherche (FNR) Luxembourg (INTER/MERA/17/11760144, FNR/PoC16/11559169). Furthermore, the project is part of the The National Centre of Excellence in Research on Parkinson's Disease (NCER-PD) which is funded by the Luxembourg National Research Fund (FNR/NCER13/BM/11264123). This project has received funding from the European Union’s Horizon 2020 research and innovation programme H2020-FETPROACT-2018-01 under grant agreement No 824070

Unilateral intrastriatal 6-hydroxydopamine lesion in mice: a closer look into non-motor phenotype and glial response

Parkinson’s disease (PD) is a prevalent movement disorder characterized by the progressive loss of dopaminergic neurons in substantia nigra pars compacta (SNpc). The 6-hydroxydopamine (6-OHDA) lesion is still one of the most widely used techniques for modeling Parkinson’s disease (PD) in rodents. Despite commonly used in rats, it can be challenging to reproduce a similar lesion in mice. Moreover, there is a lack of characterization of the extent of behavioral deficits and of the neuronal loss/neurotransmitter system in unilateral lesion mouse models. In this study, we present an extensive behavioral and histological characterization of a unilateral intrastriatal 6-OHDA mouse model. Our results indicate significant alterations in balance and fine motor coordination, voluntary locomotion, and in the asymmetry’s degree of forelimb use in 6-OHDA lesioned animals, accompanied by a decrease in self-care and motivational behavior, common features of depressive-like symptomatology. These results were accompanied by a decrease in tyrosine hydroxylase (TH)-labelling and dopamine levels within the nigrostriatal pathway. Additionally, we also identify a marked astrocytic reaction, as well as proliferative and reactive microglia in lesioned areas. These results confirm the use of unilateral intrastriatal 6-OHDA mice for the generation of a mild model of nigrostriatal degeneration and further evidences the recapitulation of key aspects of PD, thereby being suitable for future studies beholding new therapeutical interventions for this disease.This work was supported by the Foundation for Science and Technology (FCT) through PhD Fellowships attributed to B.M.P. (SFRH/BD/120124/2016), E.L.C. (SFRH/BD/131278/2017), J.C. (SFRH/BD/5813/2020), S.B.A. (PD/BDE/135568/2018), D.M.F. (SFRH/BD/147947/2019), and CEEC attributed to C.S.C. (CEECIND/03887/2017), S.D.S. (CEECIND/00685/2020), and L.P. (CEECIND/ 02855/2020). ICVS Scientific Microscopy Platform, member of the national infrastructure PPBI— Portuguese Platform of Bioimaging PPBI-POCI-01-0145-FEDER-022122; National funds, through the FCT—project UIDB/50026/2020 and UIDP/50026/2020; the projects NORTE-01-0145-FEDER-000013 and NORTE-01-0145-FEDER-000023, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF); Competitiveness Internationalization Operational Programme (POCI), and by National funds, through the FCT, under the scope of the project POCI-01-0145-FEDER-032619

Cellular Aging Secretes : a Comparison of Bone-Marrow-Derived and Induced Mesenchymal Stem Cells and Their Secretome Over Long-Term Culture

Mesenchymal stem cells (MSCs) hold promising therapeutic potential in several clinical applications, mainly due to their paracrine activity. The implementation of future secretome-based therapeutic strategies requires the use of easily accessible MSCs sources that provide high numbers of cells with homogenous characteristics. MSCs obtained from induced pluripotent stem cells (iMSCs) have been put forward as an advantageous alternative to the gold-standard tissue sources, such as bone marrow (BM-MSCs). In this study, we aimed at comparing the secretome of BM-MSCs and iMSCs over long-term culture. For that, we performed a broad characterization of both sources regarding their identity, proteomic secretome analysis, as well as replicative senescence and associated phenotypes, including its effects on MSCs secretome composition and immunomodulatory action. Our results evidence a rejuvenated phenotype of iMSCs, which is translated into a superior proliferative capacity before the induction of replicative senescence. Despite this significant difference between iMSCs and BM-MSCs proliferation, both untargeted and targeted proteomic analysis revealed a similar secretome composition for both sources in pre-senescent and senescent states. These results suggest that shifting from the use of BM-MSCs to a more advantageous source, like iMSCs, may yield similar therapeutic effects as identified over the past years for this gold-standard MSC source. Graphical Abstract: [Figure not available: see fulltext.]