Desvendando o potencial das células estaminais mesenquimatosas derivadas de iPSCs para o desenvolvimento de terapias livres de células para a doença de Parkinson

Tese de Doutoramento em Ciências da SaúdeA doença de Parkinson (DP) é a segunda doença neurodegenerativa mais comum mundialmente, caracterizada principalmente pela perda de neurónios dopaminérgicos na substancia nigra pars compacta (SNc), levando a uma desnervação do sistema nigroestriatal. A neurodegeneração na SNc e noutras áreas dopaminérgicas e não dopaminérgicas são clinicamente traduzidas em lentidão dos movimentos, rigidez e tremor em repouso, bem como manifestações não-motoras, como a depressão. Devido à sua complexidade e causa desconhecida, terapias modificadoras da doença, capazes de abrandar, interromper ou reverter a neurodegeneração inerente à DP, constituem uma necessidade não atendida na área. As células estaminais mesenquimatosas (MSCs) são uma população adulta de células estaminais que tem sido sugerida como uma abordagem terapêutica promissora para a DP, particularmente devido à libertação de fatores solúveis e vesículas, coletivamente designados como secretoma. Apesar da crescente evidência do potencial neuroprotetor do secretoma das MSCs, MSCs derivadas de tecidos, tais como a comumente utilizada medula óssea (BM-MSCs), têm uma capacidade proliferativa limitada e estão sujeitas a variabilidade do dador, dificultando assim a recolha de volumes de secretoma significativos para uma aplicação clínica abrangente. O principal objetivo desta tese é avaliar o potencial terapêutico do secretoma recolhido de uma fonte MSCs emergente e clinicamente adequada – as células estaminais pluripotentes induzidas (iPSCs). Para isso, gerámos iPSCs de sujeitos saudáveis e de pacientes com DP, através da reprogramação de fibroblastos e células mononucleares do sangue periférico. Posteriormente, as iPSCs obtidas de sujeitos saudáveis foram diferenciadas em MSCs (iMSCs) e o seu secretoma foi recolhido e comparado com o secretoma das BM-MSCs. Tanto o secretoma das BM-MSCs como o das iMSCs atenuaram os défices motores e a degeneração nigrostriatal num modelo de DP de rato baseado na exposição a 6-OHDA, apesar de não protegerem de maneira significativa neurónios dopaminergicos derivados de células estaminais embrionárias de murganho do mesmo insulto tóxico in vitro. A semelhança entre as duas fontes foi também revelada numa análise proteómica direcionada do secretoma das duas fontes, mostrando um perfil de secreção comparável de citocinas e fatores de crescimento, que podem mediar os seus efeitos neuroprotetores in vivo. Deste modo, o nosso estudo demonstra o potencial terapêutico do secretoma das iMSCs no contexto da DP, abrindo caminho para o uso desta fonte de MSCs emergente em testes clínicos subsequentes.Parkinson’s disease (PD) is the second most common neurodegenerative disease worldwide, mainly characterized by the loss of dopaminergic neurons from the substantia nigra pars compacta (SNc) leading to the denervation of the nigrostriatal tract. Neurodegeneration at the SNc and other dopaminergic and non-dopaminergic areas are clinically translated into slowness of movement, rigidity and tremor at rest, as well as nonmotor manifestations, such as depression. Due to its complexity and unknown etiology, disease-modifying therapies, able to slow, halt or reverse PD underlying neurodegeneration, are a large unmet need in the field. Mesenchymal stem cells (MSCs) are a population of adult of stem cells that has been put forward as a promising therapeutic approach for PD, particularly due to the release of soluble factors and vesicles, collectively known as secretome. Despite the growing evidence of the neuroprotective potential of MSCs secretome, tissue-derived MSCs, such as the commonly used bone marrow-derived MSCs (BM-MSCs), have limited proliferative capacity and are subjected to donor variability, thereby hindering the collection of significant volumes of secretome for a widespread clinical application. The main goal of this thesis was to address the therapeutic potential of the secretome collected from an emerging and clinically appropriate source of MSCs – induced pluripotent stem cells (iPSCs). For that, we have firstly generated iPSCs from heathy subjects and PD patients, through the reprograming of fibroblasts and peripheral blood mononuclear cells. Subsequently, iPSCs obtained from healthy subjects were differentiated into MSCs (iMSCs) and their secretome was collected and compared to BM-MSCs secretome. Both BM-MSCs and iMSCs secretome were able to attenuate motor deficits and nigrostriatal degeneration in a rat model of PD based on 6-OHDA exposure, although they did not significantly protect mouse embryonic stem cells (mESCs)-derived dopaminergic neurons from the same toxic insult in vitro. The similarity between both sources was further revealed in a targeted proteomic analysis of the secretome from both sources, showing a comparable secretion profile of neuroregulatory cytokines and growth factors, which might mediate their neuroprotective effects in vivo. Therefore, our study demonstrates the therapeutic potential of iMSCs secretome in the context of PD, paving the way for the use of this emerging MSC source in subsequent clinical testing.The work presented in this thesis was performed at the Life and Health Sciences Research Institute (ICVS) and at the Bn’ML – Behavioral & Molecular Lab, at the School of Medicine, University of Minho. Financial support was provided by Portuguese Foundation for Science and Technology (FCT): Doctoral fellowship (PDE/BDE/113598/2015) to A. Marote; IF Development Grants to A. J. Salgado (IF/00111/2013) and L. Pinto (IF/01079/2014). This work was also funded by FEDER, through the Competitiveness Internationalization Operational Programme (POCI), and by National funds, through the FTC, under the scope of the projects POCI-01-0145-FEDER-007038; POCI-01-0145-FEDER-029751 and POCI-01-0145-FEDER-032619. This project has also 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). Part of the work was also developed at Department of Experimental Medical Science at Lund University and financially supported by the Strategic Research Environment MultiPark—multidisciplinary research on Parkinson's disease and Crafoord Foundation Sweden