Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects upper and lower motor neurons, the loss of which causes weakness and progressive muscle atrophy. ALS often has an onset in the muscles of the limbs and subsequently spreads to different regions of the body, where insufficiency of the respiratory muscles typically limits survival to 2-5 years after the onset of the disease. ALS has an incidence rate of about 2 per 100,000 people-year globally, with a prevalence of 6-9 cases per 100,000 people. It is a complex multifactorial disease in which genetic and environmental factors, age and male sex increase the risk. To date, more than 20 genes have been associated with ALS, but the most frequent pathogenic variants have been identified in the genes SOD1, TARDBP, FUS and C9ORF72: in some ALS patients, family history suggests an autosomal dominant model of heredity, while most have no affected family members and are classified as sporadic ALS (sALS). To date, there is no cure or effective treatment for ALS: the only drug approved by the European Medicines Agency is Riluzole for symptom management. Many mechanisms involved in the pathogenesis of ALS have been identified, including neuroinflammation, which is characterized by activation of microglia and astrocytes, elevated production of inflammatory cytokines, and infiltration of T cells into nervous tissue. In particular, elevated levels of certain cytokines such as IL-1β, IL-6 e IL-8, TGF-β, TNF-α, IFN-γ and GM-CSF were found in ALS patients. In this thesis, a protocol for the generation of spinal organoids (SCOs) from sALS patients and healthy controls (CTRL) was developed. The SCOs were characterized and the levels of some cytokines present in the culture medium were analyzed, with the aim of evaluating any deregulations in their concentration both over time and between SCOs sALS and SCOs CTRL. Organoids are 3D cell cultures that in recent years have proved very useful as in vitro models, as an alternative to 2D cultures and animal models. The SCOs sALS and SCOs CTRL were obtained by differentiating induced pluripotent stem cells derived from peripheral blood mononuclear cells. Morphological characterization by microscopy demonstrated that SCOs sALS were smaller, with more irregular shape and shorter neurites than SCOs sALS. Cellular characterization by immunofluorescence highlighted that SCOs sALS had more undifferentiated cells and fewer mature motor neurons than SCOs CTRL. Lastly, in SCOs sALS astrocytes formed a thicker outer layer than in SCOs CTRL. Cytokines present in the culture medium were also quantified using an array of 105 cytokines. Among the most deregulated ones, we found cytokines and proteins that play a fundamental role in the remodeling of the extracellular matrix. We then confirmed what we observed with the array using the innovative ELISA technique for high-throughput quantifications (ELLA - Biotechne, USA). Altered levels of IL8, OPN, VCAM and MMP9 were found in SCOs sALS compared to SCOs CTRL. These findings reflect the main characteristics of ALS, such as the loss of motor neurons, gliosis, neurite degeneration and alteration of the production of cytokines involved in extracellular matrix remodeling. Organoids can therefore provide a useful model for studying the pathological mechanisms of ALS, such as neurodegeneration and neuroinflammation, and the identification of these pathways may also lead to the discovery of future new therapeutic targets.
La sclerosi laterale amiotrofica (ALS) è una malattia neurodegenerativa che colpisce i motoneuroni superiori e inferiori, la cui perdita provoca debolezza e atrofia muscolare progressiva. L’ALS ha spesso un esordio a livello dei muscoli degli arti e successivamente si diffonde a diverse regioni del corpo, dove l'insufficienza dei muscoli respiratori tipicamente limita la sopravvivenza a 2-5 anni dopo l'esordio della malattia. L’ALS ha un'incidenza di circa 2 ogni 100.000 persone-anno a livello globale, con una prevalenza di 6-9 casi ogni 100.000 persone. Si tratta di una complessa malattia multifattoriale, in cui fattori genetici, ambientali, età e sesso maschile ne aumentano il rischio. Ad oggi, più di 20 geni sono stati associati all’ALS, ma le varianti patogenetiche più frequenti sono state identificate nei geni SOD1, TARDBP, FUS e C9ORF72: in alcuni pazienti affetti da ALS, l'anamnesi familiare suggerisce un modello di ereditarietà autosomica dominante, mentre la maggior parte non ha familiari affetti e viene classificata come ALS sporadica (sALS). Ad oggi non esiste una cura o un trattamento efficace per l’ALS: l’unico farmaco approvato dall’agenzia europea per i medicinali è il Riluzolo per la gestione dei sintomi. Sono stati individuati molti meccanismi coinvolti nella patogenesi dell’ALS, tra cui la neuroinfiammazione che è caratterizzata dall'attivazione di microglia e astrociti, elevata produzione di citochine infiammatorie e infiltrazione di linfociti T nel tessuto nervoso. In particolare, nei pazienti affetti da ALS sono stati riscontrati elevati livelli di alcune citochine, come IL-1β, IL-6 e IL-8, TGF-β, TNF-α, IFN-γ e GM-CSF. In questo elaborato di tesi, è stato sviluppato un protocollo di generazione di organoidi spinali (SCOs) ottenuti da pazienti sALS e di controlli sani (CTRL). Gli SCOs sono stati caratterizzati e sono stati analizzati i livelli di alcune citochine presenti nel terreno di coltura, con lo scopo di valutare eventuali deregolazioni nella concentrazione sia nel tempo che tra SCOs sALS e SCOs CTRL. Gli organoidi sono colture cellulari 3D che negli ultimi anni si sono rivelati molti utili come modello in vitro, in alternativa alle colture 2D e ai modelli animali. Gli SCOs sALS e gli SCOs CTRL sono stati ottenuti dal differenziamento di cellule staminali pluripotenti indotte derivate da cellule mononucleate del sangue periferico. La caratterizzazione morfologica mediante microscopia ha dimostrato che gli SCOs sALS erano più piccoli e irregolari, con neuriti più corti rispetto agli SCOs CTRL. La caratterizzazione cellulare tramite immunofluorescenza ha evidenziato che gli SCOs sALS avevano più cellule indifferenziate e meno motoneuroni maturi rispetto agli SCOs CTRL. Inoltre, negli SCOs sALS gli astrociti formavano uno strato esterno più spesso rispetto agli SCOs CTRL. Sono state infine quantificate le citochine presenti nel terreno di coltura degli SCOs utilizzando un pannello di 105 citochine. Tra le più deregolate, abbiamo trovato citochine e proteine che svolgono un ruolo fondamentale nel rimodellamento della matrice extracellulare. Abbiamo successivamente confermato quanto osservato con l’array tramite una tecnica ELISA di ultima generazione per quantifiche high-throughput (ELLA - Biotechne, USA). Sono stati riscontrati alterati livelli di IL8, OPN, VCAM e MMP9 negli SCOs sALS rispetto agli SCOs CTRL. Questi dati rispecchiano le principali caratteristiche dell’ALS, come la perdita dei motoneuroni, gliosi, degenerazione dei neuriti e alterazione della produzione di citochine coinvolte nel rimodellamento della matrice extracellulare. Gli organoidi possono quindi fornire un utile modello per lo studio dei pato-meccanismi dell’ALS, come neurodegenerazione e neuroinfiammazione, e l’identificazione di questi pathway può inoltre portare alla scoperta di futuri nuovi target terapeutici.
Caratterizzazione di organoidi spinali come modello per lo studio di citochine coinvolte nella Sclerosi Laterale Amiotrofica
PELLEGRINI, ELISA
2022/2023
Abstract
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects upper and lower motor neurons, the loss of which causes weakness and progressive muscle atrophy. ALS often has an onset in the muscles of the limbs and subsequently spreads to different regions of the body, where insufficiency of the respiratory muscles typically limits survival to 2-5 years after the onset of the disease. ALS has an incidence rate of about 2 per 100,000 people-year globally, with a prevalence of 6-9 cases per 100,000 people. It is a complex multifactorial disease in which genetic and environmental factors, age and male sex increase the risk. To date, more than 20 genes have been associated with ALS, but the most frequent pathogenic variants have been identified in the genes SOD1, TARDBP, FUS and C9ORF72: in some ALS patients, family history suggests an autosomal dominant model of heredity, while most have no affected family members and are classified as sporadic ALS (sALS). To date, there is no cure or effective treatment for ALS: the only drug approved by the European Medicines Agency is Riluzole for symptom management. Many mechanisms involved in the pathogenesis of ALS have been identified, including neuroinflammation, which is characterized by activation of microglia and astrocytes, elevated production of inflammatory cytokines, and infiltration of T cells into nervous tissue. In particular, elevated levels of certain cytokines such as IL-1β, IL-6 e IL-8, TGF-β, TNF-α, IFN-γ and GM-CSF were found in ALS patients. In this thesis, a protocol for the generation of spinal organoids (SCOs) from sALS patients and healthy controls (CTRL) was developed. The SCOs were characterized and the levels of some cytokines present in the culture medium were analyzed, with the aim of evaluating any deregulations in their concentration both over time and between SCOs sALS and SCOs CTRL. Organoids are 3D cell cultures that in recent years have proved very useful as in vitro models, as an alternative to 2D cultures and animal models. The SCOs sALS and SCOs CTRL were obtained by differentiating induced pluripotent stem cells derived from peripheral blood mononuclear cells. Morphological characterization by microscopy demonstrated that SCOs sALS were smaller, with more irregular shape and shorter neurites than SCOs sALS. Cellular characterization by immunofluorescence highlighted that SCOs sALS had more undifferentiated cells and fewer mature motor neurons than SCOs CTRL. Lastly, in SCOs sALS astrocytes formed a thicker outer layer than in SCOs CTRL. Cytokines present in the culture medium were also quantified using an array of 105 cytokines. Among the most deregulated ones, we found cytokines and proteins that play a fundamental role in the remodeling of the extracellular matrix. We then confirmed what we observed with the array using the innovative ELISA technique for high-throughput quantifications (ELLA - Biotechne, USA). Altered levels of IL8, OPN, VCAM and MMP9 were found in SCOs sALS compared to SCOs CTRL. These findings reflect the main characteristics of ALS, such as the loss of motor neurons, gliosis, neurite degeneration and alteration of the production of cytokines involved in extracellular matrix remodeling. Organoids can therefore provide a useful model for studying the pathological mechanisms of ALS, such as neurodegeneration and neuroinflammation, and the identification of these pathways may also lead to the discovery of future new therapeutic targets.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/17397