Joubert syndrome (JS) is a genetically heterogeneous disorder characterized by a distinctive cerebellar malformation, the molar tooth sign, associated with a wide range of neurological phenotypes and multiorgan involvement. JS is a ciliopathy, i.e., a pathological condition related to mutations of genes that code for proteins involved in the development and functioning of the primary cilium. To clarify the molecular mechanisms underlying JS, induced pluripotent stem cells (iPSCs) derived from 3 JS patients were produced, and subsequently characterized, two of which carrying a mutation in the gene, and one with a mutation in the AHI1 gene. The JS patients-derived cell lines were analyzed in parallel with cells from a healthy control (CTRL-1). The characterization of the iPSC lines was carried out through a series of molecular analyses, such as RT-qPCR and immunofluorescence, aimed at verifying both the expression of standard stem markers in the reprogrammed lines and the ability of these cells to differentiate in the three embryonic germ layers as proof of pluripotency. Subsequently, their ability to differentiate into neural stem cells (NSCs), from which neurons derive, was evaluated. Both patients and control (CTRL-1) iPSCs demonstrated characteristics of stemness and pluripotency; however, in the case of a line deriving from a patient mutated in the CPLANE1 gene, the iPSCs showed an anomalous differentiation into NSCs. The generated iPSC lines offer the opportunity to study the formation of neurons deriving from patients with JS, thus allowing both the future study of in vitro pathogenesis and the development of possible therapeutic strategies.
La sindrome di Joubert (JS) è una patologia geneticamente eterogena, caratterizzata da una peculiare malformazione cerebellare, il segno del dente molare, associata ad una vasta gamma di fenotipi neurologici e coinvolgimenti multiorgano. La JS è una ciliopatia, ovvero una condizione patologica correlata a mutazioni di geni che codificano per le proteine coinvolte nello sviluppo e nel funzionamento del ciglio primario. Al fine di chiarire i meccanismi molecolari correlati alla JS, sono state prodotte, e successivamente caratterizzate, cellule staminali pluripotenti indotte (iPSCs) derivanti da 3 pazienti affetti, due dei quali con mutazione del gene CPLANE1, ed uno con la mutazione del gene AHI1, in parallelo ad un controllo sano (CTRL-1). La caratterizzazione è stata effettuata mediante una serie di analisi molecolari, come RT-qPCR e immunofluorescenze, atte a verificare sia l’espressione dei marcatori di staminalità nelle linee riprogrammate, sia la capacità di queste cellule di differenziarsi nei tre foglietti embrionali. Successivamente è stata valutata la loro capacità di differenziarsi in cellule staminali neurali (NSCs), dalla quale derivano i neuroni. Sia le iPSCs prodotte dai pazienti che quelle controllo (CTRL-1) hanno dimostrato caratteristiche di staminalità e pluripotenza; solo nel caso di una linea derivante da un paziente mutato nel gene CPLANE1, le iPSCs hanno mostrato un anomalo differenziamento in NSCs. Le iPSCs generate offrono l’opportunità di studiare la formazione di neuroni derivanti da pazienti con la JS, permettendone quindi sia lo studio della patogenesi in vitro, sia lo sviluppo di eventuali strategie terapeutiche.
Caratterizzazione di cellule staminali pluripotenti indotte (iPSCs) derivate da pazienti affetti da sindrome di Joubert con mutazioni in geni diversi.
BIANCA, LORENZO
2021/2022
Abstract
Joubert syndrome (JS) is a genetically heterogeneous disorder characterized by a distinctive cerebellar malformation, the molar tooth sign, associated with a wide range of neurological phenotypes and multiorgan involvement. JS is a ciliopathy, i.e., a pathological condition related to mutations of genes that code for proteins involved in the development and functioning of the primary cilium. To clarify the molecular mechanisms underlying JS, induced pluripotent stem cells (iPSCs) derived from 3 JS patients were produced, and subsequently characterized, two of which carrying a mutation in the gene, and one with a mutation in the AHI1 gene. The JS patients-derived cell lines were analyzed in parallel with cells from a healthy control (CTRL-1). The characterization of the iPSC lines was carried out through a series of molecular analyses, such as RT-qPCR and immunofluorescence, aimed at verifying both the expression of standard stem markers in the reprogrammed lines and the ability of these cells to differentiate in the three embryonic germ layers as proof of pluripotency. Subsequently, their ability to differentiate into neural stem cells (NSCs), from which neurons derive, was evaluated. Both patients and control (CTRL-1) iPSCs demonstrated characteristics of stemness and pluripotency; however, in the case of a line deriving from a patient mutated in the CPLANE1 gene, the iPSCs showed an anomalous differentiation into NSCs. The generated iPSC lines offer the opportunity to study the formation of neurons deriving from patients with JS, thus allowing both the future study of in vitro pathogenesis and the development of possible therapeutic strategies.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/20.500.14239/15857