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Tolomeo, Anna Maria (2018) Integration of transcriptional programming and micro-technologies for in vitro modelling of neural development. [Ph.D. thesis]

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Abstract (italian or english)

The goal of this thesis is the downscaling of neuronal differentiation, starting from hiPSCs, using microfluidic and transcriptional programming technologies. The microfluidic lab-on-chip platform, used for the validation of neuronal induction protocols, offers the possibility to work in a context of tight control of culture conditions, high-throughput, efficient delivery of soluble factors. Instead, the induced pluripotent stem cells (iPSCs) technology allows to reprogram somatic cells, such as skin fibroblasts, to an embryonic-like phenotype and to obtain, in this way, a clonal expansion of undifferentiated cells that can then be differentiated in the desired phenotype (e.g. neuronal lineage). The combination between cell culture in microfluidics and iPSCs provides a very important contribution in the understanding of those unknown molecular mechanisms, responsible for specific pathologies, which could be useful for finding effective therapies.
This work finds its basis and strong motivation in a contest that, to date, does not provide exhaustive in vitro or in vivo models, because a large amount of neurological diseases and human brain studies are performed on post-mortem biopsies or on tissues collected at very late disease stages. The animal models, instead, could represent a possibility to understand some neurological mechanisms, but they are limited and sometimes do not fully recapitulate the patient phenotype.
In this scenario, we have focused our attention on the downscaling of in vitro neuronal differentiation protocols starting from hiPSCs, taking advantage first of all of lentiviral vectors for the overexpression of a proneural transcription factors, Ngn2, After the definition of the best culture condition, we focus our attention on the regulation of the expression pattern of Ngn2 in hiPSCs. Ngn2 is characterized by an oscillatory expression, in the early stages of the neurogenesis, and becomes constantly expressed on mature neurons. So, we evaluated weather we could reproduce in vitro this oscillatory expression pattern.
Furthermore, since the generation of neurons with high efficiency is influenced by the correct delivery of exogenous factors to the cells with a precise timing, we developed a protocol of neuronal induction using an automated microfluidic platform.
Lastly, to avoid any genetic aberration caused by lentiviral vectors and to have a system that could be easily modulated in term of dose and frequency of administration, we induced the generation of neurons by introducing synthetic modified mRNA encoding for Ngn2 into hiPSCs and coupled this method with microfluidic technology. We focused on increasing the differentiation efficiency of neuronal differentiation, working on cellular signallings that play important roles during the in vivo development of the central nervous system.

Abstract (a different language)

L'obiettivo di questa tesi è il downscaling di protocolli di differenziazione neuronale, a partire da hiPSC, utilizzando tecnologie di programmazione trascrizionale, in microfluidica. La piattaforma microfluidica lab-on-chip, utilizzata per la validazione dei protocolli, offre la possibilità di lavorare in un contesto di stretto controllo delle condizioni della coltura cellulare, come ad esempio la consegna efficiente di fattori solubili. Invece, la tecnologia delle cellule staminali pluripotenti indotte (iPSCs) consente di riprogrammare le cellule somatiche, come i fibroblasti, in un fenotipo simile a quello embrionale e di ottenere, in questo modo, un'espansione clonale di cellule indifferenziate che possono quindi essere differenziate nel fenotipo desiderato (ad esempio quello neuronale). La combinazione tra coltura cellulare in microfluidica e iPSC fornisce un contributo molto importante nella comprensione di quei meccanismi molecolari, responsabili di patologie specifiche, utili all’individuazione di efficaci terapie.
Questo lavoro trova le sue basi ed una forte motivazione in un contesto che, ad oggi, non fornisce modelli esaurienti in vitro o in vivo, perché una grande quantità di malattie neurologiche e studi sul cervello umano vengono eseguiti su biopsie post-mortem o su tessuti raccolti a fasi della malattia molto tardive. I modelli animali, invece, potrebbero rappresentare una possibilità di comprendere alcuni meccanismi neurologici, ma sono limitati e talvolta non ricapitolano completamente il fenotipo del paziente.
In questo scenario, abbiamo focalizzato la nostra attenzione sul downscaling dei protocolli di differenziazione neuronale in vitro a partire da hiPSC, sfruttando prima di tutto i vettori lentivirali per la sovraespressione di fattori di trascrizione, nel nostro caso la Neurogenina 2, un gene proneurale. Inoltre, abbiamo usato questo approccio con una prospettiva innovativa: regolando la durata e la frequenza dell’espressione di Ngn2, attraverso la somministrazione controllata di doxiciclina, è possibile imitare, in vitro, il profilo oscillatorio di Ngn2 osservato in vivo. In vivo, infatti, i progenitori neurali sono caratterizzati da un pattern di espressione di Ngn2 oscillatorio, mentre i neuroni mostrano un'espressione sostenuta e costante di Ngn2.
Inoltre, poiché la generazione di neuroni, con un’elevata efficienza, è influenzata da un corretto rilascio di fattori esogeni alle cellule, in modo time-dependent, abbiamo sviluppato un protocollo di induzione neuronale utilizzando una piattaforma microfluidica automatizzata.
Infine, per evitare qualsiasi aberrazione genetica causata dai vettori lentivirali e per avere un sistema che potrebbe essere facilmente modulato in termini di dose e frequenza di somministrazione, abbiamo indotto la generazione di neuroni, in hiPSC, introducendo sintetici mRNA modificati codificanti per Ngn2 e accoppiato questo metodo con la tecnologia microfluidica. Ci siamo, quindi, concentrati sull'aumento dell'efficienza di differenziazione neuronale, lavorando sul signaling cellulare, importante per lo sviluppo in vivo del sistema nervoso centrale.

EPrint type:Ph.D. thesis
Tutor:Muraca, Maurizio
Supervisor:Elvassore, Nicola
Ph.D. course:Ciclo 31 > Corsi 31 > MEDICINA DELLO SVILUPPO E SCIENZE DELLA PROGRAMMAZIONE SANITARIA
Data di deposito della tesi:29 November 2018
Anno di Pubblicazione:29 November 2018
Key Words:Neural development, neuronal induction, pluripotent stem cells, microfluidics, transcriptional programming, signaling pathways
Settori scientifico-disciplinari MIUR:Area 05 - Scienze biologiche > BIO/11 Biologia molecolare
Struttura di riferimento:Dipartimenti > Dipartimento di Salute della Donna e del Bambino
Codice ID:11441
Depositato il:06 Nov 2019 12:08
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