Systematic, genome-wide interrogations have identified hundreds of genes, including several transcription factors, which have expression patterns tightly correlated with ES cell differentiation. OCT4, SOX2 and NANOG constitute a triad of transcription factors, identified as crucial for the maintenance of ES cells self-renewal and pluripotent state.The principal aim of our project was to induce the differentiation process of embryo-derived stem cells into neural cells (neurons, glial cells), to follow during the differentiation process the changing in the expression of characteristic stemness markers (OCT4, SOX2 and NANOG) responsible for the regulatory networks involved in embryo-derived stem cells pluripotency, whose understanding is fundamental for any potential therapeutic application. For this reason the use of advanced spectroscopic techniques, such as time-resolved fluorescence correlation spectroscopy (FCS), could allow to follow protein changes and to analyze different aspects such as the molecular dynamics and intracellular translocation of some selected transcription factors tightly bound to the activation of the ESCs differentiation processes into neural cells. Our Results show that the pluripotency circuit is known to act as a unit that strongly represses lineage specific gene expression in ESCs. However, rather than being a monolithic entity, the pluripotency circuit components have lineage specific roles, so that the same proteins can also be used for lineage selection.
Scopo di questo studio è stata l'analisi delle caratteristiche correlate alla pluripotenza delle human embryonic stem cells (hES) durante il differenziamento in coltura, dopo trattamento con specifici fattori di trascrizione, perseguendo l'obiettivo dell'induzione verso cellule della linea neurale, essendo nota la scarsa capacità autoriproduttiva delle cellule del sistema nervoso centrale. Durante il differenziamento è stata valutata e monitorata la presenza e la traslocazione intracellulare di fattori di trascrizione come Oct4, Sox2, e Nanog che regolano l espressione di proteine che svolgono un ruolo cruciale nel mantenere lo stato di self renewal e la pluripotenza. Tale indagine è stata condotta con l uso di tecniche analitiche di frontiera come la spettroscopia di correlazione di fluorescenza risolta nel tempo, Fluorescence Correlation Spectroscopy (F.C.S.) con particolare riferimento alla tecnica di Number of Molecules and Brightness (N&B), una applicazione innovativa delle tecniche di imaging molecolare. Questo tipo di analisi ha consentito di monitorare eventi molecolari attraverso l osservazione diretta intracellulare di macromolecole targate , responsabili dell insorgenza di trasformazioni fisiologiche e non, legate a modificazioni dimensionali e strutturali durante il tempuscolo del loro reale accadimento. I nostri risultati hanno dimostrato come l'interazione tra questi fattori di trascrizione giochi un ruolo chiave durante il mantenimento dello stato indifferenziato di self renewal delle hES, di contro durante il processo di induzione verso la linea neurale hanno mostrato una differente espressione e localizzazione intracellulare.
New analytical scenarios and new approaches in the embryonic genetic investigation of the macromolecular alterations responsible for the neurodegenerative diseases / Bonaventura, Gabriele. - (2013 Dec 10).
New analytical scenarios and new approaches in the embryonic genetic investigation of the macromolecular alterations responsible for the neurodegenerative diseases
BONAVENTURA, GABRIELE
2013-12-10
Abstract
Systematic, genome-wide interrogations have identified hundreds of genes, including several transcription factors, which have expression patterns tightly correlated with ES cell differentiation. OCT4, SOX2 and NANOG constitute a triad of transcription factors, identified as crucial for the maintenance of ES cells self-renewal and pluripotent state.The principal aim of our project was to induce the differentiation process of embryo-derived stem cells into neural cells (neurons, glial cells), to follow during the differentiation process the changing in the expression of characteristic stemness markers (OCT4, SOX2 and NANOG) responsible for the regulatory networks involved in embryo-derived stem cells pluripotency, whose understanding is fundamental for any potential therapeutic application. For this reason the use of advanced spectroscopic techniques, such as time-resolved fluorescence correlation spectroscopy (FCS), could allow to follow protein changes and to analyze different aspects such as the molecular dynamics and intracellular translocation of some selected transcription factors tightly bound to the activation of the ESCs differentiation processes into neural cells. Our Results show that the pluripotency circuit is known to act as a unit that strongly represses lineage specific gene expression in ESCs. However, rather than being a monolithic entity, the pluripotency circuit components have lineage specific roles, so that the same proteins can also be used for lineage selection.File | Dimensione | Formato | |
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