The Amniotic Fluid (AF) surrounds and protects the human fetus during gestation. This solution is rich in cells, shed from a number of developing fetal tissues. Consequently, containing secretions and cells from various tissues, the AF is highly heterogeneous in origin and composition. However, the precise cellular composition remains largely unexplored. This study employs single-cell RNA sequencing (scRNAseq) on fresh, uncultured AF, to create the first comprehensive single-cell atlas of human AF cells across human development. The analysis presented in this thesis showed presence in the AF of heterogeneous epithelial populations alongside multiple clusters of hematopoietic and immune cells. Interestingly, in line with a recent report from the host laboratory, epithelial progenitors specifically for the GI, kidney, and lung were also identified. This discovery enabled the generation of AF-derived organoids (AFO) resembling GI, kidney, and lung tissues. Aiming at translating this technology, the host team started working on Congenital Diaphragmatic Hernia (CDH), condition characterised by a remarkable lung hypoplasia. CDH can be partially rescued thanks to a recently developed fetal surgery procedure named Fetoscopic Endoluminal Tracheal Occlusion (FETO). This procedure enabled access to fetal amniotic and tracheal fluid (TF), which have been sequenced, and used to generate CDH organoids resembling features of the disease. In this work, I generated the first scRNAseq map of the CDH AF and improved the annotation of the CDH AFO. This project aims at investigating the potential use of the AF composition and the organoids derived from this fluid, as advanced methods to monitor human development and disease.
Il Fluido Amniotico (FA) circonda e protegge il feto umano durante la gestazione. Questo fluido è ricco di cellule provenienti da diversi tessuti fetali in via di sviluppo e, di conseguenza, è altamente eterogeneo per origine e composizione. Tuttavia, la composizione cellulare precisa rimane ancora poco esplorata. Questo studio utilizza la tecnica di single-cell RNA sequencing (scRNAseq) su FA fresco e non coltivato, con l'obiettivo di creare la prima mappa completa di cellule del FA umano durante lo sviluppo. L'analisi presentata in questa tesi ha rivelato la presenza nel FA di popolazioni epiteliali eterogenee, insieme a diversi gruppi di cellule immunitarie. È interessante notare che, in linea con un recente studio del laboratorio ospite, sono stati identificati anche progenitori epiteliali specifici per il tratto GI, il rene e i polmoni. Questa scoperta ha permesso di generare organoidi derivati dal FA (AFO) che rappresentano i tessuti GI, renale e polmonare. Con l'obiettivo di utilizzare questa tecnologia, il gruppo ospite ha iniziato a concentrarsi sull'Ernia Diaframmatica Congenita (CDH), una patologia caratterizzata da una marcata ipoplasia polmonare. La CDH può essere parzialmente trattata grazie a una recente procedura di chirurgia fetale chiamata Occlusione Tracheale Endoluminale Fetoscopica (FETO). Questa procedura ha consentito l'accesso ai fluidi amniotici e tracheali fetali (TF), che sono stati sequenziati e utilizzati per generare organoidi CDH con caratteristiche della malattia. In questa ricerca, ho creato la prima mappa scRNAseq del FA con CDH e migliorato l'annotazione di CDH LAFOs. Questo progetto ha l'obiettivo di esplorare il potenziale utilizzo della composizione del FA e degli organoidi derivati da FA come metodi avanzati per monitorare lo sviluppo umano e le malattie.
Generation of a single cell atlas of the amniotic fluid across gestation and in presence of Congenital Diaphragmatic Hernia and single cell annotation of amniotic fluid-derived organoids
Mariani, Alessandro
2023/2024
Abstract
The Amniotic Fluid (AF) surrounds and protects the human fetus during gestation. This solution is rich in cells, shed from a number of developing fetal tissues. Consequently, containing secretions and cells from various tissues, the AF is highly heterogeneous in origin and composition. However, the precise cellular composition remains largely unexplored. This study employs single-cell RNA sequencing (scRNAseq) on fresh, uncultured AF, to create the first comprehensive single-cell atlas of human AF cells across human development. The analysis presented in this thesis showed presence in the AF of heterogeneous epithelial populations alongside multiple clusters of hematopoietic and immune cells. Interestingly, in line with a recent report from the host laboratory, epithelial progenitors specifically for the GI, kidney, and lung were also identified. This discovery enabled the generation of AF-derived organoids (AFO) resembling GI, kidney, and lung tissues. Aiming at translating this technology, the host team started working on Congenital Diaphragmatic Hernia (CDH), condition characterised by a remarkable lung hypoplasia. CDH can be partially rescued thanks to a recently developed fetal surgery procedure named Fetoscopic Endoluminal Tracheal Occlusion (FETO). This procedure enabled access to fetal amniotic and tracheal fluid (TF), which have been sequenced, and used to generate CDH organoids resembling features of the disease. In this work, I generated the first scRNAseq map of the CDH AF and improved the annotation of the CDH AFO. This project aims at investigating the potential use of the AF composition and the organoids derived from this fluid, as advanced methods to monitor human development and disease.| File | Dimensione | Formato | |
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Mariani_Alessandro_executive_summary.pdf
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Mariani_Alessandro_tesi.pdf
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https://hdl.handle.net/10589/227748