10 research outputs found
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
No full textLAUREA MAGISTRALEIl 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.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
Characterising the cells of the amniotic fluid and their coupling to fetal development using RNA sequencing
No full textThe amniotic fluid (AF) has been known to contain fetal cells, however the precise cellular composition
remains overlooked. The AF has previously been used to generate mesenchymal AF stem cells (AFSCs,
also referred to as AF-MSC), which have been shown to have the multipotent capacity to generate
cells representing each embryonic germ layer. While research into the clinical translation of AFSC is
ongoing, attempts are limited by a lack of clarity on the AFSC tissue of origin and identity. Initially this
research conducted a full characterisation of the AFSC, applying bulk, spatial and single-cell RNA
sequencing (scRNAseq) to confirm their renal nephron origin. This led to the novel finding that the
AFSC precursors persist in the AF as epithelial cells, having likely shed from the nephron and been
transported through the fetal urine. These precursors then appear to undergo culture-induced
epithelial-to-mesenchymal transition (EMT) to form the widely studied AFSC.
These findings led me to investigate the fresh, uncultured AF through scRNAseq, generating the first
single-cell map of the AF cells in both human and mouse. Through this atlas, I identified a
heterogenous epithelial population, as well as macrophages, monocytes, dendritic cells, neutrophils,
B-cells, T-cells, natural killer cells, eosinophils, erythroblasts, granulocytes, common myeloid
progenitors and haematopoietic stem cells (HSC). The epithelial population was then shown to contain
cells from the kidney, lung, skin, eye and gastrointestinal tract (GI), with epithelial progenitors
identified for the GI, kidney and lung. The identification of these progenitors led to the generation of
AF-derived clonal epithelial organoids of GI, kidney and lung identity. The ability of these organoids to
recapitulate a disease phenotype was preliminarily demonstrated with congenital diaphragmatic
hernia (CDH). Overall, this research provides the first in-depth characterisation of the AF cellular
content and identifies novel cells with clinical capacity, highlighting the potential of this overlooked
source of fetal cells
Author correction: Single-cell guided prenatal derivation of primary fetal epithelial organoids from human amniotic and tracheal fluids
No full textCorrection to: Nature Medicinehttps://doi.org/10.1038/s41591-024-02807-z, published online 4 March 2024. In the version of this article initially published, there were inconsistences, where in the Methods “AF collection and isolation of the viable cell fraction” paragraph, the first sentence, now reading “AF samples (amniocenteses and amniodrainages) were collected from UCLH FMU and UZ Leuven as part of standard patient clinical care”, was initially preceded by “Euploid,” though the sample contained two chromosomal abnormalities; and in Supplementary Table 1, where for sample code HO680, the sex was listed as female following prenatal clinical data, whereas the sample was identified and processed in further analysis as male. The changes have been made in Supplementary Table 1 and in the HTML and PDF versions of the article
Single-cell guided prenatal derivation of primary fetal epithelial organoids from human amniotic and tracheal fluids
No full text: Isolation of tissue-specific fetal stem cells and derivation of primary organoids is limited to samples obtained from termination of pregnancies, hampering prenatal investigation of fetal development and congenital diseases. Therefore, new patient-specific in vitro models are needed. To this aim, isolation and expansion of fetal stem cells during pregnancy, without the need for tissue samples or reprogramming, would be advantageous. Amniotic fluid (AF) is a source of cells from multiple developing organs. Using single-cell analysis, we characterized the cellular identities present in human AF. We identified and isolated viable epithelial stem/progenitor cells of fetal gastrointestinal, renal and pulmonary origin. Upon culture, these cells formed clonal epithelial organoids, manifesting small intestine, kidney tubule and lung identity. AF organoids exhibit transcriptomic, protein expression and functional features of their tissue of origin. With relevance for prenatal disease modeling, we derived lung organoids from AF and tracheal fluid cells of congenital diaphragmatic hernia fetuses, recapitulating some features of the disease. AF organoids are derived in a timeline compatible with prenatal intervention, potentially allowing investigation of therapeutic tools and regenerative medicine strategies personalized to the fetus at clinically relevant developmental stages
Author Correction: Single-cell guided prenatal derivation of primary fetal epithelial organoids from human amniotic and tracheal fluids
No full textCorrection to: Nature Medicine https://doi.org/10.1038/s41591-024-02807-z, published online 4 March 2024.
In the version of this article initially published, there were inconsistences, where in the Methods “AF collection and isolation of the viable cell fraction” paragraph, the first sentence, now reading “AF samples (amniocenteses and amniodrainages) were collected from UCLH FMU and UZ Leuven as part of standard patient clinical care”, was initially preceded by “Euploid,” though the sample contained two chromosomal abnormalities; and in Supplementary Table 1, where for sample code HO680, the sex was listed as female following prenatal clinical data, whereas the sample was identified and processed in further analysis as male. The changes have been made in Supplementary Table 1 and in the HTML and PDF versions of the article
Teenage Pregnancy and Fertility in English Communities: Neighbourhood, Family and Peer Influences on Behaviour
No full textPhDThe British government established the Teenage Pregnancy Unit in 1999 to
reduce early pregnancy. Current policy initiatives have a significant geographic
dimension: specific English neighbourhoods have been identified as the sites
where most early pregnancy occurs and have been targeted for intervention.
The aim of this thesis is to explore the factors that influence teenage sexual and
reproductive behaviour by drawing on the neighbourhoods effects literature.
Within this body of research, teenage reproduction is believed to be affected by a
multiplicity of factors operating within different domains. The analysis (of survey
data and qualitative material collected in three locations) was guided by two
research questions: which factors within neighbourhoods, family and peer
contexts are the most important in elucidating the causal pathways to teenage
sex, pregnancy and fertility; and do the importance of these factors vary between
neighbourhoods?
Overall, factors within neighbourhood and peer contexts were found to be less
significant than family and individual-level factors. The analysis of British Cohort
Study data showed that, for example, women who experience teenage
pregnancy or birth lived in deprived areas at age 16, but other neighbourhood
variables were not significant in multivariate analysis. There were some
differences between neighbourhoods, but the cohort member's attitude to school
was, generally, the most important factor associated with teenage sexual and
reproductive behaviour. The qualitative data supported these statistical results.
There was little evidence that women had been influenced by either their friends
or others within their neighbourhoods (though some women reported knowing
high numbers of teenage mothers), and nearly all the young mothers had low
educational attainment. In conclusion, individual and family-level influences on
sexual and reproductive outcomes are paramount, but behaviour is also subtly
informed by wider social factors
Single-cell guided prenatal derivation of primary fetal epithelial organoids from human amniotic and tracheal fluids
No full textIsolation of tissue-specific fetal stem cells and derivation of primary organoids is limited to samples obtained from termination of pregnancies, hampering prenatal investigation of fetal development and congenital diseases. Therefore, new patient-specific in vitro models are needed. To this aim, isolation and expansion of fetal stem cells during pregnancy, without the need for tissue samples or reprogramming, would be advantageous. Amniotic fluid (AF) is a source of cells from multiple developing organs. Using single-cell analysis, we characterized the cellular identities present in human AF. We identified and isolated viable epithelial stem/progenitor cells of fetal gastrointestinal, renal and pulmonary origin. Upon culture, these cells formed clonal epithelial organoids, manifesting small intestine, kidney tubule and lung identity. AF organoids exhibit transcriptomic, protein expression and functional features of their tissue of origin. With relevance for prenatal disease modeling, we derived lung organoids from AF and tracheal fluid cells of congenital diaphragmatic hernia fetuses, recapitulating some features of the disease. AF organoids are derived in a timeline compatible with prenatal intervention, potentially allowing investigation of therapeutic tools and regenerative medicine strategies personalized to the fetus at clinically relevant developmental stages
Single-cell guided prenatal derivation of primary fetal epithelial organoids from human amniotic and tracheal fluids.
No full textIsolation of tissue-specific fetal stem cells and derivation of primary organoids is limited to samples obtained from termination of pregnancies, hampering prenatal investigation of fetal development and congenital diseases. Therefore, new patient-specific in vitro models are needed. To this aim, isolation and expansion of fetal stem cells during pregnancy, without the need for tissue samples or reprogramming, would be advantageous. Amniotic fluid (AF) is a source of cells from multiple developing organs. Using single-cell analysis, we characterized the cellular identities present in human AF. We identified and isolated viable epithelial stem/progenitor cells of fetal gastrointestinal, renal and pulmonary origin. Upon culture, these cells formed clonal epithelial organoids, manifesting small intestine, kidney tubule and lung identity. AF organoids exhibit transcriptomic, protein expression and functional features of their tissue of origin. With relevance for prenatal disease modeling, we derived lung organoids from AF and tracheal fluid cells of congenital diaphragmatic hernia fetuses, recapitulating some features of the disease. AF organoids are derived in a timeline compatible with prenatal intervention, potentially allowing investigation of therapeutic tools and regenerative medicine strategies personalized to the fetus at clinically relevant developmental stages
Single-cell guided prenatal derivation of primary fetal epithelial organoids from human amniotic and tracheal fluids
No full textIsolation of tissue-specific fetal stem cells and derivation of primary organoids is limited to samples obtained from termination of pregnancies, hampering prenatal investigation of fetal development and congenital diseases. Therefore, new patient-specific in vitro models are needed. To this aim, isolation and expansion of fetal stem cells during pregnancy, without the need for tissue samples or reprogramming, would be advantageous. Amniotic fluid (AF) is a source of cells from multiple developing organs. Using single-cell analysis, we characterized the cellular identities present in human AF. We identified and isolated viable epithelial stem/progenitor cells of fetal gastrointestinal, renal and pulmonary origin. Upon culture, these cells formed clonal epithelial organoids, manifesting small intestine, kidney tubule and lung identity. AF organoids exhibit transcriptomic, protein expression and functional features of their tissue of origin. With relevance for prenatal disease modeling, we derived lung organoids from AF and tracheal fluid cells of congenital diaphragmatic hernia fetuses, recapitulating some features of the disease. AF organoids are derived in a timeline compatible with prenatal intervention, potentially allowing investigation of therapeutic tools and regenerative medicine strategies personalized to the fetus at clinically relevant developmental stages.</p
Configurations of mothering in post-war British women's playwriting
No full textWhile examining a selection of plays centred on the phenomenon of mothering, my
thesis also investigates the interaction between theatre and feminism in post-war
Britain, aiming to highlight mutual correspondences between women's theatre
making and feminist agendas. I focus mainly on the period of second-wave
feminism, but I also discuss the decade preceding the appearance of the Women's
Liberation Movement, as well as its aftermath up to the mid-nineties. Scrutinising
proto-feminist, feminist and post-feminist stances, I argue that several fifties women
dramatists anticipated key concerns of the late sixties and seventies; and equally, that
many playwrights active after the heyday of second-wave feminism revisited the
climate of the seventies in an attempt to evaluate the transformations that have since
occurred in women's lives. In this manner, I not only contextualise some of the
major achievements and shortcomings of successive feminist interventions, but also
elaborate on key changes that have taken place in the negotiation of dramatic form
and content.
Rather than privileging one dominant theoretical position and adopting its
perspective for the purposes of my analysis, I connect the work of playwrights
informed by different artistic positions and political convictions, in order to pinpoint
the principle of co-existence and multiplicity. This aesthetic and ideological diversity
in women's writing for the stage, characteristic of the past five decades, has been
confirmed not only by the primary and secondary sources that I drew upon but also
by the playwrights themselves, whom I interviewed. For most present-day female
dramatists, as this thesis argues, contemporary British women's theatre is a space of
experimentation and of confluence - in which the broad range of individual voices
can situate themselves next to one another, without the urge to replicate an ultimate
direction imposed by hegemonic political constraints or artistic platforms
