Beesley, Max Arran; (2024) Characterising the cells of the amniotic fluid and their coupling to fetal development using RNA sequencing. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The 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.

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