The maintenance of the airway/respiratory epithelium during adult homeostasis and repair and its construction during embryonic development require tightly regulated cell fate decisions. This regulation takes the form of complex transcription factor and signalling cascades, much of which are unknown, particularly in human lung development. Multiciliogenesis describes the process of specification/differentiation of airway epithelial progenitors/stem cells into mature multiciliated cells (MCCs). Here, I have identified 2 novel transcription factors, Fank1 and Jazf1 which form part of the transcription factor cascade regulating multiciliogenesis in adult and embryonic mouse tracheas. Mouse tracheal epithelium is representative of epithelium lining the entire human airway and it is possible that we will also be able to extrapolate these findings to the human airway. It is not until we fully understand the regulation of multiciliogenesis that it will be possible to look at ways of pushing basal cells towards a MCC fate for purposes of cell replacement therapy, for example in patients with mucociliary disease. As well as exploring cell fate decisions in the mouse upper airway epithelium using embryonic tracheal explants and mouse tracheal epithelial cell (MTEC) cultures, I have also explored the regulation of cell fate decisions in distal human lung epithelium at the pseudoglandular stage of development. At this stage SOX9+ distal tip cells are self-renewing and multipotent and give rise to SOX2+ stalk descendents, which differentiate into airway epithelium. The regulation of SOX9+ lung tip cell multipotency and migration of SOX2+ stalk descendents during human lung development is poorly understood. I have compared human tip (SOX9+) versus stalk (SOX2+) transcriptomes using gene ontology (GO), which has highlighted some key signalling pathways enriched in tip cells which could be important in maintaining distal tip cell multipotency. These pathways have been utilised in optimising conditions for propagating self-renewing tip-derived organoids. These organoids have the potential to be differentiated into bronchiolar and alveolar fates and as such are an invaluable research tool for studying human lung epithelial development, whilst minimising the use of human embryos and its associated ethical implications. I have also performed human tip versus mouse tip transcriptome GO analysis which highlights that although there are many similarities, there are also differences between human and mouse lung epithelium development, emphasising the need for research on human tissue.

Human pluripotent stem cells (hPSCs) have the ability to self-renew and differentiate into all specialized body cells, providing material suitable for studying basic biology, modeling disease, and for regenerative medicine. The differentiation of hPSCs into functional cell types has been further enhanced by the production of organoids, miniature 3D organ-like structures that mimic the architecture and function of their in vivo counterparts, representing more physiologically relevant models of native tissues than monolayer cultures. Our initial aim was to differentiate hPSCs into lung epithelial organoids in vitro, and we hypothesized that applying knowledge of signaling cues during embryonic development to the dish would produce lineage-specific tissue. Using a multi-stage differentiation strategy, we derived organoids sharing properties with the developing lung as well as intestine. From this work, we learned the importance of purification, selection, and using singularized precursor cells to produce populations of bona fide lineage-restricted organoids. Upon developing a type of intestinal organoid technology from hPSCs not reported before, we shifted our focus to the intestine. We generated cystic intestinal epithelial organoids called enterospheres (hEnS) in vitro from hPSCs, which mimic structural and cell type properties of the native small intestinal epithelium. hEnS growth, differentiation, and long-term culture can be controlled by modulating media conditions. Importantly, hEnS are functional in that they elicit an innate immune response upon treatment with enteric pathogens. We established hEnS as an attractive in vitro model system for studying human gastrointestinal biology. We then developed an automated hEnS imaging assay to measure responses to growth factors, bacterial products, and enteric bacteria themselves. In doing so, we demonstrated the utility of hEnS as a germ-free system for studying host-microbe interactions and intestinal maturation. Finally, we investigated the expression of protein markers of intestinal maturation in tissue sections of primary human intestine spanning gestation, and made observations that are different from those reported in mice. Overall, our work provides new and important insights into hPSC differentiation, organoid technologies, and intestinal development in humans. / Thesis / Doctor of Philosophy (PhD)