Alsehli, Haneen

English

During gastrulation, the pluripotent stem cells of the epiblast differentiate, ultimately shaping the embryo's body plan. This process is tightly associated with extensive cellular movements that induce breaking symmetry to form anterior-posterior axial elongation giving rise to the three germ layers. Morphogenesis events and lineage specifications of the tri-germ layers are regulated by distinctive interactions, including cell-cell contacts that transmit biochemical signals and cell-ECM interplay with physical forces exerted from the extra-embryonic environment. Nonetheless whether tissue rearrangements are regulated mechanically (tissue environment) or biochemically (cellular response) is poorly understood. Cultures of human induced pluripotent stem cells (hiPSC) offer unprecedented scope to profile and screen conditions affecting cell fate decisions and self-organisation during early embryonic development. The impact of biochemical signalling (BMP4, NODAL, and WNT) in 2D micropattern systems has been wellestablished, which recapitulates the three germ layers patterning in vivo.I have proposed here a 3D hiPSC platform to induce gastrulation upon BMP4 treatment. My aim is to investigate how biochemical cues and physical confinement separately influence morphogenesis and differentiation. To investigate the biochemical effect, I used BMP4 to trigger symmetry breaking and elongation of hiPSC spheroids in suspension culture. Having different medium conditions representing self-renewing (E8 medium) and differentiation (KSR BMP4) and their controls (E8 BMP4 and KSR) gave rise to distinct morphologies in suspension. I also postulated that morphogenesis and changes in shape are regulated by cellular tension in response to biochemical cues. Hence, I hypothesised that PEG-peptide hydrogel systems inhibit elongation and thus enable us to interrogate if physical confinement affects not just morphogenesis but also cell fate specification. High content analysis (HCA) was used as readouts obtained from live imaging to observe morphological changes and immunostaining techniques to detect the expression of pluripotency and differentiation markers. Our approach enables us to investigate changes in shape and patterning of the three germ layers using hiPSC and sheds light on the interplay between chemical signals and physical forces.<br/

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Characterisation of human induced pluripotent stem cells in 3D artificial microenvironment using high content analysis

https://kclpure.kcl.ac.uk/portal/files/229476138/2023_Alsehli_Haneen_1786987_ethesis.pdf

Characterisation of human induced pluripotent stem cells in 3D artificial microenvironment using high content analysis

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