Synthetic cell-based patterning systems and their applications in stem cell differentiation

The contents of this file relate to the data generated during the PhD research (Biomedical Sciences) of Fokion Glykofrydis, under the supervision of Professors Jamie Davies and Elaine Dzierzak, over the 4-year period between September 2016 and August 2020. All data within this file were generated during the aforementioned period, and mainly (but not exclusively) for the purposes of the PhD research and production of the thesis. The data are contained within a compressed .rar file, which can be opened and extracted using the free decompression software WinRAR. Within the compressed file, two folders can be found (‘Data Hyperuniformity’ and ‘Data PhaseSeparationWnt3a’ folders), each containing data relating to the project stated in the folder name. Within each of the two main folders, data are further categorized into sub-folders, each of which is titled according to the primary goal, aim or experimental finding that the data address/illustrate. Data are then organized based on an experiment ID, a brief description of the experiment, and/or whether files are original, processed or the final output. Data files are: illustrations and figures made using Adobe Illustrator (.ai), images and figures (such as .png .bmp .tif), GraphPad datasets (.pzfx), Excel datasets (.xlsx), DNA sequences (.fasta), flow cytometry data (original .fcs or FlowJo-processed .wsp), imaging files made using specialized software (Nikon .nd2, Zeiss .zvi or .czi, Leica .lif), and others. The PhD thesis is provided as a PDF file for reference and guidance. For any questions, please contact Fokion Glykofrydis or Jamie A. Davies.

## Access ##
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Pattern formation underpins cell specification, tissue organization, and morphogenesis during embryonic development. Tissue engineering strategies based on stem cell differentiation and organoid formation attempt to recapitulate developmental patterning, but suffer from variable efficiency, limited complexity, and anatomical disorganization. Whereas considerable research has focused on understanding patterning in vivo, bottom-up efforts in engineering synthetic niches to control patterning in vitro have been limited. This thesis (and associated data files) explores a synthetic biology-based approach to create self-organizing mammalian cell systems, and use them as signalling centres for stem cell applications. The first half of the thesis explores whether integrin-ligand heterotypic adhesions can drive the formation of hyperuniform patterns. HEK-293 derivative cell lines were bio-engineered to overexpress heterophilic cell adhesion molecules in a drug-inducible manner using CRISPR genome editing. Transgenic systems were characterized in input sensitivity, output strength, adhesion properties, and their ability to form hyperuniform distributions. The second half of the thesis explores applications of self-organizing cell systems to control stem cell decisions. A pre-established phase-separation patterning system was modified to produce WNT3A from multicellular groups, which self-organize with mouse embryonic stem cells and elicit polarized signalling events. When differentiated in self-organizing co-cultures, embryonic stem cells exhibit enhanced and spatially controlled mesoderm induction in 2D and 3D conditions respectively, due to exogenous, localized Wnt signalling. WNT3A producers can also trigger nephrogenesis in mouse metanephric mesenchyme, demonstrating their wider applicability. This work provides novel prototype platforms and conceptual approaches to control developmental cell fate decisions and organization in vitro, contributing to the generation of synthetic niches.

Glykofridis F; Popravko A; Dzierzak E; Davies J. "Synthetic cell-based patterning systems and their applications in stem cell differentiation - Fokion Glykofridis Datasets". Edinburgh DataVault (2021).