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Azis, Rizal (2024) Development of a human induced pluripotent stem cells (hiPSCs)-derived, universal platform, for generation of immunocompetent multi-tissue organoids. PhD thesis, University of Nottingham.

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Abstract

Human induced pluripotent stem cells (hiPSCs) provide a platform that can be used for disease modelling and developing a better understanding of developmental biology. Traditional two-dimensional (2D) co-culture of different cell types derived from hiPSCs is the simplest platform for modelling multicellular tissue similar to an in vitro model, however, these 2D co-cultures are not always physiologically relevant and fail to reproduce tissue complexity, especially in terms of 3D architecture. The interplay or cell-cell interaction between non-parenchymal cells including macrophages, fibroblasts, and endothelial cell and parenchymal cells (tissue-specific epithelial cells) exhibits crucial regulatory activity of tissue homeostasis, yet current in vitro models to not capture this and therefore it needs to rely on non-human animal models to better understand this interplay. To bridge this gap, highly efficient protocols are required to generate different cell types and combined them as 3D organoids for more accurate and human-specific disease modelling and other applications are desperately needed.

Improved methods for generating these cell types that are xeno and serum-free, and minimise cellular heterogeneity are important advancements that will be important for developing more complex 3D stem-cell-based tissue models. Recently protocols to generate hematopoietic progenitor cells are based on either the embryoid bodies (EBs) formation or stem cell colonies-specific size as the starting point of the differentiation. As a result, it is often inefficient, has low reproducibility and requires undefined media components that can limit clinical application. To address this issue, a chemically defined, xeno-free differentiation platform have been optimised that can generate macrophages (MФ), dendritic cells (DCs), fibroblast like cells (FLCs), and vascular endothelial cells (VECs) from the same starting population of human induced pluripotent stem cells (hiPSCs) with a minimal seeding density by inducing mesoderm differentiation over 2 days and then changing growth factor composition which can drive the mesoderm towards fibroblasts, vascular endothelial cells or hemogenic endothelial cells and monocytic cells. The resulting cell types have been validated via mRNA-seq as well as using cell type specific functional assays including polarisation of MФ into M1 and M2 cells endocytosis assay, FBs activation into MyoFBs using TGF-ß and ECs tube formation assay. Furthermore, to demonstrate how our approach works, these non-parenchymal cells (MФ, DCs, FLCs, and VECs) were combined with parenchymal cells (PCs) representative of liver hepatocytes which were generated using our established protocol in chemically defined and xeno-free medium to generate multi-tissue organoids. This work showed that multi-tissue liver organoids retained their cellular composition over time and showed gene expression profiles consistent with either healthy or fatty liver conditions using flow cytometry and scRNAseq technology.

In summary, isogenic MФ, DCs, FLCs and VECs were generated in a chemically defined, xeno free, single cell differentiation protocol that had gene expression and functional profiles similar to their primary equivalents, which could be combined with PCs from different tissues including heart, lung, kidney, gut and liver. These cells are an important step towards development of more complex in-vitro developmental and disease models and could also be applicable to translational clinical applications.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Hannan, Nicholas Ghaemmaghami, Amir
Keywords:	induced pluripotent stem cell, organoid, differentiation, fibroblast, macrophages, dendritic cells, endothelial cells, hematopoietic stem cells, hepatocytes
Subjects:	QS-QZ Preclinical sciences (NLM Classification) > QU Biochemistry
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Medicine
Item ID:	79975
Depositing User:	Rizal, Rizal
Date Deposited:	12 Dec 2024 08:31
Last Modified:	12 Dec 2024 08:31
URI:	https://eprints.nottingham.ac.uk/id/eprint/79975

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