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Reed, Liam (2024) Developing a chemically defined, xeno-free platform to differentiate human induced pluripotent stem cells to type 2 alveolar epithelial cells of the lung. PhD thesis, University of Nottingham.

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Abstract

Type 2 alveolar epithelial cells (AECs) are the facultative progenitors of the lung alveolar epithelium, as they self-renew and dedifferentiate into type 1 AECs under homeostatic conditions and in response to chronic injury. They are responsible for the synthesis and secretion of a lipoprotein rich surfactant to aid in the mechanics of breathing, preventing alveolar collapse at periods of low lung volume during respiration. Surfactant protein C (SFTPC) is synthesised exclusively in type 2 AECs and enhances phospholipid adsorption and spreading. Damage to type 2 AECs is implicated in diseases such as idiopathic pulmonary fibrosis (IPF), and several genetic and environmental risk factors have been identified. This includes mutations in SFTPC identified in familial cases of pulmonary fibrosis. Access to type 2 AECs for research is limited, and so animal models have primarily been used. However, animal models lack many features of human development and disease and as a result newly developed therapeutics for lung disease often fail in clinical trials.

The advent of human induced pluripotent stem cell technology and directed differentiation has circumvented some of these problems, providing an unlimited source of adult cells to model human development and disease, and for clinical translation. However, one major limitation of current protocols to differentiate human pluripotent stem cells (hPSCs) to type 2 AECs is the presence of animal-derived components in medium and the widespread use of basement membrane matrices like Matrigel™. This can cause significant biological variability and limits the therapeutic application of derived cells.

To address these issues, we reprogramme fibroblasts from a patient diagnosed with familial idiopathic pulmonary fibrosis (fIPF) harbouring a heterozygous mutation in the BRICHOS domain of SFTPC to human induced pluripotent stem cells (hiPSCs). Using base-editing technology, we generate homozygous-corrected and homozygous-mutant isogenic control hiPSC lines. We describe the development a novel, xeno-free protocol for the directed differentiation of hiPSCs to type 2 AEC organoids using chemically defined medium and human recombinant laminin. To overcome the need for fluorescence reporter cell lines, we develop a strategy to enrich for lung epithelial progenitor cells and type 2 AECs via cell surface markers by fluorescence activated cell sorting (FACS). Importantly, type 2 AEC organoids derived from healthy and mutant hiPSCs are able to recapitulate features of human lung disease. We perform detailed transcriptomic profiling across the differentiation and confirm the enrichment of genes related to major stages of embryonic lung development. Finally, we benchmark our type 2 AECs to primary lung samples and confirm similar gene expression profiles to late human fetal lung. These findings provide a platform to investigate lung disease and generate type 2 AECs with clinical applicability.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Hannan, Nicholas Merry, Cathy Cuevas Ocaña, Sara
Keywords:	Type 2 alveolar epithelial cells; Human induced pluripotent stem cells; Cell culture; Culture media; Lung alveolar epithelium
Subjects:	W Medicine and related subjects (NLM Classification) > WF Respiratory system
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Medicine
Item ID:	77961
Depositing User:	Reed, Liam
Date Deposited:	17 Jul 2024 04:40
Last Modified:	17 Jul 2024 04:40
URI:	https://eprints.nottingham.ac.uk/id/eprint/77961

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