Studying lung development in vitro: expanding the genetic toolbox and analysis of SOX2 and SOX9 function

Respiratory disease is a growing threat to human health worldwide. Induced pluripotent stem cell (iPSC) based stem cell therapy is promising for end-stage respiratory disease patients. Generation of mature/functional lung cells from iPSCs heavily relies on understanding of human lung development. However, analysis of lung development has largely been based on mouse models. Given the differences between mouse and human lungs in terms of size, cell types and gene expression profiles, mouse models cannot fully recapitulate human lung development. Recent advances in 3-D in vitro culture systems have allowed the establishment of human foetal lung organoids from fresh foetal lung tissues. These organoids recapitulate key features/gene expression signatures of in vivo foetal lung multipotent progenitor cells which are capable of giving rise to all epithelial lineages. The extrinsic factors that regulate human lung progenitor cell self-renewal and differentiation are extensively studied by manipulating the culture conditions. However, the functions of cell intrinsic genes, particularly transcription factor regulatory networks, are largely unexplored. For the purpose of studying intrinsic gene function using a tissue-derived organoid system, I have systematically explored and developed a series of genetic manipulation tools. With these tools, gene knock-ins, knock-outs, conditional over-expression and knock-downs can all be easily achieved. As proof of principle, I have used these tools to systematically characterise the function of two SRY-related HMG-box (SOX) genes, SOX2 and SOX9, in human foetal lung progenitor cells. I show that although SOX2 is expressed in early stage progenitor cells it is dispensable for progenitor cell survival and self- renewal. By contrast, SOX9 is crucial for tip progenitor cell self-renewal by regulating progenitor cell proliferation and epithelial layer organisation. Through analysis of upstream signals and downstream targets I show that SOX9 connects WNT and receptor tyrosine kinase (RTK) signalling pathways, both of which drive lung epithelium development. SOX9 expression is regulated by canonical WNT signalling and SOX9 modulates the expression of ETV4 and ETV5, which are RTK signalling effectors, thereby influencing RTK signalling. This study has marked the first effort to study gene function in human lung development using tissue derived organoids. I envision that human foetal lung organoids empowered by gene perturbations will enable high throughput genetic/drug screens to benchmark gene functions with the Human Cell Atlas as reference. This will provide new insights into human lung development.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2020

Author dc:creator

• Sun, Dawei

Advisor dc:contributor.advisor

• Rawlins, Emma

• Lung development
• Organoid
• CRISPR
• SOX proteins

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/16627e62-08b4-4e98-ab76-a821349b3200/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

Author Identifier

0000-0003-1551-4349
0000-0001-7426-3792

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/316481