Hypoxia signalling regulates human lung epithelial cell fates

Human embryos develop in an environment with low oxygen tension (< 5% O2) during the first trimester. Hypoxia substantially impacts mammalian development and tissue homeostasis. However, it remains unclear whether and how hypoxia directly affects human lung epithelial development. In this PhD thesis, I present my investigation of the effects of chronic hypoxia on the human lung epithelium using organoid models. Human first-trimester lung epithelial progenitors can be isolated from embryonic and fetal lungs and expanded in an undifferentiated state when culturing in the self-renewal medium under normoxia (around 20% O2). In physiological hypoxia (2-5% O2), however, the progenitors spontaneously differentiated towards multiple airway cell types, and simultaneously inhibited alveolar differentiation. Time-series single-cell transcriptomic analysis revealed unexpected organoid heterogeneity and delineated hypoxia-induced differentiation processes. The self-renewing organoids under normoxia consisted of tip and primed progenitors, the potential counterparts of tip and stalk cells in vivo, respectively. Under hypoxia, the tip progenitors decelerated proliferation, while the primed progenitors potentially gave rise to basal, neuroendocrine, secretory-like, and hillock-like cells. Hypoxia-inducible factors (HIFs) are the central regulators mediating cellular response to hypoxia. I found that the HIF pathway was rapidly activated upon exposure to hypoxia with a genetic HIF-DNA binding reporter. Chemical stabilisation of HIF-α subunits (HIF-1α and HIF-2α) under normoxia recapitulated hypoxia-induced airway differentiation. With CRISPRi and ectopic expression systems, I demonstrated that HIF-1 and HIF-2 differentially regulated airway and alveolar fate decisions of lung epithelial progenitors. Combining transcriptomic and targeted DamID-sequencing analyses, my collaborators and I identified direct HIF target genes, including KLF-family transcription factors. I further examined functions of KLF4 and KLF5 in lung epithelial progenitors with CRISPRi. KLF4 and KLF5 facilitated differentiation of progenitors to basal and secretory cells in both hypoxia-induced and chemical-induced airway differentiation processes. These results suggest that hypoxia signalling might act through a core genetic program to regulate progenitor differentiation. Local hypoxia is a prevailing signature of fibrotic lungs with aberrant airway cells frequently occurring in alveoli. Consistent with this observation, I showed that chronic hypoxia directly reprogrammed human alveolar type 2 cells into airway cells. HIF pathway activation largely recapitulated this phenotype. These findings together reveal roles for hypoxia and HIF signalling during human lung development. Activation of the developmental program due to local hypoxia may also contribute to aberrant cell fate decisions in chronic lung disease.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2024

Author dc:creator

• Dong, Ziqi

Advisor dc:contributor.advisor

• Rawlins, Emma

• Lung
• Development
• Progenitor cells
• Cell differentiation
• Hypoxia

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/8a6e7e41-2aa6-4682-ae60-3d41b8064721/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.113753

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/376561