Search

The balance between self‐renewal and differentiation in human foetal lung epithelial progenitors controls the size and function of the adult organ. Moreover, progenitor cell gene regulation networks are employed by both regenerating and malignant lung cells, where modulators of their effects could potentially be of therapeutic value. Details of the molecular networks controlling human lung progenitor self‐renewal remain unknown. We performed the first CRISPRi screen in primary human lung organoids to identify transcription factors controlling progenitor self‐renewal. We show that SOX9 promotes proliferation of lung progenitors and inhibits precocious airway differentiation. Moreover, by identifying direct transcriptional targets using Targeted DamID, we place SOX9 at the centre of a transcriptional network, which amplifies WNT and RTK signalling to stabilise the progenitor cell state. In addition, the proof‐of‐principle CRISPRi screen and Targeted DamID tools establish a new workflow for using primary human organoids to elucidate detailed functional mechanisms underlying normal development and disease.

Human organoid systems recapitulate key features of organs offering platforms for modelling developmental biology and disease. Tissue-derived organoids have been widely used to study the impact of extrinsic niche factors on stem cells. However, they are rarely used to study endogenous gene function due to the lack of efficient gene manipulation tools. Previously, we established a human foetal lung organoid system (Nikolić et al., 2017). Here, using this organoid system as an example, we have systematically developed and optimised a complete genetic toolbox for use in tissue-derived organoids. This includes ‘Organoid Easytag’, our efficient workflow for targeting all types of gene loci through CRISPR-mediated homologous recombination followed by flow cytometry for enriching correctly targeted cells. Our toolbox also incorporates conditional gene knockdown or overexpression using tightly inducible CRISPR interference and CRISPR activation which is the first efficient application of these techniques to tissue-derived organoids. These tools will facilitate gene perturbation studies in tissue-derived organoids facilitating human disease modelling and providing a functional counterpart to many ongoing descriptive studies, such as the Human Cell Atlas Project.

Recent studies have revealed biologically-significant differences between human and mouse lung development and highlighted new in vitro systems to allow experimental manipulation of human lung models. At the same time, emerging clinical data suggests that the origins of some adult lung diseases are found in embryonic development and childhood. The convergence of these research themes has fuelled a resurgence of interest in human lung developmental biology. This review discusses our current understanding of human lung development, which has been profoundly influenced by studies in mice and, more recently, by experiments in human in vitro lung developmental models and RNA-sequencing of human foetal lung tissue. Together, these approaches are helping to shed light on mechanisms of human lung development and disease, and may help pave the way for new therapies to be developed and tested., Rutherford Fund Fellowship allocated by the Medical Research Council and the UK Regenerative Medicine Platform MR/5005579/1 to MZN; Academic Clinical Lectureship, University of Cambridge to MZN; Wellcome Trust PhD studentship 109146/Z/15/Z to DS; Medical Research Council G0900424 to ELR.