Abstract
Basal cells are multipotent stem cells responsible for the repair and regeneration of all the epithelial cell types present in the proximal lung. In mice, the elusive origins of basal cells and their contribution to lung development were recently revealed by high-resolution, lineage tracing studies. It however remains unclear if human basal cells originate and participate in lung development in a similar fashion, particularly with mounting evidence for significant species-specific differences in this process. To address this outstanding question, in the last several years differentiation protocols incorporating human pluripotent stem cells (hPSC) have been developed to produce human basal cells in vitro with varying efficiencies. To facilitate this endeavour, we introduced tdTomato into the human TP63 gene, whose expression specifically labels basal cells, in the background of a previously described hPSC line harbouring an NKX2-1GFP reporter allele. The functionality and specificity of the NKX2-1GFP;TP63tdTomato hPSC line was validated by directed differentiation into lung progenitors as well as more specialised lung epithelial subtypes using an organoid platform. This dual fluorescent reporter hPSC line will be useful for tracking, isolating and expanding basal cells from heterogenous differentiation cultures for further study.
Highlights
Cell identity as mice without functional Trp[63] lack basal cells and die at b irth[22,23,24]
As ΔNp63α is the most highly expressed isoform in airway epithelial cells[44,45], we chose to generate a ΔNp63α reporter allele in which tdTomato is inserted at the 3′ end of exon 14 in the previously described BU3 NKX2-1GFP human induced pluripotent stem cells (hiPSCs) line (Fig. 1A)[46] that allows the specific isolation of lung basal cells (Fig. 1B)[47]
We report the generation of an NKX2-1GFP;TP63tdTomato hiPSC reporter line using CRISPR/Cas[9] gene editing
Summary
Cell identity as mice without functional Trp[63] lack basal cells and die at b irth[22,23,24]. Given the important role these cells play in lung homeostasis and repair, elucidating the molecular mechanisms of their development can potentially inform the development of protocols to direct hPSC differentiation into basal cells, which will be invaluable in applications such as disease modelling, regenerative medicine as well as for the understanding of normal human lung development. To this end, we have generated an NKX2-1GFP;TP63tdTomato dual fluorescence reporter line that will facilitate the investigation of human lung basal cell biology
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