An NKX2-1GFP and TP63tdTomato dual fluorescent reporter for the investigation of human lung basal cell biology

Sci Rep. 2021 Feb 25;11(1):4712. doi: 10.1038/s41598-021-83825-6.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Green Fluorescent Proteins / analysis*
  • Green Fluorescent Proteins / genetics
  • Humans
  • Luminescent Proteins / analysis*
  • Luminescent Proteins / genetics
  • Lung / cytology*
  • Lung / metabolism
  • Organoids / cytology
  • Organoids / metabolism
  • Pluripotent Stem Cells / cytology*
  • Pluripotent Stem Cells / metabolism
  • Red Fluorescent Protein
  • Thyroid Nuclear Factor 1 / analysis*
  • Thyroid Nuclear Factor 1 / genetics
  • Transcription Factors / analysis*
  • Transcription Factors / genetics
  • Tumor Suppressor Proteins / analysis*
  • Tumor Suppressor Proteins / genetics

Substances

  • Luminescent Proteins
  • NKX2-1 protein, human
  • TP63 protein, human
  • Thyroid Nuclear Factor 1
  • Transcription Factors
  • Tumor Suppressor Proteins
  • Green Fluorescent Proteins