4 Transgenic porcine model reveals two roles for LGR5 in lung development and homeostasis

LGR5, a transmembrane receptor, has been a distinguished marker of regenerative adult stem cells in organs with high turnover, such as the intestines, stomach, and liver. However, the role of LGR5-expressing cells in the lung, another organ with high turnover and regenerative capacity, has been relatively unstudied. The objective of this research, therefore, was to understand the role of LGR5 in lung development and homeostasis. In addition, much of the research on these populations has been driven by murine models, which are limited by anatomical and physiological differences from humans. To overcome these limitations, we generated a transgenic porcine model for the study of LGR5. Transgenic pigs were generated by CRISPR/Cas9 mediated knock-in of the H2BGFP gene into the endogenous LGR5 locus, and correctly edited cells were used to obtain boars by somatic cell nuclear transfer. Cells and tissues from embryonic Day 80 or juvenile lungs from clones and F₁ generation offspring were used as follows. Tissues were collected in paraformaldehyde and frozen in OCT compound for cryosectioning and immunostaining analyses. Single cells were isolated using an enzymatic digest of dispase II, collagenase, and DNaseI. Cell suspensions were sorted for presence of LGR5-H2B-GFP by fluorescence-activated cell sorting on a MoFlo (Beckman Coulter), and positive or negative populations were either seeded in Matrigel (Corning) for organoid formation or sent to Genewiz (https://www.genewiz.com/en) for bulk RNA-sequencing. Organoids were grown in Dulbecco’s modified Eagle medium/F10 supplemented with a growth factor cocktail and antibiotics, and at least three biological replicates were used for each experiment. RNA-seq results were analysed using DeSEqn 2, and differentially expressed genes were tested by a Wald test and corrected by a Benjamini-Hochberg correction for multiple comparisons. Analyses of fetal lungs show robust expression of LGR5, with two anatomically distinct subsets: one with epithelial expression in developing bud tips, and one with mesenchymal expression around developed airways. Immunohistochemistry and RNA-seq analyses showed that the mesenchymal LGR5+ populations co-expressed fibroblast markers such as vimentin and PDGFRa, whereas the epithelial populations expressed markers such as SOX9 and EPCAM. Cell culture experiments showed that the epithelial LGR5+ population was capable of forming organoids from single cells, whereas the mesenchymal subset was not. We next asked where LGR5 was expressed at homeostasis in juvenile lungs. Remarkably, only the mesenchymal population was present, with no LGR5 expression detectable in any epithelial cells. Although juvenile mesenchymal LGR5+ cells also do not form organoids, the organoids formed by LGR5− epithelial cells showed robust LGR5-H2BGFP expression after 5 days in culture. These results describe two previously unreported roles for LGR5 in lung development and homeostasis, suggesting that LGR5 has a role early in airway development, and later in airway maintenance. Because injury repair often recapitulates development, it is likely that these same pathways could be important in regeneration.