Identification of a multipotent lung progenitor for lung regeneration

We recently showed that a suspension of mouse or human fetal or adult lung cells infused intravenously following appropriate conditioning of recipient mice leads to marked lung chimerism within alveolar and bronchiolar lineages, in distinct 'patches'. A large proportion of these donor-derived 'patches' contain both epithelial and endothelial cells. We show here, using R26R-Confetti mice as donors, that these multi-lineage patches are derived from a single lung progenitor. Fluorescence activated cell sorting of adult mouse lung cells revealed that the putative patch-forming progenitors co-express the endothelial marker CD31 (PECAM-1) and the epithelial marker CD326 (EPCAM). Transgenic Cre/lox mice expressing GFP under different promoters support this duality by demonstrating expression of additional epithelial and endothelial markers in this double-positive lung subpopulation. These findings could pave the way not only for effective lung regeneration but also for better understanding of lung vasculature and airways development and maintenance in health and disease. Overall design: Long term chimerism in recipient lungs after transplantation of td-tomato labeled cells: In our previous studies we followed by immuno-histology (IH) lung chimerism up to 4 months. We have now performed 6-8 months follow up of chimerism using and combining results of immunohistology with single cell RNA transcriptome analysis , showing donor derived epithelial (ciliated cells, club cells, AT1 and AT2 cells,) and endothelial clusters (lymphatics, endothelial progenitors, as well as the newly described gCap -“general”, and aCap -“aerocytes” capillary cells). The resulting clusters were defined based on marker genes and gene set analysis results, using publicaly available LGEA (Lung Gene Expression Analysis) Web Portal. For definition of each cluster we used well definied hallmark gene sets. We conducted single cell transcriptome analysis of the chimeric lung, using FACS purified donor- and recepient-derived lung cells. Three chimeric lungs were first verified to exhibit significant chimerism by fluorescent microscopy, pooled, enzymatically dissociated and FACS separated after gating on CD45-, single, live cells into donor and recipient compartments based on Td-Tomato expression.