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. 2019 Jun 21;9:9027. doi: 10.1038/s41598-019-45195-y

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Lung repopulation potential of mouse iLEC. (a) Hematoxylin and eosin staining of a rat decellularized lung scaffold. (b) Flow cytometric analysis of rat embryonic lung fibroblasts (RELF) for Epcam, epithelial and vimentin, stromal cell markers. (c) Recellularized lungs with iLEC and RELF to provide paracrine support show airway reconstitution and evidence of cilia (blue arrowheads) protruding into the luminal space (right). (d) Immunofluorescence characterization of the recellularized scaffolds show donor mouse iLEC (Gfp+) reconstituting the airway structures with lung (Nkx2-1+) epithelial (panKrt+) cells. Further characterization shows Club (Ccsp+), Ciliated (Acetylated tubulin+, Foxj1+), Basal (P63+), and cells expressing Type II alveolar epithelial cell marker proSPC+ (white arrows). Of note, pan cytokeratin-expressing Gfp+ cells do not express mesenchymal marker Vimentin. No airway epithelial reconstitution was observed when scaffolds were seeded with rat embryonic lung fibroblasts only. Respective non-immune immunoglobulins were used for staining controls. N ≥ 3 recellularization experiments with separate batches of mouse iLEC, RELF and decellularized scaffolds. Scale bars represent 22–45 μm. Yellow hatched indicate GFP+ iLEC areas co-expressing lung markers.