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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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Generation of mouse iLEC by directed lineage conversion. (a) Illustrative scheme of the directed lineage conversion process of mouse embryonic fibroblasts from Nkx2-1-mCherry e15.5 embryos into induced lung epithelial progenitors (iLEP). (b) Representative DIC images of colonies in various stages of the conversion process. Only fibroblasts transduced with OSKM and Nkx2-1 showed evidence of epithelial-like cells (yellow hatched area). (c) At the end of the conversion, epithelial-like cells express nkx2-1-driven mCherry transgene. Note: mCherry fluorescence was relatively dim. This image exposure was increased to show the dim fluorescence. Representative DIC image of (d) OSKM only or (e) Nkx2-1 only transduced cells showing no morphological change into epithelial-like cells. Representative DIC image Cd326 + FACS-sorted cells (f) after freeze-thawed twice and (g) maintained in BEGM for 20 passages. (h) Flow cytometric contour plots depicting epithelial cell surface marker (Cd326) acquisition and down-regulation of stromal cell surface marker Fsp1 during the cell conversion process. (i) Real-time qRT-PCR analysis of pluripotency (Oct4, Klf4, cMyc and Sox2), lung epithelial (Nkx2-1, Trp63, Foxa2) and mesenchymal (Vimentin) gene expression. MEF + 5F represents MEFs transduced with OSKM and Nkx2-1. The cells were harvested 24 hours after Nkx2-1 transduction. DE to iLEC groups represents cells that were transduced with all 5 factors and subsequently differentiated into the respective cell types. N = 4 independent conversion experiments. Scale bars represent 100–150 μm.