1 Foxa2 lineage + mesendoderm forms a competitive pulmonary mesenchyme niche crucial for generating the entire lungs Akihiro Miura 1, 2 , Hemanta Sarmah 1 , Junichi Tanaka 1 , Youngmin Hwang 1 , Anri Sawada, 1 Yuko Shimamura 1 , Yinshan Fang 1 , Dai Shimizu 2 , Zurab Ninish 1 , Jake Le Suer 3, 4 , Nicole C. Dubois 5 , Jennifer Davis 6 , Shinichi Toyooka 2 , Jun Wu 7 , Jianwen Que 1 , Finn J. Hawkins 3, 4 , Chyuan-Sheng Lin 8 , Munemasa Mori 1 ♰ 1 Columbia Center for Human Development and Division of Pulmonary, Allergy, Critical Care, Department of Medicine, Columbia University Medical Center, New York, NY 10032, USA. 2 Department of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, 7008558, Japan. 3 The Pulmonary Center and Department of Medicine, Boston University School of Medicine, Boston, MA 02118, USA 4 Center for Regenerative Medicine, Boston University and Boston Medical Center, Boston, MA 02118, USA 5 Department of Cell, Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. 6 Department of Pathology, University of Washington, Seattle, WA 98109, USA. 7 Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA 8 Bernard and Shirlee Brown Glaucoma Laboratory, Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University Irving Medical Center, New York, NY 10032, USA. ♰ To whom correspondence should be addressed: Munemasa Mori, M.D., Ph.D. 650W 168 th St, New York, NY 10032, USA E-mail: mm4452@cumc.columbia.edu Summary Millions worldwide suffer from incurable lung diseases, and organ transplantation remains their only hope. However, there has been an absolute shortage of donor lungs and unmet needs for transplantable lung generation composed of tissue-specific mesenchyme, epithelium, and endothelium. Elucidation of lung precursor traits during development can lead to solving this issue. Using lineage-tracing mice, we discovered that gastrulating Foxa2 lineage + Pdgfrα + mesendoderm forms the competitive mesenchymal lung niche. We further evidenced that Foxa2 + Pdgfrα + mesendoderm is an evolutionarily-conserved niche during human iPSC-derived lung differentiation. The Fgfr2 gene depletion, specifically in the Foxa2 lineage, showed lung agenesis phenotype. Strikingly, donor iPSCs injection into those blastocysts complemented endodermal and mesodermal defective lung organ niches that efficiently led to the entire lung generation. Together, targeting Foxa2 lineage for lung generation is a novel paradigm, holding a grand promise for future human whole lung generation in large animals using human iPSCs. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint this version posted November 2, 2022. ; https://doi.org/10.1101/2022.10.31.514628 doi: bioRxiv preprint