Differentiation of human induced pluripotent stem cells into functional airway epithelium

Rationale Highly reproducible in vitro generation of human bronchial epithelium from pluripotent stem cells is an unmet key goal for drug screening to treat lung diseases. The possibility of using induced pluripotent stem cells (hiPSC) to model normal and diseased tissue in vitro from a simple blood sample will reshape drug discovery for chronic lung, monogenic and infectious diseases. Methods We devised a simple and reliable method that drives a blood sample reprogrammed into hiPSC subsequently differentiated within 45 days into air-liquid interface bronchial epithelium (iALI), through key developmental stages, definitive-endoderm (DE) and Ventralized-Anterior-Foregut-Endoderm (vAFE) cells. Results Reprogramming blood cells from one healthy and 3 COPD patients, and from skin-derived fibroblasts obtained in one PCD patient, succeeded in 100% of samples using Sendai viruses. Mean cell purity at DE and vAFE stages was greater than 80%, assessed by expression of CXCR4 and NKX2.1, avoiding the need of cell sorting. When transferred to ALI conditions, vAFE cells reliably differentiated within 4 weeks into bronchial epithelium with large zones covered by beating ciliated, basal, goblets, club cells and neuroendocrine cells as found in vivo . Benchmarking all culture conditions including hiPSCs adaptation to single-cell passaging, cell density and differentiation induction timing allowed for consistently producing iALI bronchial epithelium from the five hiPSC lines. Conclusions Reliable reprogramming and differentiation of blood-derived hiPSCs into mature and functional iALI bronchial epithelium is ready for wider use and this will allow better understanding lung disease pathogenesis and accelerating the development of novel gene therapies and drug discovery. ### Competing Interest Statement AB reports grants, personal fees, non-financial support and other from AstraZeneca; grants, personal fees, non-financial support and other from Boeringher Ingelheim; grants, personal fees, non-financial support and other from GlaxoSmithKline; personal fees, non-financial support and other from Novartis; personal fees and non-financial support from Teva; personal fees, non-financial support and other from Regeneron; personal fees, non-financial support and other from Chiesi Farmaceuticals; grants, personal fees, non-financial support and other from Actelion; personal fees from Gilead; non-financial support and other from Roche; other from Nuvaira, outside the submitted work. JDV reports personal fees and other from Stem Genomics; personal fees and other from MedXCell Science; personal fees from Gilead; personal fees from Celgene, outside the submitted work. In addition, JDV and SA have a patent EP20150306389 pending. SA reports personal fees and other from Stem Genomics, outside the submitted work. JPG is employee of Boehringer Ingelheim. EA, MF, CB, JM, AP, CV, MJ, CC, HB, GM, IV declare no conflict of interest.