Identification of AAV serotypes for lung gene therapy in human embryonic stem cell-derived lung organoids

摘要

Key stages of lung bud organoid generation. Representative images of the key stages in lung bud organoid generation are shown together with the key respective differences in culture, plate type/coating and cellular factors. d0: hESC are routinely cultured in adherent mode on Matrigel-coated plates. d0–d4: Transition to suspension culture allows differentiation (via embryoid bodies) to definite endoderm. d4–d6: Transition to adherent culture on fibronectin-coated plates and a two-step treatment with cellular factors induces anterior foregut endoderm. d6–d28: Transition to suspension culture allows the generation of nascent organoids. d28–d45+: mature LBOs are generated after nascent organoids are placed in a Matrigel sandwich, with ‘buds’ starting to emerge after a few days as highlighted by the arrow. Mature LBOs are typically used for experiments from d45 onwards (in this study d59 and d79). LBO image shown is representative of d59–79. Images are representative of > 4 cycles of LBO generation. Scale bars 200 μm