Journal Article

In vitro generation of human pluripotent stem cell derived lung organoids

Ferguson, Michael AH, White, Eric S, Tsai, Yu-Hwai, Hill, David R, Dye, Briana R, Dyal, Rachel, Klein, Ophir D, Mayhew, Christopher N, Spence, Jason R, Deutsch, Gail H, Nagy, Melinda S, Nattiv, Roy, Wells, James M

2015

Overview

Recent breakthroughs in 3-dimensional (3D) organoid cultures for many organ systems have led to new physiologically complex in vitro models to study human development and disease. Here, we report the step-wise differentiation of human pluripotent stem cells (hPSCs) (embryonic and induced) into lung organoids. By manipulating developmental signaling pathways hPSCs generate ventral-anterior foregut spheroids, which are then expanded into human lung organoids (HLOs). HLOs consist of epithelial and mesenchymal compartments of the lung, organized with structural features similar to the native lung. HLOs possess upper airway-like epithelium with basal cells and immature ciliated cells surrounded by smooth muscle and myofibroblasts as well as an alveolar-like domain with appropriate cell types. Using RNA-sequencing, we show that HLOs are remarkably similar to human fetal lung based on global transcriptional profiles, suggesting that HLOs are an excellent model to study human lung development, maturation and disease. Cell behavior has traditionally been studied in the lab in two-dimensional situations, where cells are grown in thin layers on cell-culture dishes. However, most cells in the body exist in a three-dimensional environment as part of complex tissues and organs, and so researchers have been attempting to re-create these environments in the lab. To date, several such ‘organoids’ have been successfully generated, including models of the human intestine, stomach, brain and liver. These organoids can mimic the responses of real tissues and can be used to investigate how organs form, change with disease, and how they might respond to potential therapies. Here, Dye et al. developed a new three-dimensional model of the human lung by coaxing human stem cells to become specific types of cells that then formed complex tissues in a petri dish. To make these lung organoids, Dye et al. manipulated several of the signaling pathways that control the formation of organs during the development of animal embryos. First, the stem cells were instructed to form a type of tissue called endoderm, which is found in early embryos and gives rise to the lung, liver and other several other internal organs. Then, Dye et al. activated two important developmental pathways that are known to make endoderm form three-dimensional intestinal tissue. However, by inhibiting two other key developmental pathways at the same time, the endoderm became tissue that resembles the early lung found in embryos instead. This early lung-like tissue formed three-dimensional spherical structures as it developed. The next challenge was to make these structures develop into lung tissue. Dye et al. worked out a method to do this, which involved exposing the cells to additional proteins that are involved in lung development. The resulting lung organoids survived in laboratory cultures for over 100 days and developed into well-organized structures that contain many of the types of cells found in the lung. Further analysis revealed the gene activity in the lung organoids resembles that of the lung of a developing human fetus, suggesting that lung organoids grown in the dish are not fully mature. Dye et al.'s findings provide a new approach for creating human lung organoids in culture that may open up new avenues for investigating lung development and diseases.

• Publisher: eLife Sciences Publications Ltd,eLife Sciences Publications, Ltd
• Subject: Alveoli / Basal cells / Cell Culture Techniques - methods / Cell Differentiation - genetics / Cell Line / Cells, Cultured / Children & youth / Cluster analysis / Developmental biology / Developmental Biology and Stem Cells / Embryo cells / Embryonic Stem Cells - cytology / Embryonic Stem Cells - metabolism / endoderm / Endoderm - cytology / Endoderm - metabolism / Epithelium / Fetuses / Foregut / Gene Expression Profiling / Genomes / Hospitals / Humans / Induced Pluripotent Stem Cells - cytology / Induced Pluripotent Stem Cells - metabolism / lung / Lung - cytology / Lung - embryology / Lung - metabolism / Lung diseases / Mesenchyme / Microscopy, Confocal / Microscopy, Electron, Transmission / Organogenesis / organoid / Organoids / Organoids - cytology / Organoids - metabolism / Organoids - ultrastructure / Pluripotency / pluripotent stem cell / Pluripotent Stem Cells - cytology / Pluripotent Stem Cells - metabolism / Principal components analysis / Reproducibility of Results / Respiratory tract / Reverse Transcriptase Polymerase Chain Reaction / Ribonucleic acid / RNA / Smooth muscle / spheroid / Spheroids / Spheroids, Cellular - cytology / Spheroids, Cellular - metabolism / Stem cells / Tissue Engineering - methods / Transcription