Generation of functional mature type II pneumocytes from human induced pluripotent stem cells in 3D dynamic culture

Abstract

Lung diseases have become a more prominent problem worldwide due to smoking and pollution and they are increasingly responsible for elevated mortality and morbidity. Pro-gressive fibrosis followed by a subsequent loss of functional lung tissue and gas-exchange units, are the main causes for chronic lung diseases. In addition, failure to maintain endo-thelial and epithelial regeneration can also lead to the pathological fibrosis. More research is needed to find an alternative solution for the effective repair of lung tissue (Moodley et al., 2013). For patients with chronic lung disease, lung transplantation is the only option available at present. However, the limited availability of lung donors and possible immu-nological and surgical complications constrain the wide application of transplantation. Alt-hough implantation of ex-vivo-produced lung tissue could address the limitations of trans-plantation, their development has been limited by the lack of models to study the necessary developmental roadmaps for ex-vivo production of the required tissues. In this thesis, these issues were addressed through a tissue engineering approach whereby human induced pluripotent stem cells (human IPSCs) were used to produce type-II pneumocyte. This ap-proach offers the promise of cure and regeneration of injured and diseased lungs. Several researchers attempted differentiation of human IPSCs into type-II pneumocytes using 2-Dimensional (2D) cell cultures. The novelty of this work resides in the use of 3-Dimensional (3D) cultures motivated by the ability to mimic the in-vivo microenvironment. We show that alginate beads provide a reduced oxygen environment, better cellular expansion, dif-ferentiation and functional capacities ideal for the production of Type II pneumocytes. Dif-ferentiation was induced by stimulating the signalling pathways involved in alveolar epi-thelia development, forming human IPSC-derived SFTPC+ cells in 3D cultures without the need for mesenchymal support and xeno-free contamination (i.e. Matrigel™). These cells exhibit the capacity of trans-differentiation of alveolar epithelial type II to type I cells, and show additional functional features such as the lamellar bodies specific of type II pneumo-cytes, and confirmed by immuno-histology using electron microscopy. Furthermore, the 3D cultures herein developed were comparatively easy to scale up to bioreactors. Overall, our approach has proved able to promote IPSCs differentiation into Type II pneumocytes in a 3D environment using an engineered scaffold in a scalable manner. In the future, the strategy herein presented can be used for the generation of distal lung tissue for drug screening and disease modelling.