A Rapid Lung De-cellularization Protocol Supports Embryonic Stem Cell Differentiation In Vitro and Following Implantation

Abstract

Pulmonary diseases represent a large portion of neonatal and adult morbidity and mortality. Many of these have no cure, and new therapeutic approaches are desperately needed. De-cellularization of whole organs, which removes cellular elements but leaves intact important extracellular matrix (ECM) proteins and three-dimensional architecture, has recently been investigated for ex vivo generation of lung tissues. As specific cell culture surfaces, including ECM composition, profoundly affect cell differentiation, this approach offers a potential means of using de-cellularized lungs to direct differentiation of embryonic and other types of stem/progenitor cells into lung phenotypes. Several different methods of whole-lung de-cellularization have been reported, but the optimal method that will best support re-cellularization and generation of lung tissues from embryonic stem cells (ESCs) has not been determined. We present a 24-h approach for de-cellularizing mouse lungs utilizing a detergent-based (Triton-X100 and sodium deoxycholate) approach with maintenance of three-dimensional lung architecture and ECM protein composition. Predifferentiated murine ESCs (mESCs), with phenotypic characteristics of type II alveolar epithelial cells, were seeded into the de-cellularized lung scaffolds. Additionally, we evaluated the effect of coating the de-cellularized scaffold with either collagen or Matrigel to determine if this would enhance cell adhesion and affect mechanics of the scaffold. Finally, we subcutaneously implanted scaffolds in vivo after seeding them with mESCs that are predifferentiated to express pro-surfactant protein C (pro-SPC). The in vivo environment supported maintenance of the pro-SPC-expressing phenotype and further resulted in vascularization of the implant. We conclude that a rapid detergent-based de-cellularization approach results in a scaffold that can maintain phenotypic evidence of alveolar epithelial differentiation of ESCs and support neovascularization after in vivo implantation.