Abstract
Pediatric lung disorders are a leading cause of illness and death in infants and children. These diseases are poorly understood and there are few treatment options available. For example, infants born with hereditary surfactant protein-B (SP-B) deficiency will die within a year after birth without a lung transplantation. SP-B is an essential component of pulmonary surfactant, secreted by alveolar epithelial type II cells (AEC II). Potential therapeutic strategies under development to correct SP-B deficiency include the use of human induced pluripotent stem cell (hiPSC)-derived lung precursors if methods can be optimized to derive AEC II cells of sufficient purity and quantity. The goal of this project was to optimize differentiation and expansion of hiPSC-derived definitive endoderm (DE), the first stage for AEC II specificity, using novel scaffold culture systems. A lentiviral vector was used to transduce hiPSC to express the reporter genes firefly luciferase and the enhanced green fluorescent protein (eGFP). An established well-tested bank of hiPSC expressing these reporter genes were used for these studies. Monolayer cultures or embryoid body (EB) cultures were supplemented with Activin A (100 ng/mL) for 4 days to direct cells to definitive endoderm (DE). Directed differentiation of hiPSC in EB culture increased expression of DE markers SOX17 (19-fold; p<0.05), FOXA2 (13-fold), CXCR4 (81-fold), and GATA4 (38-fold) compared to standard monolayer cultures. To enhance differentiation and expansion to DE, biologically inert, biodegradable hyper-crosslinked carbohydrate polymer scaffolds (HCCP) were used to evaluate cellular attachment. These results showed that decellularized lung scaffolds were more effective for cellular attachment and migration compared to HCCP scaffolds. Analysis of protein expression showed a rapid upregulation of DE markers by day 3 in recellularized lung scaffolds. Previous studies in the lab show that bioactive molecules are retained in the decellularized lung scaffold other than proteins of the extracellular matrix. Since HCCP scaffolds provide a controlled environment, they will be important in future studies to determine which bioactive molecules are important for enhancing DE differentiation. Overall, our studies demonstrate the importance of cell – cell interactions for DE differentiation in vitro, which are not captured in standard monolayer cultures. In addition, our studies provide a basis for optimizing AEC II from hiPSC. Ultimately, the goal is to optimize each stage of AEC II differentiation for use in pre-clinical studies of engraftment and function for the treatment of disorders such as hereditary SP-B deficiency and other pediatric lung disorders.