Stem cell differentiation and biomaterial processing for the engineering of pulmonary epithelial tissue

Abstract

The standard approach of tissue engineering is to develop a cell-seeded scaffold which has morphology and mechanical properties similar to those of the target tissue. The scaffold material should possess surface properties so that the seeded cells can maintain proper activity, function and morphology. In terms of engineering of pulmonary epithelial tissue, a major challenge is to obtain sufficient cells because of the difficulty in isolating and culturing primary pulmonary epithelial cells in vitro, especially type II pneumocytes. To overcome this problem, a method to differentiate pulmonary progenitors, which possessed most of the features of type II pulmonary epithelial cells, from murine embryonic stem cells (mESCs) was investigated in this study. The first aim of this project was to increase the differentiation efficiency of type II pneumocyte progenitors from mESCs by enhancing the efficiency of an established differentiation protocol using extracellular matrix (ECM), growth factors and bioactive peptides. In the first study, mESCs were differentiated on tissue culture plastic (TCP) and poly(D,L-lactide) (PDLLA) films coated with ECM proteins, collagen, laminin, fibronectin and Matrigel. The results demonstrated that all protein coatings can enhance the wettability of the TCP and PDLLA films and, moreover, laminin and Matrigel can enhance the differentiation of mESCs into pulmonary progenitors. In the second study, growth factors that are commonly thought to affect the development of embryonic lung, including fibroblast growth factors (FGFs) 1, 2, 7 and 10, were added to the differentiation culture at various concentrations and the subsequent expression of surfactant protein C, a marker of type II pneumocytes and their progenitors, was measured. It was found that FGF1 alone and FGF10 in combination with Matrigel coating enhanced the differentiation of mESCs into pulmonary progenitors. In another study, mESCs were differentiated on PDLLA films grafted with the bioactive peptides RGD and YIGSR. Preliminary result showed that YIGSR enhanced the differentiation of mESCs into pulmonary progenitors. The second aim of this project was to develop 2D environments and 3D scaffolds made of PDLLA suitable for the culture of human pulmonary epithelial cells (A549 line). PDLLA has advantages of biocompatibility and biodegradability, but a major drawback is its hydrophobic nature. To make the surface of PDLLA films hydrophilic, it was modified using a variety of methods, i.e. by grafting the bioactive peptides RGD and YIGSR, by introducing amines using aminolysis and by creating amine-terminated and carboxylic acid-terminated tree-like branched architectures on to the surface. The surface properties of modified PDLLA films were evaluated using various techniques. The culture of A549 cells on PDLLA films demonstrated that surface modifications can affect the attachment, focal adhesion point formation, activity and population size, depending on the type and the concentration of the bioactive peptides or functional groups presented on the surface of PDLLA films. The challenge of culturing pulmonary epithelial cells in 3D is to generate scaffolds with proper porous structures which allow sufficient medium diffusion in and waste disposal out of the scaffolds. The influence of the preparation conditions, i.e. coarsening time and coarsening period of a liquid-liquid phase separation system and freezing temperature of a solid-liquid phase separation system, on the porous morphology and the subsequent pulmonary epithelial cell culture were examined. Scaffolds that possessed alveolus-like round pores and ladder-like pores were prepared using liquid-liquid phase separation and solid-liquid phase separation, respectively. Culture of A549 cells on the PDLLA scaffolds demonstrated that cell penetration into and activity on the scaffolds are influenced by the pore size and the pore throat size of the scaffolds. In conclusion, the results of this project demonstrated that the differentiation of mESCs into pulmonary epithelial progenitors can be enhanced by external signals i.e. from ECM proteins, FGFs and bioactive peptides. The responses of pulmonary epithelial cells to the PDLLA scaffolds can be enhanced by surface modifications using bioactive peptides and functional groups and scaffolds that can serve as a culture environment for pulmonary epithelial cells were prepared accordingly. Taken together, the results of these studies provide a basis for future engineering of pulmonary epithelial tissue, an area of tissue engineering that lags behind that of other major organs.