Human embryonic stem cells (hESCs) show significant therapeutic potential in treating degenerative disorders. This is in part because of their ability to produce a limitless supply of starting cells and their potential to differentiate into more than 200 different cell types. The aim of the current research was to generate a robust stage wise protocol for the differentiation of hESCs to respiratory epithelial cells. The epithelial cells could then be used either for transplantation studies or, as an in vitro model for drug toxicity testing. In order to achieve this goal, we must identify the key steps in lung development and apply these to the differentiation protocol.In this study, we maintained Shef3 hESCs in their undifferentiated pluripotent state to expand the cells prior to the differentiated towards the definitive endoderm (DE) lineage. I used a two-stage protocol based on culture with a novel glycogen synthase kinase-3 (GSK-3) inhibitor (termed 1m), along with Activin-A. We confirmed the status of the cells by a combination of immunostaining and PCR. We showed loss of the pluripotency markers (Sox2 and Oct3/4) and gain of DE markers (Sox17, FoxA2 and CXCR4). After the induction of DE from hESCs, we then treated the cells with transforming growth factor (TGF)-β and bone morphogenetic protein (BMP) pathway inhibitors (SB431542 and Noggin respectively). This combinatorial treatment resulted in the differentiation into the anterior foregut endoderm (AFE) lineage based on expression of Pax9 and FoxA2 plus the up-regulation of Sox2. Further differentiation of AFE derivatives into more mature epithelial cells, termed lung progenitor cells (LPCs), was achieved following the treatment of AFE cells with a cocktail of trophic factors (BMP4, EGF, bFGF, FGF10, KGF and Wnt3a) yielded a population of NKX2.1-positive and FoxA2-positive cells that potentially corresponded to the lung lineage. Finally, prolonged treatment with FGF10 and FGF2 on LPC derived hESCs induced proximal (CC10, MUC5AC) and distal (SPB, SPC) airway epithelial cells. In addition, we also utilised the ectopic expression of an adenovirus expressing NKX2.1 to promote lung maturation.In conclusion, we have generated a protocol for the differentiation of hESCs into mature lung-like cells. The generation of these cells in vitro could potentially lead to a better in vitro model for toxicity testing and the development of novel therapies for promoting regeneration of lungs in patients with severe lung disorders.
|Date of Award||11 Dec 2017|
|Sponsors||The Government of Kuwait|
|Supervisor||David Tosh (Supervisor)|