The generation of human lung progenitors from human embryonic stem cells
thesisposted on 2017-02-17, 03:55 authored by Jenny, Robert Alexander
Respiratory epithelial cells generated from pluripotent stem cells differentiated in vitro represent a resource for research into a variety of human conditions in which this cell type is implicated. With the aid of a suite of hESC reporter lines, we have developed a novel system for assessing the effect of different growth factor combinations on hESC differentiation towards a range of mesendodermal lineages. Using this system in conjunction with an NKX2.1-GFP reporter hESC line we have developed a serum free protocol for the generation of NKX2.1+ respiratory endoderm which, when transplanted into immunodeficient mice, has the capacity for further maturation into definitive end cell types identified by expression of CC10, MUC5AC, P63, TUBB-IV and surfactant proteins. Generation of this population was found to be dependent on specific concentrations of BMP4 and activin A during the first 4 days of differentiation and on FGF1 between differentiation day 4 and 7. Gene profiling experiments indicated that NKX2.1+ endoderm expressed markers indicative of early foregut but lacked genes associated with later stages of respiratory epithelial cell differentiation. Interestingly, when purified NKX2.1+ progenitors were cultured further in vitro they rapidly lost NKX2.1 expression and up-regulated SOX2 and CDX2, suggesting adoption of a dorsal (oesophagus) or posterior (stomach) fate in the absence of appropriate instructive signals. Collectively, these findings suggest that early hESC-derived NKX2.1+ cells can give rise to a number of endodermal cell types. Finally, to further our ability to investigate respiratory endoderm differentiation from hESCs we have generated an additional reporter line in which sequences encoding GFP were inserted into the SFTPC locus using homologous recombination. Collectively, the tools and protocols described in this thesis will contribute to efforts aimed at establishing hESCs as a reliable source of human respiratory epithelial cells.