Stem cell therapies and tissue engineering strategies are required for the
clinical treatment of respiratory diseases. Previous studies have established protocols
for the differentiation of airway epithelium from stem cells but have involved costly
and laborious culture methods. The aim of this thesis was to achieve efficient and
reproducible maintenance and differentiation of embryonic stem cells to airway
epithelium, in 2D and 3D culture, by developing appropriate bioprocessing
technology.
Firstly, the 2D differentiation process of human and murine ES cells into
pulmonary epithelial cells was addressed. The main finding in was that the
proportion of type II pneumocytes, the major epithelial component of the gas-exchange
area of lung, differentiated with this method was higher than that obtained
in previous sudies, 33% of resultant cell expressed the specific marker surfactant
protein C (SPC) compared with up to 10%.
Secondly, the maintenance and differentiation was carried out in 3D. A
protocol was devised that maintained undifferentiated human ES cells in culture for
more than 200 days encapsulated in alginate without any feeder layer or growth
factors. For ES cell differentiation in 3D, a method was devised to provide a
relatively cheap and simple means of culture and use medium conditioned by a
human pneumocyte tumour cell line (A549). The differentiation of human and murine
ES cells into pulmonary epithelial cells, particularly type II pneumocytes, was found
to be upregulated by culture in this conditioned medium, with or without embryoid
body formation.
The third step was to test whether this differentiation protocol was amenable
to scale-up and automation in a bioreactor using cell encapsulation. It was possible to
show that encapsulated murine ES cells cultured in static, co-culture or rotating wall
bioreactor (HARV) systems, differentiate into endoderm and, predominantly, type I
and II pneumocytes. Flow cytometry revealed that the mean yield of differentiated
type II pneumocytes was around 50% at day 10 of cultivation.
The final stage of the work was to design and produce a perfusion system
airlift bioreactor to mimic the pulmonary microenvironment in order to achieve large
scale production of biologically functional tissue. The results of these studies thus
provide new protocols for the maintenance of ES cells and their differentiation
towards pulmonary phenotypes that are relatively simple and cheap and can be
applied in bioreactor systems that provide for the kind of scale up of differentiated
cell production needed for future clinical applications.