Modelling the differentiation and development of human tissues using mammalian stem cells and advanced cell technologies

Abstract

Human tissue development is a complex process which remains poorly understood. A variety of different techniques are currently used to study the events involved, yet each has associated limitations. Human embryo work is subject to ethical and logistical issues, animal studies are not necessarily representative of human processes, traditional cell culture lacks complexity and embryoid body formation suffers from viability and size related issues. The study of this process is crucial to the better understanding of this topic, as well as allowing for the development of reproducible differentiation protocols for regenerative medicine applications and the study and characterisation of pluripotent stem cells.
The overall aim of this project was to develop a novel in vitro model to be used to study the differentiation of human tissues. Based on work from the literature and previous work carried out within the group, it was hypothesised that the culture of pluripotent stem cell derived embryoid bodies (EBs) on the surface of a porous scaffold to enhance cell maturation over time and for the formation of complex tissue structures not previously possible. EBs show self-organisation, recapitulating some events of early embryogenesis, but have limited viability on increasing EB size and agglomeration, preventing long term studies. It was theorised that the reduction of cellular diffusion distances by changing the structure of the EB into a flattened tissue disc structure on the scaffold allows for improved nutritional and oxygen support, as well as the influence of attachment mediated signalling, which in turn would allow sufficient time for complex structure formation.
Proof of concept was confirmed using embryonal carcinoma cells, as was the ability to study specific signalling pathways through directing the differentiation of EBs using known morphogens. The incorporation of additional microenvironmental cues to the model was also undertaken, with co-culture and perfusion successfully introduced into the tissue disc model, impacting on the structure and phenotype of the cells. Long term culture with murine ES cell derived EBs resulted in the formation of a variety of complex structures which have not previously been seen in vitro. Tissue discs were successfully directed towards specific germ layers using various combinations of morphogens, and the introduction of physiological conditions impacted significantly on tissue disc morphology, with perfusion in particular showing an enhancement of differentiation. Validation against teratomas formed from the same cell populations confirmed the complexity of the in vitro structures and their similarity to the in vivo samples, and pilot studies confirmed the ability to culture human pluripotent stem cell derived tissue discs within the system also.
The model developed in this project has shown the ability to form varied and complex tissue structures in vitro in response to a variety of microenvironmental cues. As well as being used to study the events of human tissue development, this model also offers a controllable and reproducible in vitro alternative to the teratoma assay.