Assessment of stem cell pluripotency using an "in vitro" 3D perfusion-based culture model

Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have the capacity to self-renew while maintaining the ability to differentiate into cell types derived from all three germ layers (endoderm, mesoderm, ectoderm). They have a high potential as a cell source in the field of regenerative medicine, drug development, disease modelling and early embryonic development. Multiple criteria have been proposed to evaluate hPSCs such as morphology, karyotype, cell surface phenotype, gene expression profile, in vitro differentiation as well as in vivo differentiation. However, there are many inconsistencies in the way of generating and reporting the results, most particularly concerning the formation of teratoma in vivo, which is the gold standard for the demonstration of pluripotency. The place of implantation, the number of injected cells as well as the duration of the assay introduce high level of variability with various consequences, including absence of detectable teratoma formation.
In the first part of the thesis I report the generation of four hESC lines under the same conditions. They were characterized for their pluripotency status, their gene expression profile and their differentiation behaviour in vitro. We showed that although derived and cultured in identical conditions, the 4 hESC lines exhibit differences in their gene expression pattern and their propensity to spontaneously commit during their maintenance. We also demonstrated that these differences have further consequence for their directed differentiation into neuronal tissue.
In the second part of the thesis I present the establishment of a new perfusion-based 3D in vitro culture system, which allows the formation of teratoma-like structures derived from all three germ layers. We show that the perfused culture allows spontaneous differentiation of hPSCs into the three embryonic lineages in a more efficient, balanced and reliable way than previously used in vitro systems. This leads to the formation of teratoma-like tissue structures similar to those observed in vivo. Finally, we defined a quantitative system to grade teratomas, which indicates that our in vitro culture is as efficient but more controlled and reproducible than the in vivo assay. This grading method could also potentially be used to compare hPSC line properties in our system.
Taken all results together, my thesis illustrates the complexity of hPSCs and the extensive need of standardized processes to derive, to maintain and characterize their pluripotency status.