Study and optimization of mouse embryonic stem cell culture

Perestrelo, Ana Rubina

http://hdl.handle.net/10400.1/497

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Authors

Perestrelo, Ana Rubina

Advisor(s)

Belo, José António

Abstract(s)

Stem cell research has grown from unexplored to an important field in biomedical sciences today. Indeed, a deeper understanding of the basic biology of stem cells holds the key to unlock new hopes to various incurable human diseases. Embryonic stem (ES) cells have the unique ability to proliferate indefinitely in an undifferentiated state and to give rise to any type of somatic cell lineage depending on specific signals. ES cells have been typically studied in two-dimension (2D) Petri dishes, 2D multi-well plates or 2D glass slides coated with different substrates which differ radically from the three-dimension (3D) microenvironment in the body. Consequently, cells isolated from higher organisms frequently modify their metabolism, morphology and gene expression profile. The substitution of the 2D systems by a 3D micro or nanomolecular network (scaffold) is a very promising approach in replicating the architecture of the in situ environment of a cell in a living organism. However, the existing 3D culture systems formed from animal-derived biomaterials pose problems for replacement therapies. The goal of this dissertation was to study the possibility of using a new artificial 3D nanowire scaffold formed from self-assembly of amphiphilic biodegradable peptidecopolymers, instead of conventional mitotically inactivated mouse embryonic fibroblasts, for culture of mouse ES cells (mES cells). Undifferentiated mouse ES cells were cultured using the bioconjugated polymeric nanowire scaffold and control conditions. The potential of the scaffold for applications in ES cell research was evaluated by assessing the morphology, proliferation, survival rate, self-renewal and pluripotency of mES cells. The results obtained suggest that, in low concentrations (50 and 25 Eg/ml) of Arginine– Glycine–Aspartate (RGD) bioconjugated polymeric nanowires scaffold the cells retain their viability and pluripotency. However, an improvement of the scaffold would be required in order to promote better survival rates and making this 3D system in an alternative substrate for mES cell culture, which saves time and is easy to use.