Design of cellular microarrays to study mesenchymal stem cell differentiation

RODRÍGUEZ SEGUÍ, S.A.; PLA-ROCA, M.; ENGEL, E.; PLANELL, J.A.; MARTÍNEZ, E.; SAMITIER, J.

Sant Feliu de Gixols

Conferencia; European Science Foundation (ESF)-EMBO conference; 2007

European Science Foundation

The differentiation of Mesenchymal Stem Cells (MSCs) into several cell fates has been widely explored during the last decade. As a result, a lot of information is now available on different ways of inducing its differentiation to many cell fates. Usual differentiation strategies, involving the culture of MSC in tissue culture plates with medium enriched by a combination of soluble differentiation factors and animal serum, lead to the induction of predominant phenotypes for the desired fate after periods ranging from 1 to 3 weeks. However, this process is not always effective enough to provide a straightforward extrapolation to the medical field. In fact, non-desired cell fates can also be found in the culture plates despite the addition of differentiation-specific factors. Moreover, complex cellular interactions can be envisaged coming from cell-cell signalling, thus appearing a fraction of arbitrary cell phenotypes whose distribution within the monolayer culture is independent of the extracellular factors added in the culture medium.In order to get an insight into the bio molecular processes that take place during stem cell differentiation and effectively develop new therapies based on them, there is a need to develop new tools that enable a much more accurate evaluation of the response of individual cells to certain combination of factors. New techniques based on bionanotechnology can contribute to this objective, as novel ?bottom up approach?-based tools can be implemented to mimic at the biomolecular level individual MSCs microenvironments on flat surfaces.The objective of this work is to expose our progress in the development and evaluation of cellular microarrays to study the differentiation of rat MSCs to either osteoblasts or adipocytes. Our strategy involved the immobilisation of fibronectin (Fn) within the spots, as an extracellular matrix protein that allows initial MSC attachment when seeded, in combination with BMP2 and Wnt3a growth factors. These factors have been widely reported in the literature to stimulate osteoblast commitment in MSCs. We used of completely defined medium (CDM) for subsequent cell culture. We have characterized the cellular microarray formation as a result of printing Fn in the array spots on chemically activated glass and PMMA slides. Once the arrays were printed, the surfaces were passivated with either PEG polymer or BSA. For this purpose, a microarray plotter machine is used to perform arrays where protein mixtures can be covalently immobilised, thus controlling their concentration and spatial distribution. In order to keep this biomolecular structure unaltered, as well as to ensure that cells response is due to known factors, we used CDM in our cell cultures.As future work, we plan to immobilise mixtures of factors also affecting MSC differentiation to the adipocyte fate. The combination of the surface topography (in modified PMMA substrates) with the protein printed spots will also be analysed as a way to create 3D microenvironments.