STUDY OF THE APPLICATION POTENTIAL OF A NEW CLASS OF 3D SCAFFOLD POLYMERIC NANOFIBERS IN STEM CELL RESEARCH

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Locust bean gum as an alternative polymeric coating for embryonic stem cell culture

Publication . Perestrelo, Ana Rubina; Grenha, Ana; Costa, Ana M. Rosa da; Belo, José A.

Pluripotent embryonic stem cells (ESCs) have self-renewal capacity and the potential to differentiate into any cellular type depending on specific cues (pluripotency) and, therefore, have become a vibrant research area in the biomedical field. ESCs are usually cultured in gelatin or on top of a monolayer of feeder cells such as mitotically inactivated mouse embryonic fibroblasts (MEFsi). The latter is the gold standard support to maintain the ESCs in the pluripotent state. Examples of versatile, non-animal derived and inexpensive materials that are able to support pluripotent ESCs are limited. Therefore, our aim was to find a biomaterial able to support ESC growth in a pluripotent state avoiding laborious and time consuming parallel culture of MEFsi and as simple to handle as gelatin. Many of the new biomaterials used to develop stem cell microenvironments are using natural polymers adsorbed or covalently attached to the surface to improve the biocompatibility of synthetic polymers. Locust beam gum (LBG) is a natural, edible polymer, which has a wide range of potential applications in different fields, such as food and pharmaceutical industry, due to its biocompatibility, adhesiveness and thickening properties. The present work brings a natural system based on the use of LBG as a coating for ESC culture. Undifferentiated mouse ESCs were cultured on commercially available LBG to evaluate its potential in maintaining pluripotent ESCs. In terms of morphology, ESC colonies in LBG presented the regular dome shape with bright borders, similar to the colonies obtained in co-cultures with MEFsi and characteristic of pluripotent ESC colonies. In short-term cultures, ESC proliferation in LBG coating was similar to ESC cultured in gelatin and the cells maintained their viability. The activity of alkaline phosphatase and Nanog, Sox2 and Oct4 expression of mouse ESCs cultured in LBG were comparable or in some cases higher than in ESCs cultured in gelatin. An in vitro differentiation assay revealed that mouse ESCs cultured in LBG preserve their tri-lineage differentiation capacity. In conclusion, our data indicate that LBG coating promotes mouse ESC growth in an undifferentiated state demonstrating to be a viable, non-animal derived alternative to gelatin to support pluripotent mouse ESCs in culture.

2014-07-01Journal article

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Novel triblock co-polymer nanofibre system as an alternative support for embryonic stem cells growth and pluripotency

Publication . Perestrelo, Ana Rubina; Mouffouk, Fouzi; Costa, Ana M. Rosa da; Belo, José A.

Conventionally, embryonic stem cells (ESCs) are cultured on gelatin or over a mitotically inactivated monolayer of mouse embryonic fibroblasts (MEFsi). Considering the lack of versatile, non-animal-derived and inexpensive materials for that purpose, we aimed to find a biomaterial able to support ESC growth in a pluripotent state that avoids the need for laborious and time-consuming MEFsi culture in parallel with mouse ESC (mESC) culture. Undifferentiated mESCs were cultured in a new nanofibre material designed for ESC culture, which is based on the self-assembly of a triblock co-polymer, poly(ethyleneglycol-β-trimethylsilyl methacrylate-β-methacrylic acid), conjugated with the peptide glycine-arginine-glycine-aspartate-serine, to evaluate its potential application in ESC research. The morphology, proliferation, viability, pluripotency and differentiation potential of mESCs were assessed. Compared to conventional stem cell culture methodologies, the nanofibres promoted a higher increase in mESCs number, enhanced pluripotency and were able to support differentiation after long-term culture. This newly developed synthetic system allows the elimination of animal-derived matrices and provides an economic method of ESC culture, made of a complex network of nanofibres in a scale similar to native extracellular matrices, where the functional properties of the cells can be observed and manipulated.

2016Journal article

Open Access