Abstract

As the central nervous system shows very little capability for self-repair following injury, regenerative medicine approaches are increasingly interested in the use of stem cells for cell replacement strategies. Biomaterials are an interesting tool to carry out this type of therapies. They allow three-dimensional cultures for stem cells differentiation and are helpful in order to obtain cells at the right developmental stage for transplantation. Moreover, they could help to enhance and control cell survival after transplantation, minimizing cell death. Stroke is a very severe form of brain injury and one of the leading causes of death worldwide, as no effective cures are available. Several studies show that neural stem cells (NSCs) are able to integrate and improve functional recovery once transplanted in stroke animal models. However, the majority of the grafted NSCs die within weeks after transplantation, limiting treatment efficacy. Tissue engineering approaches aim to restore tissue functions combining principles of cell biology and engineering, using designed and tailored three-dimensional biomaterial scaffolds. In this study we tested alginate as candidate biomaterial for neural tissue repair. We studied its ability to support mouse embryonic stem cells (mESCs) neural differentiation in vitro. We evaluated whether changes in its concentration or modifications with extracellular matrix components could influence cell differentiation, analysing the mechanical and physical properties of the generated scaffolds. In the first part, we evaluated the suitability of alginate as a scaffold for three-dimensional cultures able to enhance differentiation of mESCs towards neural lineages. We tested whether encapsulation of mESCs within alginate beads could support and/or enhance neural differentiation with respect to two-dimensional cultures. We encapsulated cells in beads of alginate at two different concentrations, with or without modification by fibronectin, RGD peptide or hyaluronic acid. Cells survive and differentiate inside our scaffolds, forming clusters. Gene expression analyses showed that cells grown in alginate scaffolds increase differentiation toward neural lineages with respect to the two dimensional controls. Immunocytochemistry analyses confirmed these results, further showing terminal differentiation of neurons by the expression of synaptic markers. Cells showed also the capability to form networks among themselves and with cells of other clusters. All the scaffolds we prepared resembled brain tissue characteristics, thus we decided to test alginate as potential support for tissue engineering approaches in the injured brain. In the second part of the work we tested alginate as support for NSCs injection in the brain. We evaluated in vivo crosslinking of alginate after injection, and verified inflammation levels due to its presence in mouse brain tissue. Our preliminary studies suggest that alginate polymerizes in vivo, forming a hydrogel, and that it does not elicit any inflammatory response following injection. Our data show that alginate, alone or modified, is a suitable biomaterial to promote in vitro differentiation of pluripotent cells toward neural fates. Moreover, it could be used as injectable hydrogel for brain tissue regeneration. We plan to co-inject alginate with NSCs in stroke mouse models in order to enhance viability and integration of the engrafted cells in the damaged tissue. We plan to study alginate permanence in the brain and NSCs viability, integration and capability to stimulate regeneration after ischemic injury.