Hybrid microenvironments for the ex-vivo expansion of Hematopoietic Stem Cells

Background

Type I diabetes is a genetically based chronic autoimmune disease that is associated with deficient pancreatic beta cell function and insufficient insulin production. The self-duplication mediated rather than self-renewal maintenance of the beta cell niche in the pancreas and their targeting by the immune system creates the need for external insulin intake or pancreatic islet cell transplantation in patients suffering from the disease (Murtaugh, 2007). However, the inadequate supply of functional insulin producing cells as well as the increased risk for autoimmune rejection of the transplanted tissue pose major obstacles to a successful surgical transplantation of islet cells. By bypassing the possibility of transplant rejection, the differentiation of mesenchymal stem cells to insulin producing beta cells for autologous transplantation may provide an alternative treatment of type I diabetes.

Aims of the project

In recent years, the development of peptide hydrogels has opened up new possibilities for tissue culture in regenerative medicine (Chawla et al., 2012). The biodegradability and biocompatibility of these gels have also suggested the possibility of this injectable material (in vivo or in situ) to enable patients to avoid surgical transplantation procedures (Castillo Diaz et al., 2016). Previous studies have confirmed the ability of MSCs to differentiate into insulin producing pancreatic beta cells (Xin et al., 2016). The differentiation into insulin secreting cells has been achieved both by microenvironmental manipulation and by gene manipulation methods using viral vectors (Xie et al., 2009 and Allahverdi et al., 2015). However, these studies have been performed in tissue culture plates and therefore represent a less physiological view of the whole process in the way it occurs at an organism level. Consequently, a peptide hydrogel would allow the 3D culture of MSCs and their differentiation into beta cells, providing us with both a better overview of the whole process and the possibility to inject the terminally differentiated cells in organisms suffering from type I diabetes for clinical trials.

Methods

The project will initially involve the preparation of the hydrogel and the initiation of the mesenchymal cell culture. I intend to perform a cell viability assay (e.g. LIVE/DEAD assay) to confirm the suitability of the gel for a 3D MSC culture. In order to achieve MSC differentiation into pancreatic beta cells, I will introduce the appropriate culturing media and differentiation stimuli into the hydrogel (eg. to include the expression of relevant transcription factors such as Neurod1, Ngn3, Pdx1 etc). Finally, it is important that I confirm the production of insulin and beta cell specific markers by the cells by immunofluorescence. At the end of the project, I will test the degradability of the hydrogel to suggest the possibility and safety of its potential introduction to an organism in situ (possibly using the method for evaluating the hydrogel proteolytic degradability described by Castillo Diaz et al., 2016).