Investigating the recruitment of MSC from whole blood flow & Generation and characterisation of mIPS

Abstract

Project 1: Mesenchymal stem cells (MSCs) are a population of adult stem cells located in the bone marrow. They are able to differentiate into cartilage, muscle, bone and fat. MSCs are an attractive therapeutic treatment option as they have been shown to be immunosuppressive and can be isolated and then expanded in culture. However delivering the MSCs to a site of damage or disease is not ideal as currently the best method is direct injection into the site which is highly invasive. Delivering the MSCs by injection into the circulation and then recruited to the damaged or diseased site would be a much safer and less invasive option. The aim of this study was to gain an insight into the ability of MSCs to be recruited to sites of damage or inflammation from the circulation by using an in vitro whole blood flow system. This study showed that MSCs were not able to become recruited from whole blood flow in this system when using shear rates equivalent to human circulation. Project 2: The use of embryonic stem cells to treat human disease has not reached its potential due to a number of problems including ethical issues and limited supply. The development of induced pluripotent stem (iPS) cells aims to address these issues by being ethically sound and unlimited in supply. Several methods for reprogramming have been developed. This project employed a recently described technique to generate iPS cells with and without the c-Myc gene (Sommer et al., 2009). This method was chosen because the pluripotency cassette is flanked by loxP sites which allowed for Cre recombinase mediated excision from the genome to produce insert free iPS cells. iPS cells were generated with a 3 gene and 4 gene approach, the cells were tested for pluripotency by Immunofloursecense of pluripotency proteins including Nanog, oct4 and SSEA-1 in the iPS cells. The differentiation potential was studied as further confirmation of the generated iPS cells pluripotency. Expression of the proteins \(\alpha\)-Smooth Muscle Actin (mesoderm), \(\beta\)-III-Tubulin (ectoderm) and Troma1 (endoderm) were analysed by Immunofloursecense to show that the iPS cells could differentiatiate into cells from each of the three germ layers. All tested iPS clones that were generated were observed to be pluripotent by these methods of measuring pluripotency.

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