Detela, G; (2015) Enhancing Functional Responses of MSCs for Ischemic Injury Using Ex Vivo Pre-conditioning Strategies. Doctoral thesis , UCL (University College London).

Abstract

Autologous hMSCs are a promising therapeutic candidate for ischemic tissue injury such as that resulting from myocardial infarction. In recognition of the poor functional characterisation of hMSCs used in clinic and lack of priming methods to enhance cell function, the work presented in this thesis describes several ex vivo strategies designed to assess and enhance hMSC responses. Firstly, growth kinetics of hMSCs isolated from 10 healthy donors were mapped over 5 passages and inter-donor variability was determined. A process operating window was identified between passage 2 and passage 3 where different donors displayed no PDL and cell density differences. Expansion potential at the end of passage 2 was 17.29x106 ± 5.19x106 and at the end of passage 3 was 50.40x106 ± 8.59x106. Next, functional effects of sJag1 and sDll4 pre-conditioning on hMSCs attachment and migration responses were studied. The effect of low oxygen was also assessed, to more closely reflect the physiologic environment. In low oxygen conditions, sJag1 pre-conditioning significantly increased hMSC attachment to fibronectin after just 20 minutes compared with unconditioned cells. However, in ambient oxygen, the stimulatory effect was not seen until 40 minutes attachment time. sJag1 pre-conditioning also significantly enhanced hMSC chemotaxis through fibronectin in response to SDF-1α in low oxygen. Comparable effects were not detected when using a type I collagen substrate. Finally, sJag1 also enhanced the production of angiogenic factors VEGF-A165 and HGF by hMSCs on fibronectin. We assessed the effects of sJag1 and sDll4 on vascular support capacity of hMSCs using in vitro co-culture assays with endothelial cells. Pre-conditioning hMSCs with sJag1 resulted in significantly increased branching within co-culture networks and enhanced vessel-like network formation and structural maintenance over 96 hours in the low oxygen environment. Finally, the requirement for functional Nrp1 in MSCs was studied using Nrp1 cytoKO mice. Although cell attachment was impaired in MSCs lacking fully functional Nrp1, sJag1 was still able to enhance cell attachment. Since hMSCs are widely used in clinical trials with limited positive outcomes, pre-conditioning with sJag1 may be a tool for priming immature cells to improve retention and therapeutic efficacy in ischemic tissues.

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