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Developing a stem cell based therapy to replace nephrons lost through reflux nephropathy

Final Report Summary - RENALSTEM (Developing a stem cell based therapy to replace nephrons lost through reflux nephropathy)

Summary of project objectives

The long-term aim of this proposal was to explore the potential of resident kidney stem cells for renal reparative and replacement therapy in order to prevent the development of end stage renal disease (ESRD). The initial projective objectives were to isolate resident stem cells from mouse kidney and investigate their potential for generating nephrons by transplantation into mouse embryonic kidneys ex vivo. Additional objectives included developing a suitable biomaterial scaffold to support the growth and differentiation of the stem cells, and developing a magnetic nanoparticle (MNP)-based cell tracking technique to enable the stem cells to be monitored in vivo using magnetic resonance imaging.

Description of work performed since the start of the project

During the first six months of the project, renal cell populations were isolated from adult mouse kidney and clonal lines were generated. Following expansion of the clonal lines, they were tested for expression of the renal progenitor markers, Pax2 and Wt1, but none of the lines were found to express these markers. Therefore, for the remainder of the project, Dr Palona used Pax2+ Wt1+ kidney stem cells isolated from neonatal mouse kidneys that had previously been isolated in our lab, and also performed experiments using mouse embryonic stem cell (ESC)s and mesenchymal stem cell (MSC)s. From months 7 - 12, Dr Palona worked with researchers in the Department of Chemistry at the University of Liverpool (UoL) to generate MNPs for stem cell tracking. Cytotoxicity tests were performed to determine if the MNPs had any effect on cell viability. The retention time of the MNPs was compared to that of the commercially available MNP, Eudorem; this was achieved by fixing cells at defined time points following labelling, and staining with Prussian blue. To investigate if the MNPs had any effect on the differentiation potential of the different stem cell lines, MNP-labelled keratinocyte stem cell (KSC)s were incorporated into mouse kidney rudiments ex vivo to determine if there was any effect on their nephrogenic potential. To determine if MNPs had any effect on the differentiation potential of mouse ESCs, labelled cells were cultured in suspension to generate embryoid bodies and their ability to generate derivatives of the three embryonic germ layers was investigated using quantitative PCR. To determine if MNPs had any effect on the differentiation potential of MSCs, labelled cells were cultured in osteogenic and adipogenic media, and their ability to generate osteocytes and adipocytes, respectively, was investigated. From months 13 to 17, the signal intensity of the MNPs was investigated using magnetic resonance imaging (MRI) (in collaboration with Prof. Williams, University of Manchester), and transmission electron microscopy (in collaboration with Dr Ian Prior, UoL) was performed to determine if the MNPs were present inside the cells and to determine their intracellular localisation. Dr Palona also developed an additional collaboration with the chemists that was focused on developing MNPs for drug / gene delivery. In month 18 of the project, Dr Palona began a period of maternity leave, from which she did not return.

Main results achieved

The MNPs prepared by the Rosseinsky group, Department of Chemistry, UoL, caused no significant cytotoxicity at concentrations of 30 micrograms per ml / 100 000 cells. At concentrations of 50, 100 and 200 micrograms per ml / 100 000 cells, the viability was approximately 80, 75 and 60 %, respectively. KSCs labelled with MNPs appeared to integrate to a similar extent to unlabelled cells, suggesting that the MNPs had no adverse effect on the nephrogenic potential of KSCs. ESCs labelled with MNPs expressed similar levels of the pluripotency markers, Oct4 and Nanog, as unlabelled ESCs. Furthermore, labelled ESC-derived embryoid bodies express markers of the three germ layer derivatives, indicating that the MNPs had no adverse effect on ESC pluripotency. MSCs labelled with MNPs were able to undergo osteogenesis and adipogenesis to a similar extent as unlabelled MSCs, indicating that MNPs had no adverse effect on the differentiation potential of MSCs. The most interesting result was the finding that following short-term culture, MNPs prepared by the Rosseinsky group were more readily retained by the stem cells than the commercial product, Eudorem. MRI showed that MNP-labelled cells embedded in agarose could be detected by the instrument. Electron microscopy showed that the MNPs appeared to be within endosomes. Furthermore, in a collaboration with the chemists, Dr Palona was involved in a project that focussed on using nanomaterials as agents to deliver nucleic acid into cells, which has led to two publications, and a third publication has just been submitted.

Potential impact and use of project outcomes

The ability to monitor stem cells non-invasively following transplantation would be of great benefit, as it would make it possible to see if the stem cells populated non-target organs, and for how long the stem cells were retained by the target organ. MRI offers distinct advantages over other imaging techniques, because unlike the situation with fluorescence, the magnetic signal does not diminish with tissue depth, meaning that internal organs such as the liver and kidneys, can be imaged effectively with MRI. For this reason, over recent years, there has been much interest in developing MNPs for stem cell labelling, which would enable the cells to be tracked using MRI following transplantation. However, a major problem with most MNPs that have been designed to date is that they tend to be quickly degraded within the cellular environment. An aim of this project was to design MNPs with protective coatings that would protect them from the cellular environment and enable them to be retained by the cells for longer periods. It was found that some of the MNPs generated during this project, were more readily retained inside the cells than were commercial MNPs. These results formed the basis of a major funding proposal to a United Kingdom (UK) stem cell initiative (GBP 1.5 million) that was funded in March 2010. Two publications evaluating the potential for iron-oxide based MNPs as agents to deliver nucleic acids into cells have been published in the journals of IEEE Transactions on Nanobioscience (2010) and the Journal of Nanoscience and Nanotechnology (2011). A further publication has recently been submitted to the Journal of Materials Chemistry B.