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Title: Characterization of renal CD133+ cells and their therapeutic efficacy in a model of acute kidney injury
Author: Santeramo, I.
ISNI:       0000 0004 6422 1435
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2016
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Renal ‘progenitor’ cells expressing CD133 have been proposed as cellular therapeutics for treating patients with kidney disease. In the literature, CD133+ cells isolated from adult kidneys showed the expression of nephron progenitor markers (Pax2), and broad differentiation plasticity, being able to differentiate towards epithelial cells and podocytes. Most importantly, when injected into preclinical models of kidney injury, CD133+ cells integrated into damaged renal tissue and improved renal health. Nevertheless, the evidence for CD133 being a bona fide renal progenitor marker is conflicting. In this study, five renal biopsies belonging to children (from 6 months to 10 years old) were used. The localization of CD133 was consistent with previous studies, as the expression of CD133 was demonstrated on the cells of the Bowman’s capsule and in scattered tubular cells. From each sample, a bulk renal population was isolated and was initially characterized for the presence of the CD133 marker. Once placed in culture, most of the renal cells started expressing CD133. A further phenotypical characterization revealed that the vast majority of the cells expressed epithelial (EpCam, E-Cadherin, CD24), and some mesenchymal (CD73, CD44) markers. Also, the CD133+ population appeared heterogeneous for the expression of other markers. Most notably, CD13, a marker of fully differentiated tubular cells, was found to be significantly expressed in part of the CD133+ cells, suggesting that either fully differentiated cells started expressing CD13 de novo, or the CD133+ were committed towards a tubular fate. The bulk population was sorted by FACS into CD133+ and CD133- sub-populations which were compared in additional experiments to explore the progenitor-like features of the CD133+ cells. First, the ability of both CD133+ and CD133- sub-populations to differentiate towards podocytes in vitro was investigated. Both sub-populations were found to express the podocyte markers, nephrin and podocin, to a similar extent following stimulation with retinoic acid. However, the assay did not prove to be consistent and it was not used any further. Secondly, the potential of both CD133+ and CD133- sub-populations to integrate into ex vivo reaggregated mouse kidney rudiments was determined. Surprisingly, the majority of both cell types died in the chimeric rudiments within two days in culture. Neither the surviving CD133+ nor the CD133- cells showed any propensity to integrate into developing renal structures. Unexpectedly, the CD133+ cells were found to clump on top of the rudiment and had a negative impact on the developing rudiment, whereas the CD133- cells did not. Alongside with the test of the human cells in the chimeric rudiments, the assay itself was modified to suit the imaging of the rudiments in a Light-sheet fluorescent microscope (LSFM). 3D embryonic renal organoids were efficiently produced and the development of their structures could be successfully monitored through the LSFM in proof-of-concept experiments. The final aim of this work was to assess the therapeutic efficacy of the CD133+ and CD133- sub-populations in a rat model of cisplatin-induced acute kidney injury. The renal function was monitored using a non-invasive transcutaneous device that measures the half-life of an exogenously administered renal marker, FITC-Sinistrin, alongside to the measurement of conventional biomarkers, serum creatinine, and urea. Following intravenous (IV) injection, both CD133+ and CD133- cells ameliorated renal function and preserved renal histology. The data suggest that the human cells passed through the lungs, and probably reached the kidneys. However, no cells were alive at the end of the study (14 days), but traces of PKH26 were retrieved in lungs and, to a lesser extent, in the kidneys, suggesting the possible involvement of paracrine mechanisms, possible through extracellular vesicles in the observed functional amelioration. Additional biodistribution studies showed that soon after the IV injection the human cells were identified in the lungs of the animals, but not in the kidneys. Phagocytic cells, identified through the marker CD68, were observed around the human cells in the lungs as early as 1 hour after the injection. By 24 hours, clusters of CD68+ cells could be found, but not human cells. Therefore, the data suggest that the human cells die in the lungs and that the macrophages might play an active role in the disappearance. Taken together, this work shows that the expression of CD133 does not confer any advantage to the nephrogenic potential ex vivo or to the therapeutic efficacy in vivo. Moreover, since the cells were shown to be entrapped in the lungs, the renal repair is probably mediated by cell-derived factors, rather than by CD133+ cells homing to the kidneys and generating specialised renal cells. The role of macrophages in the resolution or regenerative mechanisms should be considered and further examined in future preclinical studies of cellular therapies for kidney diseases.
Supervisor: Wilm, B. W. ; Murray, P. M. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral