Characterization of renal CD133+ cells and their therapeutic efficacy in a model of acute kidney injury

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

Tumor suppressor genes promote rhabdomyosarcoma progression in p53 heterozygous, HER-2/neu transgenic mice.

Human sarcomas arise suddenly, thus preempting the study of preneoplastic and early neoplastic lesions. To explore the natural history of these tumors we studied male mice carrying a heterozygous deletion of p53 and an activated HER-2/neu transgene (BALB-p53Neu mice), that develop urethral rhabdomyosarcomas with nearly full penetrance and early onset (4 months of age). Among genes prominently upregulated in preneoplastic tissue, and more highly expressed in tumors, we found the insulin-like growth factor 2 (Igf2) and tumor suppressors, p19Arf and p21Cip1. In urethral tissues of male mice p53 was less expressed than in female mice, whereas HER-2/neu was more expressed, a combination not found in other skeletal muscles of the same mice that could contribute to the anatomic and sexual specificity of BALB-p53Neu rhabdomyosarcoma. Upregulation of p19Arf and p21Cip1 was additively determined by HER-2/neu activation and by p53 inactivation. Silencing of p19Arf or p21Cip1 in rhabdomyosarcoma cell lines can inhibit cell growth and motility, thus suggesting that these genes can contribute to growth autonomy and malignancy of tumor cells. In vivo injection of gene-silenced cells highlighted selective variations in organ-specific metastatic ability, indicating that overexpression of p19Arf and p21Cip1 controlled both tumor cell-intrinsic properties and microenvironmental interactions. The onset of pelvic rhabdomyosarcoma in BALB-p53Neu male mice is triggered by the coincidental overexpression of HER-2/neu and hypoexpression of the residual p53 allele, that foster p53 loss, Igf2 autocriny and overexpression of p19Arf and p21Cip1, a phenotype that could provide novel potential targets for cancer prevention and therapy

Vertical price transmission in the Egyptian tomato sector after the Arab Spring

This study assesses price transmission along the Egyptian tomato food marketing chain in the period that followed the Arab Spring, which accentuated economic precariousness in Egypt. Static and time-varying copula methods are used for this purpose. Results suggest a positive link between producer, wholesaler and retailer tomato prices. Such positive dependence is characterized by asymmetries during extreme market events that lead price increases to be transferred more completely along the supply chain than price declines.info:eu-repo/semantics/publishedVersio