|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Stem cell-conditioned medium does not protect against kidney failure
Yousof Gheisari*1, Naser Ahmadbeigi*†1, Mahmood Naderi*, Seyed Mahdi Nassiri‡, Samad Nadri* and Masoud Soleimani§2
*Department of Stem Cell Biology and Department of Molecular Biology and Genetic Engineering, Stem Cell Technology Research Center, Tehran, Iran, †SABZ Biomedicals, Tehran, Iran, ‡Department of Clinical Pathology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran, and §Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
Paracrine secretion of mediators may be the main route by which stem cells protect against injuries. Stem cells commonly secrete different bioactive molecules. In this study, we examined the hypothesis that administration of conditioned media of stem cells can diminish the burden of kidney injury. A mouse model of cisplatin-induced nephropathy was developed to test the putative renoprotective effects of conditioned media of human umbilical cord blood USSCs (unrestricted somatic stem cells) as well as mouse bone marrow MSCs (mesenchymal stem cells). None of these two types of conditioned medium could protect against kidney failure in terms of serum urea and creatinine, histopathologic examinations and physical activity score. Neither MSC- nor USSC-conditioned media were effective in protecting against kidney injury in our study. Possible explanations for our observations are offered, and related literature is reviewed.
Key words: conditioned culture media, kidney failure, mesenchymal stem cell, paracrine communication
Abbreviations: MSCs, mesenchymal stem cells, USSCs, unrestricted somatic stem cells
1Yousof Gheisari and Naser Ahmadbeigi contributed equally to this work.
2To whom correspondence should be addressed (email email@example.com).
Renal failure is one of the most important causes of mortality and morbidity all over the world. Treatment options in CRF (chronic renal failure) mainly consist of renal transplantation and dialysis. Kidney transplantation is hampered by shortage of donors. Dialysis is lifesaving and the main modality of treatment in these patients, but it is encumbered by several limitations. It is not a complete renal replacement therapy and is associated with several socioeconomic problems for the patients (Hammerman, 2003). Acute renal failure is also quite common and affects up to 7% of all hospitalized patients, with high mortality and morbidity rates. Currently, there is no specific therapy to alter the course of the disease significantly (Kelly and Molitoris, 2000; Nash et al., 2002).
These limitations in treatment of acute and chronic renal failure have led to the search for enhanced therapeutic options. Promising results have been reported from application of different types of stem cells for treatment of kidney failure in animal models (Behr et al., 2007; Chen et al., 2008; Curtis et al., 2008; Kinomura et al., 2008). Recent studies indicate that beneficial effects of stem cells are mainly mediated via paracrine secretion of mediators, rather than direct differentiation and substitution of damaged cells (Duffield et al., 2005; Togel et al., 2005, 2007). Different roles have been attributed to the bioactive mediators secreted by stem cells such as suppression of local immunologic reactions, inhibition of fibrosis and apoptosis and stimulation of angiogenesis and mitosis (Caplan and Dennis, 2006; Parekkadan et al., 2007). Based on the evidence, we hypothesized that for protection against kidney failure, direct transplantation of stem cells is not necessary, and administration of the conditioned media of the cells may also be effective. The rationale of this approach is that we can use the benefits of cell therapy without having to encounter the risks of tumorigenesis and immunologic reactions following cell transplantation. We evaluated the therapeutic effects of conditioned media derived from human umbilical cord blood USSCs (unrestricted somatic stem cells) and mouse bone marrow MSCs (mesenchymal stem cells) in an animal model of cisplatin-induced kidney failure.
2. Materials and methods
Normal adult male BALB/c mice, 6–8 weeks old were obtained from Pasteur Institute of Iran. Animal care and experiments were according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
2.2. Isolation and characterization of stem cells
2.3. Preparation of conditioned medium
USSCs or MSCs were cultured at the confluency of about 60%. After removing the media, the cells were washed with PBS three times and cultured in the presence of UltraCULTURE medium (Cambrex) with 7.5% BALB/c serum at 37°C and 5% CO2 in air. After 48 h, this medium was used as conditioned medium. To prepare the control medium, UltraCULTURE medium with 7.5% BALB/c serum was kept in the incubator for 48 h in empty flasks.
2.4. Induction of cisplatin-induced nephropathy and administration of conditioned medium
Kidney failure was induced in mice by intraperitoneal injection of cisplatin as previously described (Faubel et al., 2007) with some modifications. Based on an initial dose–response study, cisplatin at a single 18 mg/kg dose was chosen, as it produced a reproducible renal failure. Eight hours before cisplatin administration, food and water were withheld, and after it, the mice again had free access to food and water. Twenty-four hours after cisplatin injection (day 1), mice were randomly divided in two weight match groups and received 0.5 ml of conditioned medium or control medium through tail vein injection. (MSC- and USSC-conditioned media were evaluated in independent experiments.) Ninety hours after cisplatin injection (day 4), the physical activity score of mice was determined according to a previously described method (Castellani and Adams, 1981) (1, lazy, slow movement; 2, intermediate level of activity; 3, active movement or searching), and blood and tissue samples were extracted for biochemical and histopathological examinations.
2.5. Histopathological analysis
Haematoxylin and eosin-stained coronal sections of kidneys were analysed in a blinded manner and scored for injury as previously described (Chatterjee et al., 2001) with some modifications. Briefly, random cortical fields were analysed using a ×40 objective. One hundred tubules were examined for each kidney section, and a score from 0 to 3 was assigned for each tubule: 0 = normal histology; 1 = tubular cell swelling, brush border loss, nuclear condensation, with loss of up to one-third of the tubule nuclei; 2 = same as for score 1, but greater than one-third and less than two-thirds of the tubular profile showing nuclear loss; and 3 = greater than two-thirds of the tubular profile showing nuclear loss. The total score per kidney was calculated by addition of all 100 scores with a maximum possible injury score of 300. The mean number of hyaline casts was also determined through examination of 40 random cortical fields.
2.6. Biochemical analysis
Urea and creatinine measurements in serum samples were performed by Hitachi 717 autoanalyser.
2.7. Statistical analysis
Data are shown as mean±S.E.M. Statistical analysis was carried out using SPSS software version 13 (SPSS, Inc.). Kolmogorov–Smirnov was used to test the normal distribution of the data in each group. For comparing the groups, t test or Mann–Whitney test was exploited. A P-value <0.05 was considered statistically significant.
3.1. Experiment 1 – effect of USSC-conditioned medium
USSCs that were used have been previously characterized in our laboratory (Hashemi et al., 2009). USSC-conditioned medium or control medium was administered to mice 24 h after injection of cisplatin, and the therapeutic effect was evaluated after 3 days. To confirm the reproducibility of the results, this experiment was repeated on five occasions, and a total of 120 mice was used for this experiment (60 mice for each group). No significant difference was observed in urea, creatinine, pathology score, number of hyaline casts, activity score and weight loss parameters in USSC-conditioned medium-treated group compared with control group (Figure 1). Altogether, these results do not show any favourable effect of USSC-conditioned medium in the course of our model of kidney failure.
3.2. Experiment 2 – effect of MSC-conditioned medium
One concern with using USSCs was the human origin of these cells. In fact, some of the secreted mediators by these cells might not be recognized by mouse kidney cells. The study was extended to examine the effect of mouse bone marrow MSC-conditioned medium. The identity of these cells was determined by differentiation to osteocyte, adipocyte and chondrocyte lineages and surface marker analysis which showed that the cells were positive for CD29, CD44, CD73, CD90, CD105 and negative for CD45, CD11b, VEGFR2, CD31 and CD34 (Supplementary Figure S1 at http://www.cellbiolint.org/cbi/035/cbi0350209add.htm).
We assessed the effect of MSC-conditioned medium with the same study design of experiment 1. The results of functional and structural assessments of mice receiving MSC-conditioned medium (n = 40) or control medium (n = 40) are shown (Figure 1). MSC-conditioned medium group was not significantly better than the control group in terms of urea, creatinine, pathology score, number of hyaline casts, activity score and weight loss measures. Therefore, similar to USSC-conditioned medium, we could not detect a beneficial effect for MSC-conditioned medium in the course of cisplatin-induced nephropathy. Representative pictures of histopathologic analysis of kidneys are shown (Figure 2).
3.3. Experiment 3 – effect of repetitive administration of MSC-conditioned medium
Considering that most of the mediators in conditioned media would probably have a short half-life and are present at low concentrations, we wondered if repeated injections of conditioned medium are necessary for protection against kidney failure. In fact, it is possible that a single dose administration of conditioned medium 1 day after cisplatin injection would not be enough. Hence, we performed the third experiment by daily administration of 0.5 ml of MSC-conditioned medium (n = 17) or control medium (n = 16) in three consecutive days starting from the day of cisplatin injection (days 0, 1 and 2), and the mice were killed on day 4. Similar to experiments 1 and 2, there was no significant difference in mice receiving conditioned medium or control medium in any of the examined parameters (Figure 1). Therefore, this experiment revealed that even repetitive administration of MSC-conditioned medium was not effective for ameliorating cisplatin-induced nephropathy.
We used an animal model of kidney failure to assess the putative therapeutic effects of conditioned media of USSCs and MSCs. The selection of these two cell types was based on previous studies indicating the significant power of these cells for secretion of a wide range of growth factors and mediators (Kögler et al., 2005; Togel et al., 2007). We could not detect a beneficial effect for the conditioned media derived from these two cell types for ameliorating of cisplatin-induced nephropathy. However, we acknowledge that the assessments were at the functional and histopathological structural levels and we cannot rule out the possibility of positive effects at the cellular and molecular levels.
In agreement with our study, Dai et al. (2007) could not detect therapeutic effects for MSC-conditioned medium in a rat model of myocardial infarction. There are some explanations for these findings. First, each bioactive molecule works in a defined concentration, and in some cases, they show different functions in different concentrations. It is possible that the concentration of mediators secreted by stem cells in the culture is not in the appropriate range for a renoprotective effect. Secondly, it is reasonable to assume that in response to injury, stem cells secrete defined sets of mediators in a temporally and spatially regulated manner (Imai and Iwatani, 2007), and probably in the culture system, they secret another set of mediators. In fact, when stem cells reach the location of injury, they secrete special factors based on the specific signals received from the injury microenvironment. Therefore, one may want to mimic the injury microenvironment in the culture to direct the cells towards the secretion of special mediators before assessment of the renoprotective capability of the conditioned medium. The rationale of this hypothesis remains to be evaluated in future works. However, it should be noted that, although neither USSC- nor MSC-conditioned media revealed renoprotective features in our experiments, this does not rule out a paracrine mechanism for kidney protective properties of stem cells.
Bi et al. (2007) have examined the effect of conditioned medium on renal protection. In contrast with our data, they reported that administration of MSC-conditioned medium was very potent for ameliorating cisplatin-induced kidney failure. Their study design was very similar to ours; however, there were some differences: the conditioned medium was administered twice daily for 5 days intraperitoneally, serum-free medium was used and incubation time with the cells was 4 days, whereas we added 7.5% mouse serum to the medium and incubated it with the cells for 48 h. To test if our different results were due to these dissimilarities in study design, we repeated the experiment with the protocol of this study in our laboratory. Even under these situations, we did not detect any therapeutic effect for MSC-conditioned medium (data not shown). In fact, their findings were not reproducible in our lab. Different mouse strains used in these two studies (C57Bl/6 compared with BALB/c) can, at least, partly account for the discrepancies in the findings.
Although we could not detect any beneficial role for MSC- and USSC-conditioned media in our animal model of renal failure, the therapeutic potential of this strategy still remains an open question, and further studies with different designs, animal models and cell types are certainly required.
Yousof Gheisari and Naser Ahmadbeigi were responsible for the study design, animal experiments, urea and creatinine measurements, data analysis and writing the first draft of the manuscript. Mahmood Naderi was responsible for the study design, cell culture experiments and critically editing the manuscript. Seyed Mahdi Nassiri was responsible for the study design, histopathologic examinations and critically editing the manuscript. Samad Nadri was responsible for the study design, isolation and characterization of the stem cells and critically editing the manuscript. Masoud Soleimani was responsible for the study design, supervision of the project, designing the experiments and final edition of the manuscript.
This work was supported by a grant (
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Received 11 March 2010/7 June 2010; accepted 18 October 2010
Published as Cell Biology International Immediate Publication 18 October 2010, doi:10.1042/CBI20100183
© The Author(s) Journal compilation © 2011 Portland Press Limited
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)
Figure 1 The therapeutic effect of stem cell-conditioned media was assessed in three independent experiments