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TISSUE-SPECIFIC STEM CELLS |
aInstituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil,
bHospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil,
cDepartamento de Histologia e Embriologia, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
Key Words. Mesenchymal stromal cells • Bone marrow • Liver • Fibrosis
Correspondence: Correspondence: Regina Coeli dos Santos Goldenberg, Ph.D., Avenida Carlos Chagas Filho, 373, Edifício do Centro de Ciências da Saúde, Bloco G (Instituto de Biofísica Carlos Chagas Filho), 2° andar/sala 53, Cidade Universitária 21941-902 Rio de Janeiro, Brazil. Telephone: +55 21 25626559; Fax: +55 21 22808193; e-mail: rcoeli{at}biof.ufrj.br
Received on November 8, 2007;
accepted for publication on February 19, 2008.
First published online in STEM CELLS EXPRESS February 28, 2008.
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
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Disclosure of potential conflicts of interest is found at the end of this article.
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
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In addition to its many functions, another important characteristic of the liver is its great regenerative capacity in response to injury. However, despite this ability, diseases that cause chronic injury to the liver are capable of disturbing this regenerative process, leading to the development of a common pathology known as liver cirrhosis. This disease has many etiologies such as autoimmune hepatitis, alcohol abuse, metabolic and biliary disorders, and viral hepatitis [2]. All of them cause an unbalance between collagen synthesis and degradation [3], resulting in collagen deposition in liver parenchyma and in a progressive loss of organ function, which culminates with the development of liver failure. At this stage, the only therapeutic option is organ transplantation.
According to the World Health Organization, the three most prevalent etiologies of chronic liver disease are alcohol consumption and infection by hepatitis B and C viruses [4]. More than 450 million people will develop cirrhosis as a consequence of these diseases [4], which demonstrates the great relevance of liver pathologies as causes of mortality and public health expenses.
Hence, considering the inexistence of therapeutic alternatives other than liver transplantation, the lack of organ availability, and the magnitude of chronic liver diseases, it is urgent that new therapeutic approaches are developed. In this context, the emergence of stem cell research opened new possibilities for the treatment of chronic liver diseases.
Bone marrow cells (BMCs) seem to be a promising population. Since they are easily obtained and manipulated, rejection can be avoided by autologous transplantation, and there are no ethical and religious issues concerning their use. Many marrow-derived cell types and experimental models have been used to test the efficacy of cell therapies in liver diseases, leading to divergent results on the actual role of BMC as a new treatment alternative [5–10].
Therefore, the purpose of our study was to evaluate whether bone marrow multipotent MSCs are capable of reducing liver fibrosis and improving hepatic function in a rat model of severe chronic liver injury.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
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Female Wistar rats were obtained from Instituto de Biofísica Carlos Chagas Filho (IBCCF–Rio de Janeiro/Brazil). Animals were housed at controlled temperature (23°C) with daily exposure to a 12:12 light-dark cycle.
Experimental Model
Chronic liver injury was induced in 50 female Wistar rats, weighing 200–220 g, with low-dose injections of a 20% solution (1:5 in olive oil dose of 0.05 ml/kg) of carbon tetrachloride (CCl4) (VETEC, Rio de Janeiro, Brazil, http://www.vetecquimica.com.br) associated to an alcoholic liquid diet in accordance to AIN-93 guidelines [11]. Prior to CCl4 administration, an adaptation phase was performed with a non-alcoholic liquid diet (control diet) administered for one week followed by a second week of alcoholic diet, that was continued until the fifteenth week. CCl4 injections were performed intraperitoneally, three times a week every other day over 15 weeks.
Control diet ingredients were identical to those used in the alcoholic diet except for alcohol, which was replaced by water in the same volume. Rats were given ad libitum access to liquid diets.
After 15 weeks of injury, all rats were submitted to blood tests for the evaluation of liver injury and function. Samples obtained from 10 normal rats, referred to as control, were used to define the normal range for each blood parameter analyzed. This range was calculated by using the mean ± 2 standard deviations for each test. The specific values were: Alanine Aminotransferase (ALT) 34.02–44.34 U/L, Aspartate Aminotransferase (AST) 80.50–95.90 U/L, and Albumin 3.075–3.306 g/dL.
Only those animals presenting blood values different from the predefined range in all three parameters were used in the study (n = 14). These animals were divided into two groups: eight animals (cell-treated group) were injected into the portal vein with 1 x 107 rat bone marrow MSC diluted in 0.5 ml of Balanced Salt Solution (BSS), and six animals (placebo group) were submitted to the same protocol as the cell-treated group, however, they received only BSS injections.
Blood Analysis
Rats were anesthetized and 1 ml of blood was drawn from the tail vein. Samples were centrifuged at 3,584 x g for 10 minutes and serum was collected. The following parameters were evaluated: ALT (UV-IFCC method), AST (UV-IFCC method), and Albumin (Bromocresol Green method). Samples were analyzed by Bio 200F (BioPlus, São Paulo, SP, Brazil, http://www.bioplus.com.br). Blood analyses were performed before cell injection (referred to as baseline) and 15 days, 1 and 2 months after cell infusion.
Cell Isolation and Culture Procedures
Bone marrow cells obtained from femurs of isogenic donor Wistar rats were used in cell therapy. Femurs were harvested and thoroughly cleaned of all muscle tissue. Bone marrow was flushed using Dulbecco's Modified Eagle Medium (DMEM, Gibco–Invitrogen, Carlsbad, CA, http://www.invitrogen.com) and the cell suspension centrifuged in Ficoll gradient at 400 x g for 30 minutes (Histopaque 1,083, 1:1; Sigma-Aldrich, St Louis, http://www.sigmaaldrich.com). Mononuclear cells were collected from histopaque medium interface. Cells were washed in BSS twice, counted in hemocytometer, and checked for viability using trypan blue.
Cells were then plated in 75 cm2 flasks and maintained at 37°C in a 5% CO2 incubator for 1 week, during which medium was changed at least twice, washing away all floating hematopoietic cells. Culture medium used was DMEM supplemented with 20% fetal bovine serum (HyClone, Logan, UT, http://www.hyclone.com), 2 mM L-glutamine (Sigma-Aldrich), and antibiotics (100U/ml penicillin G and 100 µg/ml streptomycin, [Gibco]). At approximately 80–90% confluence, cells were detached from the culture flasks with 0.25% trypsin-EDTA (Sigma-Aldrich) and replated. After the seventh replating, cells were again detached and counted. Subsequently, they were labeled with Hoescht 33342 (Sigma-Aldrich) for 20 minutes and washed twice in BSS to remove the unbound Hoescht 33342. This procedure was very efficient, ensuring approximately 100% labeling of cell nuclei. Then, 1 x 107 cells were diluted in 0.5 ml of BSS and injected into the portal vein of each rat.
MSC Differentiation Protocols
A fraction of these cells were seeded in six-well plates to induce differentiation into osteoblasts and adipocytes. In the osteogenic protocol, MSC were plated at a density of 105 per well and treated for 21 days with the previously described culture medium with the addition of: 1 µM dexamethasone (Sigma-Aldrich), 0.5 µM ascorbic acid (Henrifarma, São Paulo, SP, Brazil, http://www.henrifarma.com.br) and 10 mM β-glycerol-phosphate (Sigma-Aldrich). Osteogenic differentiation was assessed by Alizarin red staining.
Adipogenic differentiation protocol was the same; however, culture medium was only added with 1 µM dexamethasone and 10 µg/ml insulin (Sigma-Aldrich). MSC differentiation into adipocytes was confirmed by oil red O staining.
Flow Cytometry
A sample of cultured cells was used to analyze the cell phenotype by flow cytometry. Briefly, 3 x 105 cells were labeled at 4°C for 20 minutes in the dark with the following antibodies: anti-CD34-phycoerythrin (PE) (ICO 115, Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scht.com) and anti-CD45-fluorescein isothiocyanate (FITC), anti-CD11b-FITC (both from Caltag Laboratories–Invitrogen, Carlsbad, http://www.caltag.com), anti-CD90-PE and anti-CD29-FITC (both from BD Biosciences, San Jose, CA, http://www.bdbiosciences.com). Cells were washed twice in phosphate buffered saline (PBS) before flow cytometry (BD FACSAria, BD Biosciences). Isotype controls used were: mouse IgG1-PE with anti-CD34 (Santa Cruz Biotechnology), mouse IgG1-FITC with anti-CD45 and mouse IgG2a-FITC with anti-CD11b (both from Caltag Laboratories), mouse IgG1-PE with anti-CD90 and hamster IgM-FITC with anti-CD29 (both from BD Biosciences). All antibody dilutions were 1:100.
Cell Labeling with Technetium-99m
In order to analyze biodistribution after injection into the portal vein, 1 x 107 BMCs were labeled with 99mTc and injected into a separate group of animals (n = 3) not included in the experimental groups [12]. In short, 500 µl of stannous chloride solution (SnCl2) were added to the cell suspension in 0.9% NaCl and the mixture incubated at room temperature for 10 minutes. Then, 45 mCi 99mTc were added and the incubation continued for another 10 minutes. After centrifugation (500 x g for 5 minutes), the supernatant was removed and cells were washed again in saline solution. The pellet was resuspended in PBS. Cell viability was assessed by trypan blue exclusion test. Labeling efficiency (%) was calculated by the activity in pellet divided by the sum of radioactivity in pellet plus supernatant. Six hours after injection, total body images were captured for qualitative biodistribution analyses using a gamma camera (GE Integra, General Electronics, Fairfield, CT, http://www.ge.com) equipped with a high resolution collimator. A 20% energy window centered on the 144 keV photo peak of 99mTc was used.
Surgery
Animals were anesthetized, a 3 cm midline vertical abdominal incision was made, and the abdominal cavity exposed. A 0.5 cm liver biopsy was obtained. Portal vein was isolated from other abdominal structures and MSC were injected directly into the circulation. After injection, the vessel was pressured until bleeding stopped. Subsequently, 1 ml of normal sterile saline was administered into the abdomen as fluid replacement. The abdominal wall was closed in two layers in a running fashion. All the procedures were performed under sterile conditions.
Histology
Liver tissue biopsies were obtained just before MSC or placebo injection and 1 month after the procedure. Animals were sacrificed 2 months after cell infusion, when the organ was excised for histology. Liver tissue slices were fixed for 5 hours in Gendre's solution followed by overnight 10% buffered formalin solution (pH 7.2) and embedded in paraffin. Liver samples were sectioned (5 µm) and stained with Hematoxilin & Eosin (H&E) and Sirius red according to standard protocols [13].
Immunofluorescence
Samples were obtained just before cell infusion and 1 and 2 months after cell infusion. Liver tissue was embedded in Tissue-Tek OCT compound (Sakura Finetek, Zoeterwoude, Netherlands, http://www.sakuraus.com) and preserved at –70°C. Eight µm liver slices were sectioned in cryostat (Leica CM1850, Leica Microsystems, Wetzlar, Germany, http://www.leica-microsystems.com) at –20°C and fixed in acetone at 4°C. Indirect immunofluorescence technique was used to reveal collagen type I (1:30, Chemicon–Millipore, Billerica, MA, http://www.millipore.com). Secondary antibody used was FITC goat anti-rabbit IgG (H+L) conjugate (1:50, Zymed–Invitrogen, Carlsbad, CA).
Histomorphometry
Histomorphometry was performed using an imaging system constituted by a digital Q-color 5 camera (Olympus, Tokyo, http://www.olympus-global.com/en/global) coupled to an epifluorescence Axiovert 100 microscope (Carl Zeiss, Thornwood, NY, http://www.zeiss.com). Randomly picked fields of Sirius red sections were captured from each animal, using a magnification 4x objective lens. Quantification was estimated by the percentage of stained area in comparison to the total area of fields examined, using Image-Pro Plus 5.0 (Media Cybernetics, Bethesda, MD, http://www.mediacy.com) image analysis software.
Statistics
Data were analyzed using one-way analysis of variance with Tukey's post-test for multiple comparisons and unpaired t-test (two-tailed). p < .05 was considered statistically significant. Data are presented as mean ± SE.
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RESULTS |
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Our cells, purified by adherence to plastic culture flasks, had a spindle-shaped fibroblastic morphology (Fig. 1A). Ninety seven percent of our cells were CD90 positive, 92% were positive for CD29, whereas CD34, CD45 and CD11b expression was negative (Fig. 1). Additionally, our cells were capable of differentiating into osteoblasts and adipocytes (data not shown). These findings are in accordance to the ISCT established criteria [14].
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Fourteen animals were submitted to surgery: eight received MSC and six received placebo. Two weeks after surgery new blood samples were obtained. ALT and AST were no longer different from control values in both groups, additionally, there were no differences between cell-treated and placebo groups. This result remained unaltered one and two months after cell infusion (Fig. 3).
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Histologic Findings of Severe Chronic Liver Injury
Normal liver tissue shows hepatocytes radially arranged in plates aligned to sinusoids and converging to centrolobular veins (Fig. 4A). Additionally, collagen deposition occurs mostly around vessels (Fig. 4E).
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After one month, new biopsies were obtained. H&E staining showed marked reduction of inflammatory infiltrate both in placebo and cell-treated groups (Fig. 4C). Collagen deposition was comparable to the observed at the moment of cell or placebo injection (Fig. 4G).
Two months after surgery, animals were sacrificed. Inflammatory infiltrate was rarely found (Fig. 4D), although collagen deposition was still present (Fig. 4H).
Abnormal Collagen Deposits Are Present in Injured Liver
To confirm type I collagen presence in liver tissue, immunofluorescence was performed. In control animals, collagen I is present in portal spaces and as tiny fibrils in liver lobules (Fig. 4I). In animals submitted to 15 weeks of chronic liver injury, collagen I deposition and formation of regenerative nodules occurred (Fig. 4J). Figure 4K and 4L show, respectively, collagen I deposition 1 and 2 months after cell infusion.
MSC Do Not Contribute to Fibrosis Reduction
In order to quantify the amount of fibrosis over time comparing cell-treated and placebo, histomorphometry was performed.
Sirius red stained areas, representing collagen deposition, were measured and expressed as a percentage of total tissue area. Control animals had 0.708 ± 0.108% of collagen in liver tissue, most of it corresponding to fibers around vessels throughout the organ. Collagen content found in the animals submitted to chronic liver injury during 15 weeks was 8.198 ± 1.108%. Thus, there was an important increase in fibrosis compared to control animals (p = .001, Fig. 5).
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When animals were sacrificed, 2 months after cell infusion, collagen area in the cell-treated group was 4.794 ± 0.640%, whereas in the placebo group it corresponded to 5.441 ± 1.121%. Once more, no significant difference was found among groups (p = .6045, Fig. 5).
No Evidence of Bone Marrow Mesenchymal Stromal Cell Retention
In order to track the cells after injection, they were labeled with Hoescht 33342 prior to infusion into the portal vein. No Hoescht labeled cells were found one or two months after cell infusion in more than 20 low power microscope sections examined from all the MSC group animals.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
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Several studies have addressed the differentiating capability of BMC into hepatocytes, although the exact mechanisms leading to this process remain to be unraveled. Zhan and co-workers [18] and Grompe's group [19, 20] have reported that hematopoietic stem cells (HSC) can differentiate into hepatocytes in vivo. Some postulate that the mechanism involved is transdifferentiation [21], whereas others propose that cell fusion to resident hepatocytes is implicated [22–25]. Moreover, there is still ongoing controversy about whether HSC are really capable of giving rise to lineages of non-hematopoietic origin [26–28].
For this reason, a different population of BMC gained attention in the past years: mesenchymal stromal cells. These cells are characterized by a significant plasticity [29–31], easy accessibility, and a marked expansion potential in vitro, which are all attractive features for clinical use.
Hepatocyte differentiation from MSC has been described in vitro. Sai-Nan Shu and co-workers [32] induced MSC differentiation into hepatocytes using hepatocyte growth factor (HGF) plus acid and basic fibroblast growth factor (FGF). Furthermore, Lange and co-workers have described hepatocytic gene expression in MSC cultured alone [33] or in co-culture with hepatocytes [34], both of which required the presence of HGF, FGF-4 and epidermal growth factor (EGF). On the other hand, Luk and colleagues [35] described MSC differentiation into hepatocytes when co-cultured with these cells without the addition of growth factors to culture medium, using either normal or injured hepatocytes in the co-culture system.
Even though BMC differentiation potential into hepatocytes in vitro has been demonstrated, it is still controversial whether BMC transplantation can recover liver function and reduce tissue scarring in vivo. Sakaida and colleagues [5] demonstrated significant fibrosis reduction and albumin increase by transplanting bone marrow mononuclear cells (BMMC) in mice. This effect was attributed to anti-Liv8 negative cells, described as of non-hematopoietic origin. In another study [6], the same group showed that the association of BMMC with FGF-2 was more efficient in reducing fibrosis in a similar animal model. Oyagi and co-workers [7] showed that MSC cultured with HGF are capable of improving albumin and reducing fibrosis in rats, an effect that was not observed in MSC cultured without HGF. Moreover, Fang et al. [8] demonstrated reduction in fibrosis, ALT and bilirubin levels when Flk+ MSC were transplanted into injured mice livers.
However, in all these studies the liver aggression period was short (between 2 and 8 weeks) and maintained for a variable number of weeks after cells were transplanted. For instance, in Oyagi's and Fang's papers, aggression was only initiated after cells were delivered. Therefore, it is possible that the period of aggression and the moment in which cell injection is performed influences the result of cell therapy. However, data by our group [36] have shown that injection of BMMC during the course of injury is also ineffective in improving function or reducing fibrosis compared to placebo when a 15 week period of aggression is used.
In another study, Popp and co-workers [9] demonstrated that transplanted MSC could not engraft into the liver of rats subject to a very mild aggression (two CCl4 injections in a 2 month-period), which had the purpose of providing a regeneration stimulus, but was not able to induce chronic liver injury.
Abdel Aziz and colleagues [10] described fibrosis and ALT reduction with albumin improvement in rats by transplanting MSC. Once more, the injury period was short, although this group demonstrated that cell transplantation could be beneficial after aggression was interrupted.
In this perspective, the beneficial effect of MSC therapy described in these previous studies could have a different explanation. It has already been demonstrated that the immune response might play a major role in fibrosis establishment since macrophages and lymphocytes participate in the fibrogenic process [37–39]. In addition, MSC possess toll-like receptors that respond to danger signals and drive their migration and immunomodulatory responses [40]. Thus, it is possible that the immunomodulatory properties of MSC [41–43] act regulating the immune response when injury is promoted concurrently with cell transplantation in the liver.
Our study focused on the therapeutic potential of MSC in a model of severe and chronic liver injury, with an aggression period twice as long as all other previously published studies. Furthermore, cell infusion was only carried out after aggression was established and interrupted, so that cells would encounter more of a fibrosis environment and less of an inflammatory process. Thus, we tried to recreate in our model the clinical setting that will be most commonly seen in chronic liver disease patients: long periods of injury and a scarred rather than an inflammatory tissue. In our study, MSC capability of improving liver function and reducing fibrosis was addressed under these conditions.
We found that ALT and AST returned to normal levels 15 days after interrupting liver injury in both cell-treated and placebo groups. This indicates that soon after aggression is interrupted, damage markers tend to normalize, which is expected since fewer cells will die and these enzymes will no longer be released in the circulation. Apparently, in a two week period, MSC could not contribute to the reduction of hepatocyte damage since differences were not found between experimental groups; although we cannot discard that cell injection might have accelerated the return to normal enzyme levels within the two week period after cell infusion.
On the other hand, functional recovery, as determined by albumin values, took longer than damage markers. Only two months after aggression was interrupted, did albumin levels return to normal values. Once again, MSC therapy did not contribute to accelerate recovery since cell-treated and placebo animals showed similar increase in albumin levels with time.
In agreement with the biochemical results, we found that MSC did not reduce liver fibrosis. Although there was a reduction in fibrosis over time, which is an expected and already described phenomenon of tissue remodeling in the murine injured liver [15–17]; no difference was found between placebo and cell-treated groups, indicating that MSC did not participate in this process. These results were corroborated by indirect immunofluorescence of liver type I collagen content.
Our results provide evidence that a single MSC injection does not improve liver regeneration potential in a rat model of severe chronic liver injury. Possible explanations are that cells failed to engraft and/or died, since we could not find labeled cells either at one or two months after injection, in agreement with Popp's findings [8]. In addition, it is possible that MSC are not capable of transdifferentiating into hepatocytes, given that this capability has already been challenged in the case of HSC and BMMC [44–46], or that this occurs at a very low frequency, as has been previously reported [47–49]. Sato and co-workers [47] describe that human MSC cultured without growth factors differentiate into hepatocytes only rarely when transplanted into immunosuppressed rats. Aurich and colleagues [48] also reported that this was a rare event, even when MSC were cultured with HGF and EGF. Moreover, they hypothesized that stem cells need a growth advantage over host hepatocytes; therefore, a high selective pressure would be required for cells to engraft and differentiate [48]. This is another possible explanation for the fact that several studies that maintain continuous injury to hepatocytes after transplantation validate BMC therapeutic potential [5, 6, 7, 8].
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CONCLUSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST |
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ACKNOWLEDGMENTS |
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REFERENCES |
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