The authors have declared that no competing interests exist.
Conceived and designed the experiments: QL XMZ YQS YH DMF. Performed the experiments: QL JGL LNC LHZ LW. Analyzed the data: ZYH YQS JZ YH. Contributed reagents/materials/analysis tools: XMZ JGL YH. Wrote the paper: QL YQS YH.
Mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) have been studied for damaged liver repair; however, the conclusions drawn regarding their homing capacity to the injured liver are conflicting. Besides, the relative utility and synergistic effects of these two cell types on the injured liver remain unclear.
MSCs, HSCs and the combination of both cells were obtained from the bone marrow of male mice expressing enhanced green fluorescent protein(EGFP)and injected into the female mice with or without liver fibrosis. The distribution of the stem cells, survival rates, liver function, hepatocyte regeneration, growth factors and cytokines of the recipient mice were analyzed. We found that the liver content of the EGFP-donor cells was significantly higher in the MSCs group than in the HSCs or MSCs+HSCs group. The survival rate for the MSCs group was significantly higher than that of the HSCs or MSCs+HSCs group; all surpassed the control group. After MSC-transplantation, the injured livers were maximally restored, with less collagen than the controls. The fibrotic areas had decreased to a lesser extent in the mice transplanted with HSCs or MSCs+HSCs. Compared with mice in the HSCs group, the mice that received MSCs had better improved liver function. MSCs exhibited more remarkable paracrine effects and immunomodulatory properties on hepatic stellate cells and native hepatocytes in the treatment of the liver pathology. Synergistic actions of MSCs and HSCs were most likely not observed because the stem cells in liver were detected mostly as single cells, and single MSCs are insufficient to provide a beneficial niche for HSCs.
MSCs exhibited a greater homing capability for the injured liver and modulated fibrosis and inflammation more effectively than did HSCs. Synergistic effects of MSCs and HSCs were not observed in liver injury.
Liver transplantation remains the definitive treatment option for end-stage liver disease. But the mismatch between the number of patients requiring transplantation and the amount of available organs is set to grow, highlighting the need to develop new strategies to reduce liver scarring and stimulate liver regeneration. Stem cell replacement strategies are therefore being investigated as an attractive alternative approach to liver repair.
To date, there are several published human clinical studies investigating the effects of stem cell therapy in patients with liver disease and most of the studies yielded positive results. The cells mostly used to transplant were derived from bone marrow including MSCs
Our aim was to evaluate the biodistribution of the stem cells after the peripheral infusion of MSCs or HSCs into liver injured mice. The anti-inflammatory and anti-fibrotic activities of these two stem cells were also evaluated. In addition, whether MSCs and HSCs exhibit synergistic effects in treating liver injury was studied. We hope that these findings contribute to better understanding of the interactions between stem cells and the environment that leads to homing and integration into livers.
Breeding pairs of C57BL/6-Ly5.1 mice and EGFP transgenic mice (C57BL/6-Ly5.2 background) were purchased from the animal center of the Fourth Military Medical University. All aspects of the animal research were approved by the Animal Care and Use Committee of the Fourth Military Medical University (Approval ID 12008) and were in compliance with Guidelines for the Care and Use of Laboratory Animals, as published by the National Academy Press.
Male EGFP transgenic mice 9 to 12 weeks old (19 to 24 grams) were used as bone marrow donors. The mice were humanely sacrificed and the bone marrow cells were flushed from the tibiae and femurs, pooled, and washed twice with Ca2+/Mg2+-free phosphate-buffered saline (PBS; Beyotime, China) containing 0.1% bovine serum albumin (BSA) (Beyotime, China). Single-cell suspensions were produced after repeated pipetting and filtering through a 50-µm nylon mesh. Bone marrow cells with densities ranging from 1.063 to 1.077 g/ml were collected by gradient separation using Nycodenz (Sigma, USA). Lineage negative (Lin_) bone marrow cells of the EGFP-expressing mice were prepared by incubating the cells with anti- macrophage-1(Mac-1), anti-granulocyte receptor-1 (Gr-1), anti-TER-119, anti-B220, anti-CD4 and anti-CD8 and then by removing the positive cells with immunomagnetic beads (Dynabeads M-450 coupled to sheep anti-rat IgG) (Dynal, Great Neck). The resulting Lin_ cells were stained with phycoerythrin (PE)-conjugated anti-Sca-1, biotin-conjugated anti-CD34, and APC-conjugated anti-c-kit and with the mouse lineage panel of antibodies, followed by streptavidin-conjugated PharRed. After the addition of propidium iodide at a concentration of 1 µg/ml, the cells were washed twice, resuspended in PBS containing 0.1% BSA, and maintained on ice for cell sorting. Five-color and cell sorting analyses were performed using a FACS Vantage (BD Biosciences, San Jose) with the appropriate isotype-matched controls. The Lin_ cells were enriched for the c-kit+/Sca-1+ population. Single Lin_, c-kit+, Sca-1+, CD34_ cells were deposited into round-bottomed 96-well plates using the CloneCyt system (Becton Dickinson, USA). The plates were incubated at 37°C in a humidified 5% CO2 atmosphere. At 20 hours after cell deposition, the wells containing single cells were marked and incubated for 7 days. We selected clones consisting of no more than 20 cells for clonal cell transplantation
Bone marrow cells were collected after flushing the tibiae and femurs of 6-week-old male EGFP-expressing mice (14 to 17 grams) using sterile PBS. The cells were aspirated with a 29-gauge needle to disrupt aggregates, and then the entire aspirate was centrifuged at 2,000 rpm for 10 min. The pellet was seeded into a 25-cm2 culture plate with alpha-MEM (Gibco, USA) medium supplemented with 10% selected fetal bovine serum (FBS; Gibco, USA), an anti-mycotic solution (Sigma, USA) and 1% antibiotics. The non-adherent cells were removed after 72 h by changing the medium, and the medium was entirely replaced every 5 days
Wild-type mice (9 to 12 weeks old, 19 to 24 grams) were housed in a sterile animal facility with a 12-hour dark/light cycle and free access to food and water. Advanced liver fibrosis was induced in adult female mice with intraperitoneal injections of 7 ml/kg body weight of a 1∶4 solution of CCl4 (Sigma, USA) and olive oil (Sigma, USA) twice a week for 3 months
A total of 1×106 cells were resuspended in 100 µL PBS and slowly infused via the tail vein (Normal, MSCs, HSCs and MSCs+HSCs treatment groups). Following transplantation, CCl4 was re-administered for four additional weeks (7 ml/kg body weight of a 1∶4 solution of CCl4 and olive oil twice a week) to enable the transplanted cells to engraft and differentiate. CCl4 was not administered to the normal group (n = 20) during this period. In the CCl4 group (n = 22), the mice were injected with CCl4 alone, without cell transplantation. In the MSCs group (n = 22), the mice were administered MSCs. In the HSCs group (n = 22), the mice were administered HSCs. In the MSCs+HSCs group (n = 22), the mice were administered 5×105 MSCs and 5×105 HSCs
The mice were sacrificed under deep anesthesia at 2 h, 24 h, 7 d and 28 d after transplantation. The lungs, livers, spleens and kidneys were isolated and were directly imaged by CCD with its excitation wavelength at 465/430 nm and emission filter at 560 nm with the following parameters: binning: 4, F/Stop: 1, exposure time: 1 min. The bio
To observe the engrafted cells in the liver, the tissues were prepared as frozen sections. The livers were dissected into pieces, immediately incubated with an optimal cutting temperature compound (OCT compound, Sakura Fine technical, Japan) and held in liquid nitrogen for 20 seconds. The frozen liver sections were directly transferred to a −70°C freezer. For observing the EGFP-expressing cells in the tissue, the frozen sections were allowed to melt in distilled water for 3 min and placed on slides.
Genomic DNA was isolated from each liver using a lysis buffer containing 1% of 0.2 mg/ml proteinase K (Sigma, USA) and SDS followed by phenol-chloroform extraction. The genomic DNA was used to detect a Y chromosome sequence with the forward primer
Serum was collected to analyze aspartate aminotransferase (AST), alanine aminotransferase (ALT), and albumin (ALB) using a chemistry analyzer (Abbott Architect c8000, USA).
For the histological examinations, formalin-fixed livers were dehydrated and embedded in paraffin. The liver sections were deparaffinized and stained using hematoxylin and the eosin stain or Sirius red. For immunohistochemical studies, after microwave-based antigen retrieval, the sections were treated with 0.3% H2O2 in PBS to quench the endogenous peroxidase and then incubated with 5% goat serum (Beyotime, China) to block the non-specific sites. Monoclonal primary antibodies against mouse Ki-67 (1∶100, Abcam, USA) were applied when incubating at 4°C for 12 h, followed by incubation with the corresponding secondary antibodies (Beyotime, China) at 37°C for 30 min. The specimens were then incubated with a diaminobenzidine peroxidase substrate and then subsequently counterstained with hematoxylin. For the immunofluorescence analyses, the tissue samples were fixed using 4% paraformaldehyde (Beyotime, China) and then permeabilized using 100% acetone. The samples were blocked using 5% BSA and then incubated overnight at 4°C with alpha-fetoprotein(AFP) (1∶200, Abcam, USA), ALB (1∶200, Abcam, USA), proliferating cell nuclear antigen(PCNA)(1∶500, Abcam, USA) and alpha-smooth muscle actin (α-SMA) (1∶300, Abcam, USA) antibodies diluted in antibody dilution solutions (Beyotime, China). The excess primary antibody was removed by washing five times in PBS, and the samples were incubated with a PE-conjugated secondary antibody (1∶500) at room temperature for 2 hours. The sections were incubated with 4′, 6-diamidino-2-phenylindole (DAPI; Beyotime, China) to label the nuclei. The slides were mounted in propidium iodide-containing mounting medium (Beyotime, China) for visualization using a confocal microscope (FV-1000, Olympus, Japan).
The MSCs and HSCs suspension was incubated with PE-labeled anti-mouse CXCR4 antibody (1∶500,BD Pharmingen, USA) on ice for 1 h, washed with staining buffer, and fixed with 2% paraformaldehyde. FACS data were acquired using a flow cytometer (BD Biosciences, USA)
Total RNA was isolated from fresh liver tissue and stem cells using Trizol reagent (Life Technologies, USA), and the ratio between the absorbance values at 260 and 280 nm provided an estimate of the RNA purity. Real-time PCR was performed using a one-step kit (Takara, Japan) with the following primers: α-SMA: forward:
Quantification of the mouse serum levels of nerve growth factor (NGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), IL-10, IL-6 and tumor necrosis factor-alpha (TNF-α) was determined using enzyme-linked immunosorbent assays kits per the manufacturer’s instructions (R&D Systems, USA), and the wells were read at 450 nm on an optical plate reader. Standard curves were prepared using purified cytokine standards. Each experimental sample was run in duplicate.
The data are expressed as the mean with the standard error of the mean. The statistical significances were determined using SPSS 12.0 software (SPSS, USA). The statistical significances between the control and test groups were determined using Student’s t-test. For the analyses of multiple groups, the P-values were adjusted using the Bonferroni method and a P<0.05 was used for statistical significance. All of the procedures were performed by blinded investigators.
The MSCs were isolated from the bone marrow of EGFP-transgenic mice. In the first passage, the cells derived from the donors emitted heterogeneous levels of green fluorescence when observed under the fluorescence microscope and were of various sizes, as observed in bright field. In the third passage, the EGFP signal intensity was uniform among the cells, and the cells exhibited a homogeneous morphology (
(A) The morphologies of the MSCs in the first (i and ii) and third (iii and iv) passages observed under bright field and fluorescence microscopy, respectively. (B) Surface molecule characterization of the MSCs performed by FACS analyses after incubation with PE-conjugated antibodies (CD90, CD29, CD105, CD45, CD34 and CD80).
After intravenous infusion, the EGFP signals first accumulated in the lung, but by 2 h, those signals began to decrease, whereas they started to accumulate in the liver and spleen at 2 h after infusion. During the following hours to days, the EGFP signal intensity gradually increased in the liver. From 24 h to 7 d, the EGFP signal intensity gradually increased in the spleen and then decreased. The EGFP signal was barely detectable in the kidney. These trends were similar in the MSCs, HSCs and MSCs+HSCs groups (
EGFP+ cells from male donors were injected into liver-injured female mice. (A) After transplantation of stem cells, the EGFP fluorescence in the lung, liver, spleen and kidney was examined using bio- imaging system. A luminescent image from red (least intense) to yellow (most intense) represents the spatial distribution of the detected photons emitted from EGFP+ cells within the organs. The EGFP signal was not detected in the control mice. (B) Average radiance was quantified in the liver after stem cells transplantation. (C) PCR-based detection of donor-derived cells in the livers of different recipients. (D and E) Liver sections were stained with DAPI, and the distribution of EGFP+ cells in the portal lobe was quantified in the different groups at 4 weeks. White arrows show the stem cells in the sinusoids (i, MSCs group; ii, HSCs group; and iii, MSCs+HSCs group). (F and G) FACS analyses of CXCR4 expression on MSCs and HSCs. (H) mRNA levels of CXCR4 on MSCs and HSCs.
pone.0062426The mice in the normal group survived the observation period. The survival of the mice in the three groups that underwent stem cell transplantation was significantly higher than in the group treated with CCl4. The survival percentage in the MSCs group (68.2%) was significantly higher than in the HSCs (36.4%) or MSCs+HSCs (45.5%) group, while the survival percentage in the MSCs+HSCs group was significantly higher than in the HSCs group (
(A) A survival curve for the injured mice that underwent intravenous cell transplantation. Representative photomicrographs of H&E-stained (B) and Sirius red-stained (C) mouse livers from the different groups (i, normal mice; ii, CCl4 group; iii, MSCs group; iv, HSCs group; and v, MSCs+HSCs group).(D) Analyses of the fibrosis percentage using Image J software. (E) mRNA levels of
(A) Immunohistochemistry analyses of PCNA expression in liver tissues (i, normal mice; ii, CCl4 group; iii, MSCs group; iv, HSCs group; and v, MSCs+HSCs group). (B) Immunohistochemistry analyses of Ki-67 expression in liver tissues (i, normal mice; ii, CCl4 group; iii, MSCs group; iv, HSCs group; and v, MSCs+HSCs group). (C) Quantitative image analyses of the percentage of Ki-67+ cells. (D) Quantitative image analyses of the percentage of PCNA+ cells.
The immunofluorescence staining in the HSCs group revealed a higher percentage of double-labeled GFP+/AFP+ and GFP+/ALB+ cells in the host livers (4.3±0.6% and 3.5±0.7%, respectively), compared with the MSCs (1.4±0.5% and 2.1±0.3%, respectively) or the MSCs+HSCs (2.8±0.4% and 2.4±0.6%, respectively,
The liver sections were observed under fluorescence microscopy. (A, B and C) The liver sections were co-stained with either AFP or ALB to detect the differentiation of transplanted cells (white arrowhead) in the different groups. (D) The expression of α-SMA in the livers of CCl4-induced injured mice in the different groups (i, normal mice; ii, CCl4 group; iii, MSCs group; iv, HSCs group; and v, MSCs+HSCs group). (E) mRNA levels of α-SMA.
Four weeks after cell transplantation, the serum showed a significant increase in NGF in the MSCs (101±12 pg/ml) group when compared with the HSCs and MSCs+HSCs groups (53±5 pg/ml and 69±7 pg/ml, respectively,
In this study, we conducted a comparison of MSCs and HSCs
Cell homing and engraftment into the host liver are integral to cell-based therapies. The mechanism that governs the recruitment of bone marrow stem cells is complicated. Several signaling pathways
Previous studies regarding the homing capacity of stem cells to the injured liver have reported conflicting conclusions. Some reported that the stem cells gradually accumulated in the liver
To overcome these limitations, for the present investigation, highly purified and functionally active EGFP+ MSCs and HSCs isolated from bone marrow were transplanted intravenously into mice with CCl4-induced chronic liver injury. Our data showed that the EGFP signals were detected not only in livers but also in other tissues, such as the lungs and spleens. The distributions of these two stem cells were similar and both migrated effectively to the liver rather than to the lung, spleen and kidney in mice with CCl4-induced liver injury. The MSCs and HSCs gradually accumulated in the liver after they were first observed there. Furthermore, we found that MSCs exhibited a more superior homing ability to the injured liver in comparison with HSCs, which would help them exert their effects there. Meanwhile, fluorescence-activated cell sorting and Real-time PCR analyses of CXCR4 expression on both stem cells revealed that CXCR4 expression in MSCs(in the third passage)was higher than in HSCs.
As for the localization of stem cells in the liver, Sakaida et al. transplanted bone marrow cells into mice with liver fibrosis and found that the cells predominantly engrafted to the periportal area
The transdifferentiation ability of bone marrow-derived stem cells into hepatocytes may play a significant role in the repair of the injured liver
The concept of stem cell transplantation exerting a paracrine proliferative effect on endogenous hepatocytes is gaining support. Hepatocytes in fibrotic livers have reached replicative senescence after many rounds of injury and repair, and they have reduced proliferative capacity
Furthermore, the immunomodulatory properties of MSCs and HSCs can also play a significant role in the extenuation of liver injury. The local down regulation of pro-inflammatory cytokines and up regulation of anti-inflammatory cytokines, such as IL-10, after MSC transplantation has been described in kidney, lung injury and fulminant hepatic failure models
Several studies have demonstrated the synergistic actions of HSCs and MSCs in tissue regeneration and engineering. Moioli et al. found that the co-transplantation of HSCs and MSCs facilitated the neovascularization process in the bioengineered bone
In conclusion, these results suggest that MSCs have a greater ability to modulate chemically induced inflammation in the fibrotic liver than do HSCs. However, liver fibrosis has different causes, including alcoholic hepatitis, allograft rejection, autoimmune hepatitis and metabolic diseases. All of these disease mechanisms are quite different. In addition, the different stem cells have a variety of putative functional roles; thus, careful thought is required as to what biological action is intended from their infusion. Accordingly, further studies are needed concerning the choice of a cell therapy for specific types of liver injury.
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