Liver fibrosis is a common chronic progressive liver disease caused by one or more etiologies such as viruses, alcohol, parasites, autoimmune reactions, long-term drug damage, or repeated effects of the formation of diffuse liver damage [
With the development of cell transplantation in recent years, mesenchymal stem cells (MSCs) have attracted more and more attention in the treatment of liver fibrosis [
MSCs are widely found in the bone marrow, adipose tissue, placenta, and cord blood as well as other tissues and organs [
In this study, we used hPMSCs with green fluorescent protein (GFP+ hPMSCs) and the corresponding imaging modality to provide a novel approach to continuously track and quantify the fate of hPMSCs in vivo, investigated the effect of GFP+ hPMSCs on hepatic fibrosis in a carbon tetrachloride- (CCl4-) induced fibrotic rat model [
hPMSCs were obtained and transduced as previously described [
Six-week-old, specific-pathogen-free, male Sprague Dawley rats weighing 180 g to 200 g were obtained from the Experimental Animal Center of Zhejiang Academy of Medical Sciences and housed in an air-conditioned animal room with 50% humidity and a 12 h daylight/darkness cycle. All rats were treated according to protocols approved by the Research Ethics Committee of the First Affiliated Hospital, School of Medicine, Zhejiang University. To induce liver fibrosis, rats were given a subcutaneous injection of 50% CCl4 (Wuxi Zhanwang Chemical Co. Ltd., Wuxi, China) mixed with olive oil (Sangon Biotech Co. Ltd., Shanghai, China) at a dose of 0.5 mL per 100 g of body weight twice per week for 8 weeks. After 8 weeks, five model rats were selected randomly to verify liver fibrosis by pathological testing of liver tissue.
The animals were randomly divided into three groups as follows: group I (saline control group,
Blood samples were acquired from rats at each time point, placed at room temperature for 30 minutes, and then centrifuged for 15 minutes at 3000 rpm (Sorvall® Biofuge Stratos, Thermo, Germany), and the serum was collected. Then, albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bilirubin (TBIL), direct bilirubin (DBIL), gamma-glutamyl transpeptidase (
First, we performed liver shear-wave elastography (Siemens AG, Berlin, Germany) to assess the degree of hepatic fibrosis in rats at various time points. In the assessment of chronic liver damage, hepatic elastography is the most reproducible. In the scoring system, liver fibrosis is evaluated semiquantitatively [
To further verify the extent of the rat liver fibrosis, the hepatic specimens were fixed in 4% paraformaldehyde and embedded in paraffin, then deparaffinized and rehydrated with distilled water, and stained with hematoxylin and eosin (H&E), Masson’s trichrome (MTC) and Sirius red using a Masson’s trichrome staining kit (Sigma-Aldrich Co. LLC, MO, USA) and Sirius red staining kit (Wuhan Goodbio Technology Co. Ltd., Wuhan, China) according to the manufacturer’s instructions. First, the Laennec fibrosis scoring system was used for quantitative analysis of fibrosis [
The paraffin sections were deparaffinized and rehydrated. To inactivate the endogenous peroxidase, the sections were incubated in 3% hydrogen peroxide-methanol solution for 10 minutes. Then, antigen retrieval was performed by microwave (Midea Group Co. Ltd., Guangdong, China) treatment in 0.01 M citrate salt buffer (pH 6.0, Wuhan Goodbio Technology Co. Ltd., Wuhan, China) for 15 minutes and blocked in 5% BSA for 45 minutes at room temperature. Sections were incubated overnight at 4°C with the diluted primary antibody against human ALB (1 : 250; Abcam, UK), alpha-fetoprotein (AFP, 1 : 100; Abcam, UK), cytokeratin 18 (CK18, 1 : 100; Abcam, UK) as well as antirat
Total RNA from each sample was extracted with the Trizol reagent (Invitrogen, USA). The RNA purity and quantity were measured by determining absorbance at 260 and 280 nm, and 1
Statistical analysis was performed using the SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). All data were presented as the mean ± standard deviation (SD). Statistical comparison among three groups was performed using a one-way ANOVA analysis, and a comparison between the groups was evaluated using Student’s
hPMSCs that were cultured in normal conditions showed a fibroblast-like morphology (see Supplementary Figure S
The results from the histologic liver evaluation showed that transplantation of hPMSCs (group II) had a more beneficial effect on the recovery of liver fibrosis than saline treatment in the untreated fibrosis group (group III), exhibited as strong H&E and MTC staining, showing regenerative nodules and progressive reduction in the amount of collagen occurred in the fibrous septa, especially after the eighth week of transplantation. The degree of fibrosis was significantly improved by hPMSC transplantation. In group II rats on twelve weeks after cell transplantation, the liver was close to the normal structure of the liver, and the architecture of the impaired hepatic lobule returned to normal, as it was no different from the liver structure of rats in the saline control group (group I) (Figure
Assessment of liver fibrosis in the different experimental groups by tissue dyes. (a) Histological analysis was evaluated by H&E and MTC staining; (b-c) representative micrograph of hepatic tissue stained with Sirius red (b), and the relative expression of collagen was quantified using ImageJ analysis (c),
Histological stage of hepatic fibrosis.
Group | Stage 0 | Stage 1 | Stage 2 | Stage 3 | Stage 4A | Stage 4B | Stage 4C | Average |
---|---|---|---|---|---|---|---|---|
Score | 0 | 1 | 2 | 3 | 4 | 5 | 6 | |
Group I | 24 | 0 | ||||||
Group II | 1 | 5 | 8 | 10 | 4.125 | |||
Group III | 9 | 15 | 5.625 |
The liver elastography showed a similar result. In the untreated fibrosis group, the stiffness score was significantly upregulated, but it was significantly decreased in the hPMSC-treated group (Figure
Semiquantitative assessment of liver fibrosis in the different experimental groups by shear-wave elastography. (a) Representative micrograph of liver elastography in the three groups. Saline control group rats (group I), untreated fibrosis animals (group III), fibrotic animals transplanted with hPMSCs (group II) at four weeks, eight weeks, and twelve weeks after transplantation (4 w, 8 w, and 12 w). Images are representative images from
Biochemical analyses were performed to assess the restoration of the liver functions and hepatic fibrosis. The results showed that ALT, AST, TBIL, DBIL, and
Biochemical analyses. (a) Alanine aminotransferase (ALT); (b) aspartate aminotransferase (AST); (c) total bilirubin (TBIL); (d) direct bilirubin (DBIL); (e) gamma glutamyl transpeptidase (
The serum levels of LN and HA in the hPMSC-treated group were lower than those in the untreated fibrosis group. The level of LN approached the level in the saline control group at the last time point examined, but the level of HA was still higher than the normal level at that time (Figures
To detect the colonization and differentiation of hPMSCs in the rat after transplantation, we used fluorescence microscopy to image the liver of the rats at the corresponding time points. As shown in Figure
Localization and fate of the transplanted hPMSCs in rat livers of different experimental groups. (a) Representative figures of liver fluorescence are from
AFP, ALB, and CK18 mRNA were all detected in the liver tissue at 4 weeks, 8 weeks, and 12 weeks after transplantation through RT-PCR analysis using human-specific primers. Human AFP, ALB, and CK18 mRNA were not detected in the livers at 1 week after hPMSC transplantation, which was also confirmed by immunohistochemistry. No expression of human AFP, ALB, or CK18 was detected in group I or group III liver samples, except that human ALB mRNA expression was detected in one rat from group I (Figure
We obtained similar results through immunohistochemistry. As shown in Figure
Hepatogenic differentiation of transplanted hPMSCs in rat livers (20x). Immunohistochemistry staining for human hepatic markers ALB, AFP, and CK18, light-brown coloration in cells means the positive staining results. Group III as a negative control; PC: human liver as positive control for ALB and CK18 and human liver cancer as a positive control for AFP; group II: fibrotic animals transplanted with hPMSCs and sacrificed one week, four weeks, eight weeks, or twelve weeks after transplantation (1 w, 4 w, 8 w, or 12 w). Scale bars: 100
It is well known that activation of hepatic stellate cells (HSCs) is currently recognized as a central event in the development of hepatic fibrosis, in which activated HSCs participate in the formation of hepatic fibrosis through proliferation and secretion of
Immunohistochemistry results of
Relative mRNA expression levels of
Recently, MSCs have been reported to potentially secrete organotrophic factors that protect cells from damage or activate endogenous restorative mechanisms to enhance fibrous matrix degradation and restore the injured liver [
Cirrhosis and advanced fibrosis are generally considered irreversible conditions and are not diagnosed until the condition has reached an advanced stage [
HSC activation and increased ECM synthesis paired with insufficient degradation play a central role in the change induced by hepatic fibrosis. The expression of
The balance between ECM deposition and degradation is regulated by a variety of cytokines, the most important of which is TGF-
In conclusion, our study showed that MSCs from the human placenta could effectively cure liver fibrosis, reduce the activation of hepatic stellate cells, and restore liver functions. However, the underlying mechanism remains to be further clarified. Therefore, hPMSC therapy may be a new and effective strategy for the treatment of fibrotic liver disease in the future.
Alpha-fetoprotein
Albumin
Alkaline phosphatase
Alanine aminotransferase
Aspartate aminotransferase
Carbon tetrachloride
Cytokeratin 18
Direct bilirubin
Extracellular matrix
Ethylenediaminetetraacetic acid
Enzyme-linked immunosorbent assay
Green fluorescent protein
Hematoxylin and eosin
Hyaluronan
Human placental mesenchymal stem cells
Hepatic stellate cell
Laminin
Multiplicity of infection
Mesenchymal stem cells
Masson’s trichrome
Phosphate-buffered saline
Polymerase chain reaction
Reverse transcription-polymerase chain reaction
Total bilirubin
Transforming growth factor
Gamma-glutamyl transpeptidase.
Green fluorescent protein gene-transduced human placenta-derived mesenchymal stem cells (hPMSCs) and the corresponding imaging modality were used to provide a novel approach to continuously track and quantify the fate of hPMSCs in vivo and to investigate the effect of their transplantation on the carbon tetrachloride- (CCl4-) induced liver fibrosis in rats.
Biochemical analyses, histology, immunohistochemistry, and liver shear-wave elastography were used to evaluate the improvement on hepatic fibrosis after hPMSC transplantation.
hPMSCs had an enhanced therapeutic effect for hepatic fibrosis compared to non-hPMSCs possibly via their differentiation and inhibition of hepatic stellate cell activation.
No competing financial interests exist.
Jiong Yu and Guangshu Hao contributed equally to this work.
The authors would like to thank Dr. Tianan Jiang and Dr. Danxia Xu of the Department of Ultrasound and Dr. Ke Sun of the Department of Pathology at First Affiliated Hospital of Zhejiang University for their professional review on the B-ultrasonography and histopathology. This study was supported by the National Natural Science Foundation of China (no. 81471794) and Stem Cell and Translational Research, the National Key Research and Development Program of China (no. 2016YFA0101001).