[Show abstract][Hide abstract] ABSTRACT: Hepatitis B-related acute-on-chronic liver failure (HBV-ACLF) is a life-threatening condition and the mechanisms of its development and progression remain unclear. The aim of this study was to define the characteristics of peripheral blood mononuclear cell microRNAs in patients with HBV-ACLF. In this study, novel microRNA (miRNA) biomarkers of peripheral blood mononuclear cells (PBMCs) in patients with HBV-ACLF were characterised by high-throughput sequencing and validated by quantitative real-time polymerase chain reaction (qRT-PCR). The results showed 78 miRNAs were significantly differentially expressed in patients with HBV-ACLF compared to patients with chronic hepatitis B (CHB) and healthy controls. Among patients with HBV-ACLF, 17 dysregulated miRNAs increased or decreased more than 4-fold, of which eight miRNAs had higher expression levels than median level. qRT-PCR validation demonstrated that six miRNAs (hsa-miR-21-5p, hsa-miR-34c-5p, hsa-miR-143-3p, hsa-miR-143-5p, hsa-miR-374a-3p and hsa-miR-542-3p) may be useful as novel biomarkers for the diagnosis of HBV-ACLF. Five novel miRNAs (L-miR-1~5) were detected and two (L-miR-1 and L-miR-3) were significantly differentially expressed in patients with HBV-ACLF.
The miRNA expression profile of PBMCs is altered in patients with HBV-ACLF, and a signature of six miRNAs may be a promising biomarker for HBV-ACLF progression.
[Show abstract][Hide abstract] ABSTRACT: Background & AimsHuman bone marrow mesenchymal stem cell (hBMSC) transplantation is expected to become an alternative regenerative technique for liver diseases. However, the mechanism by which hBMSCs differentiate into hepatocytes is still unclear. The aim of this study was to establish the specific characteristics of hBMSC-derived hepatocytes (hBMSC-Heps) for future clinical applications.Methods
Potential hBMSC-Hep biomarkers were screened using cytokine arrays. Significant biomarkers were then validated by enzyme-linked immunosorbent assay (ELISA) in vitro and in an in vivo xenotransplantation model in fulminant hepatic failure (FHF) pigs.ResultsAfter 20 days of differentiation, the expression levels of tissue inhibitor of metalloproteinases 4 (TIMP-4) and follistatin (FST) in functional hBMSC-Heps were significantly increased, whereas those of activin A, osteoprotegerin and platelet-derived growth factor α polypeptide (PDGF-A) were significantly decreased. The high levels of TIMP-4 and FST were validated by ELISA in hBMSC-Heps grown in differentiation medium. The in vivo xenotransplantation model in FHF pigs showed that the serum levels of TIMP-4 and FST were significantly increased 6 hours after hBMSC transplantation and reached their highest levels at 24 and 48 hours, respectively, after hBMSC transplantation. Immunohistochemistry confirmed that TIMP-4 and FST were expressed in cultured hBMSC-Heps and in implanted hBMSC-Heps in pig livers.Conclusions
The transdifferentiation of hBMSCs into hepatocytes is associated with the expression of TIMP-4 and FST. TIMP-4 and FST represent potential novel biomarkers for the characterisation of hBMSC-Heps and may be useful for future clinical applications.This article is protected by copyright. All rights reserved.
No preview · Article · Feb 2015 · Liver international: official journal of the International Association for the Study of the Liver
[Show abstract][Hide abstract] ABSTRACT: Objective: To clarify the precise characteristics of human hepatic progenitor cells (HPCs) for future cytotherapy in liver diseases.
Methods: Hepatic progenitor-like cells were isolated and cultured from the livers of patients who had undergone partial hepatectomy for various pathologies but displayed no sign of hepatic dysfunction. These cells were characterized by transcriptomic profiling, quantitative real-time PCR and immunocyto/histochemistry.
Results:Cultured HPCs contained polygonal, high nucleus/cytoplasm ratio and exhibited a global gene expression profile similar (67.8%) to that of primary hepatocytes. Among the genes with more than 20-fold higher expression in HPCs were a progenitor marker (CD90), a pentraxin-related gene (PTX3), collagen proteins (COL5A2, COL1A1 and COL4A2), cytokines (EGF and PDGFD), metabolic enzymes (CYBRD1, BCAT1, TIMP2 and PAM), a secreted protein (SPARC) and an endothelial protein C receptor (PROCR). Moreover, eight markers (ALB, AFP, CK8, CK18, CK19, CD90, CD117 and Oval-6) previously described as HPC markers were validated by qRT-PCR and/or immunocyto/histochemistry. Interestingly, human HPCs were also positive for the hematopoietic cell markers CD45 and CD109. Finally, we characterized the localization of HPCs in the canals of Hering and periportal areas with six previously described markers (Oval-6, CK8, CK18, CK19, CD90 and CD117) and two potential markers (CD45 and CD109).
Conclusion: The human HPCs are highly similar to primary hepatocytes in their transcriptional profiles. The CD45 and CD109 markers could potentially be utilized to identify and isolate HPCs for further cytotherapy of liver diseases.
Preview · Article · Dec 2013 · International journal of medical sciences
[Show abstract][Hide abstract] ABSTRACT: The effectiveness of human bone marrow mesenchymal stem cell (hBMSC) transplantation to treat acute and chronic liver injury has been demonstrated in animal models and in a few nonrandomized clinical trials. However, no studies have investigated hBMSC transplantation in the treatment of fulminant hepatic failure (FHF), especially in large animal (pig) models. The aim of this study was to demonstrate the safety, effectiveness, and underlying mechanism of hBMSC transplantation for treating FHF in pigs through the intraportal route. Human BMSCs (3 × 10(7) ) were transplanted into pigs with FHF via the intraportal route or peripheral vein immediately after D-galactosamine injection, and a sham group underwent intraportal transplantation (IPT) without cells (IPT, peripheral vein transplantation [PVT], and control groups, respectively, n = 15 per group). All of the animals in the PVT and control groups died of FHF within 96 hours. In contrast, 13 of 15 animals in the IPT group achieved long-term survival (>6 months). Immunohistochemistry demonstrated that transplanted hBMSC-derived hepatocytes in surviving animals were widely distributed in the hepatic lobules and the liver parenchyma from weeks 2 to 10. Thirty percent of the hepatocytes were hBMSC-derived. However, the number of transplanted cells decreased significantly at week 15. Only a few single cells were scattered in the regenerated liver lobules at week 20, and the liver tissues exhibited a nearly normal structure. CONCLUSION: Immediate IPT of hBMSCs is a safe and effective treatment for FHF. The transplanted hBMSCs may quickly participate in liver regeneration via proliferation and transdifferentiation into hepatocytes during the initial stage of FHF. This method can possibly be used in future clinical therapy.
[Show abstract][Hide abstract] ABSTRACT: Our recent study first demonstrated that human bone marrow mesenchymal stem cell (hBMSC) transplantation could prevent death from fulminant hepatic failure (FHF) in pigs. To further clarify the metabolic mechanism of hBMSC transplantation in FHF, the plasma collected from FHF pigs that received transplantation of hBMSCs was examined using metabolic analysis to identify the key molecular markers that regulate recovery. The results showed that obvious metabolic disturbance occurred during FHF, whereas the hBMSC transplantation group showed less severe liver injury. The metabolic trajectory returns to its original state at week 3 following the hBMSC transplantation. In total, the concentration of 26 metabolites, including conjugated bile acids, phosphatidylcholines, lysophosphatidylcholines, fatty acids, amino acid and sphingomyelin, are significantly different between the FHF group and the hBMSC transplantation group. Moreover, the time course of changes in the metabolites corresponded with that of the biochemical and histological analyses. Real-time PCR further confirmed that the gene expression of phospholipase A1, lecithin-cholesterol acyltransferase and lysophosphatidylcholine acyltransferase 1 decreased significantly, whereas that of phospholipase A2 remained stable, which explains the decrease of the phosphatidylcholines and lysophosphatidylcholines. These novel results have revealed a metabolic mechanism for the hBMSC transplantation in FHF, which could lead to the future development of treatment strategies for stem cell therapies.
No preview · Article · May 2012 · Journal of Proteome Research
[Show abstract][Hide abstract] ABSTRACT: To establish a rapid detection method for identifying rpoB mutations associated with rifampin (RIF) resistance in sputum specimens.
We detected rpoB mutations directly in 90 sputum specimens collected from suspected tuberculosis patients using PCR-based denaturing gradient gel electrophoresis (DGGE) and compared these results with those obtained by rpoB sequencing and conventional drug susceptibility testing.
The positive detection rate of Mycobacterium tuberculosis (M. tuberculosis) was 52.2% by Acid-Fast Bacilli staining and 72.2% by conventional mycobacterial culture. In contrast, the positive rate was significantly higher (93.3%) by PCR-based detection of the rpoB gene in the same specimens. Furthermore, 75% of the tested specimens presented abnormal patterns compared with the wild-type pattern (standard H37Rv strain) analysed by DGGE. A total of 12 different patterns, representing 12 different rpoB mutations, were observed in the 63 abnormal patterns. The match rate of rpoB mutations detected by DGGE reached 96.9% when compared to DNA sequencing.
Our findings indicate that PCR-based DGGE is a rapid and reliable bio-technique for direct detection of rpoB mutations associated with RIF resistance in the sputum of suspected tuberculosis patients.
Preview · Article · Jan 2012 · International journal of medical sciences
[Show abstract][Hide abstract] ABSTRACT: The transplantation of hepatic progenitor cells (HPCs) is a promising alternate approach to liver transplantation for patients with end-stage liver disease. Here, we report a novel technique for HPCs isolation from normal adult rat livers (no preexposure to chemicals and no injury). HPCs were isolated from normal adult rat livers using a novel four-step collagenase perfusion method followed by density gradient centrifugation. The phenotypic properties of HPCs were characterized by morphological observation, reverse-transcription polymerase chain reaction (RT-PCR), and immunocytochemistry. The results showed that HPCs formed loose colonies and possessed a round or oval shape at culture day 3. These cells proliferated slowly and exhibited progenitor-like characteristics during the 30-day culture period. RT-PCR analysis indicated that the cultured cells were positive for several HPC-specific genes, such as albumin (ALB), alpha-fetoprotein (AFP), cytokeratin 18 (CK18), cytokeratin 19 (CK19), CD45, hepatocyte nuclear factor 1-alpha (HNF-1α), hepatocyte nuclear factor 4-alpha (HNF-4α), and Thy-1. Immunocytochemical staining showed that these cells were consistently positive for ALB, AFP, CK18, CK19, Thy-1, and OV-6. HPCs can be isolated from normal adult rat livers using a simple and effective technique involving four-step collagenase perfusion, further confirming their potential as a strong candidate for hepatocyte therapy.
No preview · Article · Dec 2011 · ASAIO journal (American Society for Artificial Internal Organs: 1992)
[Show abstract][Hide abstract] ABSTRACT: With the development of high-throughput screening, a variety of genetic alterations has been found in hepatocellular carcinoma (HCC). Although previous studies on HCC methylation profiles have focused on liver tissue, studies using isolated hepatocytes are rare. The heterogeneity of liver composition may impact the genuine methylation status of HCC; therefore, it is important to clarify the methylation profile of hepatocytes to aid in understanding the process of tumorigenesis.
The global methylation profile of single hepatocytes isolated from liver tissue of hepatitis B virus (HBV) related HCC (HBHC) was analyzed using Illumina Infinium Human Methylation27 BeadChips, and combined bisulfite restriction analysis (COBRA) and bisulfite sequencing were used to validate the 20 significant hypermethylated genes identified. In this study, we found many noteworthy differences in the genome-wide methylation profiles of single hepatocytes of HBHC. Unsupervised hierarchical clustering analysis showed that hepatocyte methylation profiles could be classified according to three cell types: hepatocytes of HCC, adjacent hepatocytes and normal hepatocytes. Among the 20 most hypermethylated genes in the hepatocytes of HBHC, 7 novel genes (WNK2, EMILIN2, TLX3, TM6SF1, TRIM58, HIST1H4Fand GRASP) were found to be hypermethylated in HBHC and hypomethylated in paired adjacent liver tissues; these findings have not been reported in previous studies on tissue samples.
The genome-wide methylation profile of purified single hepatocytes of HBHC was aided in understanding the process of tumorigenesis, and a series of novel methylated genes found in this study have the potential to be biomarkers for the diagnosis and prognosis of HBHC.
[Show abstract][Hide abstract] ABSTRACT: Hepatic progenitor cell (HPC) transplantation is a promising alternative to liver transplantation for patients with end-stage liver disease. However, the precise origin of HPCs is unclear. This study aimed to determine whether bone marrow mesenchymal stem cells (BMSCs) isolated from rats in acute hepatic failure (AHF) possess hepatic potential and/or characteristics. BMSCs were isolated from normal rats as well as rats in which AHF was induced by D-galactosamine. HPCs and primary hepatocytes were isolated from normal rats for comparison. The Affymetrix GeneChip Rat Genome 230 2.0 Array was used to perform transcriptome profiling of the AHF-derived BMSCs and HPCs. The results showed that AHF-derived BMSCs had a gene expression profile significantly different from that of control BMSCs. More than 87.7% of the genes/probe sets that were upregulated more than 2-fold in AHF-derived BMSCs were expressed by HPCs, including 12 genes involved in liver development, early hepatocyte differentiation and hepatocyte metabolism. Confirmatory quantitative RT-PCR analysis yielded similar results. In addition, 940 probe sets/genes were expressed in both AHF-derived BMSCs and HPCs but were absent in control cells. Compared to the control cells, AHF-derived BMSCs shared more commonly expressed genes with HPCs. AHF-derived BMSCs have a hepatic transcriptional profile and express many of the same genes expressed by HPCs, strongly suggesting that BMSCs may be a resource for hepatocyte regeneration, and further confirming their potential as a strong source of hepatocyte regeneration during severe hepatic damage.
No preview · Article · Feb 2011 · Cells Tissues Organs
[Show abstract][Hide abstract] ABSTRACT: Liver tissue engineering with hepatic stem cells provides a promising alternative to liver transplantation in patients with acute and chronic hepatic failure. In this study, a three-dimensional (3D) bioscaffold was introduced for differentiation of rat bone marrow mesenchymal stem cells (BMSCs) into hepatocytes. For hepatocyte differentiation, third passage BMSCs isolated from normal adult F344 rats were seeded into collagen-coated poly(lactic-co-glycolic acid) (C-PLGA) 3D scaffolds with hepatocyte differentiation medium for 3 weeks. Hepatogenesis in scaffolds was characterized by reverse transcript PCR, western blot, confocal laser scanning microscopy (CLSM), periodic acid-Schiff staining, histochemistry, and biochemical assays with hepatic-specific genes and markers. A monolayer culture system was used as a control differentiation group. The results showed that isolated cells possessed the basic features of BMSCs. Differentiated hepatocyte-like cells in C-PLGA scaffolds expressed hepatocyte-specific markers [eg, albumin (ALB), alpha-fetoprotein, cytokeratin 18, hepatocyte nuclear factor 4alpha, and cytochrome P450] at mRNA and protein levels. Most markers were expressed in C-PLGA group 1 week earlier than in the control group. Results of biocompatibility indicated that the differentiated hepatocyte-like cells grew more stably in C-PLGA scaffolds than that in controls during a 3-week differentiation period. The significantly higher metabolic functions in hepatocyte-like cells in the C-PLGA scaffold group further demonstrated the important role of the scaffold. Conclusion: As the phenomenon of transdifferentiation is uncommon, our successful transdifferentiation rates of BMSCs to mature hepatocytes prove the superiority of the C-PLGA scaffold in providing a suitable environment for such a differentiation. This material can possibly be used as a bioscaffold for liver tissue engineering in future clinical therapeutic applications.
Full-text · Article · Sep 2010 · Stem cells and development
[Show abstract][Hide abstract] ABSTRACT: Liver stem cell (LSC) transplantation is a promising alternate approach to liver transplantation for patients with end-stage liver disease. However, the precise origin of LSCs remains unclear. Herein we determine if bone marrow mesenchymal stem cells (BMSCs) isolated from rats in acute hepatic failure (AHF) possess hepatic characteristics and have differentiation potential. BMSCs were isolated from AHF and sham-operated rats, and primary hepatocytes were isolated from normal rats for comparison. The transcriptomic profile of BMSCs and primary hepatocytes was analyzed using the Affymetrix GeneChip Rat Genome 230 2.0 Array. BMSCs isolated from AHF and normal rats were induced to differentiate into hepatocytes in vitro and the degree of hepatic differentiation was assessed using quantitative real time RT-PCR, immunohistochemistry, and biochemical assays. AHF-derived BMSCs had a significantly different gene expression profile compared to control BMSCs. Thirty-four gene/probe sets were expressed in both AHF-derived BMSCs and primary hepatocytes, but were absent in control-derived BMSCs, including 3 hepatocyte-specific genes. Forty-four genes were up-regulated more than 2-fold in AHF-derived BMSCs compared to controls, including 3 genes involved in hepatocyte metabolism and development. Furthermore, AHF-derived BMSCs expressed more hepatocyte related genes than control BMSCs. Additional experiments to validate the differentiation of AHF-derived BMSCs, compared to control-derived BMSCs, showed that several hepatocyte-specific genes and proteins [such as albumin (ALB) and alpha fetoprotein (AFP)] were expressed earlier, and at higher levels, after 1 week of differentiation. Hepatocyte-specific metabolic functions were also significantly higher in the AHF group compared to control cells. Conclusion: AHF-derived BMSCs had a hepatic transcriptional profile and expressed hepatocyte specific genes early during differentiation, and possessed greater hepatogenic potency in vitro compared to cells isolated from control animals, further confirming their potential as a stem cell-based therapy for end-stage liver disease.