HBV infection-induced liver cirrhosis development in dual-humanised mice with human bone mesenchymal stem cell transplantation.

OBJECTIVE: Developing a small animal model that accurately delineates the natural history of hepatitis B virus (HBV) infection and immunopathophysiology is necessary to clarify the mechanisms of host-virus interactions and to identify intervention strategies for HBV-related liver diseases. This study aimed to develop an HBV-induced chronic hepatitis and cirrhosis mouse model through transplantation of human bone marrow mesenchymal stem cells (hBMSCs). DESIGN: Transplantation of hBMSCs into Fah-/-Rag2-/-IL-2Rγc-/- SCID (FRGS) mice with fulminant hepatic failure (FHF) induced by hamster-anti-mouse CD95 antibody JO2 generated a liver and immune cell dual-humanised (hBMSC-FRGS) mouse. The generated hBMSC-FRGS mice were subjected to assessments of sustained viremia, specific immune and inflammatory responses and liver pathophysiological injury to characterise the progression of chronic hepatitis and cirrhosis after HBV infection. RESULTS: The implantation of hBMSCs rescued FHF mice, as demonstrated by robust proliferation and transdifferentiation of functional human hepatocytes and multiple immune cell lineages, including B cells, T cells, natural killer cells, dendritic cells and macrophages. After HBV infection, the hBMSC-FRGS mice developed sustained viremia and specific immune and inflammatory responses and showed progression to chronic hepatitis and liver cirrhosis at a frequency of 55% after 54 weeks. CONCLUSION: This new humanised mouse model recapitulates the liver cirrhosis induced by human HBV infection, thus providing research opportunities for understanding viral immune pathophysiology and testing antiviral therapies in vivo.

Quantitative evaluation of human bone mesenchymal stem cells rescuing fulminant hepatic failure in pigs.

OBJECTIVE: Stem cell transplantation provides a promising alternative for the treatment of fulminant hepatic failure (FHF). However, it lacks fundamental understanding of stem cells' activities. Our objective was to clarify stem cell-recipient interactions for overcoming barriers to clinical application. DESIGN: We used an in-house large-animal (pig) model of FHF rescue by human bone marrow mesenchymal stem cells (hBMSCs) and profiled the cells' activities. The control and transplantation groups of pigs (n=15 per group) both received a D-galactosamine (D-Gal) injection (1.5 g/kg). The transplantation group received hBMSCs via intraportal vein infusion (3×106 cells/kg) immediately after D-Gal administration. The stem cell-recipient interactions were quantitatively evaluated by biochemical function, cytokine array, metabolite profiling, transcriptome sequencing and immunohistochemistry. RESULTS: All pigs in the control group died within an average of 3.22 days, whereas 13/15 pigs in the transplantation group lived >14 days. The cytokine array and metabolite profiling analyses revealed that hBMSC transplantation suppressed D-Gal-induced life-threatening cytokine storms and stabilised FHF within 7 days, while human-derived hepatocytes constituted only ∼4.5% of the pig hepatocytes. The functional synergy analysis of the observed profile changes indicated that the implanted hBMSCs altered the pigs' cytokine responses to damage through paracrine effects. Delta-like ligand 4 was validated to assist liver restoration in both pig and rat FHF models. CONCLUSIONS: Our results delineated an integrated model of the multifaceted interactions between stem cells and recipients, which may open a new avenue to the discovery of single molecule-based therapeutics that simulate stem cell actions.

Human bone marrow mesenchymal stem cell-derived hepatocytes express tissue inhibitor of metalloproteinases 4 and follistatin.

BACKGROUND & AIMS: Human 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. RESULTS: After 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 h after hBMSC transplantation and reached their highest levels at 24 and 48 h, 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.

Human hepatic progenitor cells express hematopoietic cell markers CD45 and CD109.

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.

Return of the metabolic trajectory to the original area after human bone marrow mesenchymal stem cell transplantation for the treatment of fulminant hepatic failure.

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.

Immediate intraportal transplantation of human bone marrow mesenchymal stem cells prevents death from fulminant hepatic failure in pigs.

UNLABELLED: 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.

A novel technique for hepatic progenitor cell isolation from normal adult rat livers.

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.

Methylation profile of single hepatocytes derived from hepatitis B virus-related hepatocellular carcinoma.

BACKGROUND: 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. METHODS AND FINDINGS: 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. CONCLUSION: 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.

Acute hepatic failure-derived bone marrow mesenchymal stem cells express hepatic progenitor cell genes.

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.

Transcriptional profiling and hepatogenic potential of acute hepatic failure-derived bone marrow mesenchymal stem cells.

UNLABELLED: 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.

3D PLGA scaffolds improve differentiation and function of bone marrow mesenchymal stem cell-derived hepatocytes.

UNLABELLED: 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.