In Vitro Hepatitis B Virus Infection Of Human Bone Marrow Mesenchymal Stem Cells

[Background]Hepatitis B is one of the most common infectious diseases worldwide. Hepatitis B virus (HBV) is transmitted by percutaneous or mucosal exposure to infected blood or other body fluids. It has been estimated that2billion people have been infected with HBV. Hepatitis B is a leading cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Approximately,360million people have chronic HBV infection, and0.6million people die each year from HBV-related liver disease or HCC. Despite the existence of a preventative vaccine, HBV represents a substantial threat to public health. HBV naturally infects only humans and experimentally chimpanzees, successful animal model for the study of human HBV infection has not been established to date. Currently, the hepatoma-derived cell line is a well-established and successful system that has been used for the in vitro study of HBV. However, the viral genome is introduced by integration or transfection rather than infection. Therefore, these cell lines are unsuitable for studying the mechanism of the early stages of virus-cell interactions, including viral attachment, penetration, and uncoating. Primary human hepatocytes are susceptible to HBV. However, the use of this model is hampered by limited resources and the technical difficulties that are associated with primary hepatocyte cultures. A convenient in vitro assay for HBV natural infectivity is currently unavailable, and the early steps of the viral life cycle are not well understood. In recent years, liver-related stem cells have attracted intense attention due to their proliferative capabilities and inherent characteristics. Bone marrow cells have been hypothesized as the third recruitment source in liver regeneration besides hepatocytes and endogenous liver stem cells. Previous studies have shown that human bone-marrow mesenchymal stem cells (BMSCs) can differentiate into functional hepatocyte-like cells in vitro, and restore liver function in animal models of liver failure. However, the susceptibility of human BMSCs to HBV infection is poorly understood. In the present investigation, human BMSCs were isolated from bone marrow and cultured with HBV-positive serum to investigate the infection and replication of HBV in cultures of human BMSCs.Section I Isolation, cultivation and purification of human bone-marrow mesenchymalstem cells in vitro[Objective]To investigate how to isolate, cultivate and purify the human BMSCs in vitro and provide a cell model for the further study.[Methods]1. Human BMSCs were isolated, cultured and purified using the gradient density centrifugation, plastic adherence and trypsin control method.2. The cells were passaged five times prior to further analysis to ensure the removal of contaminating hematopoietic cells. The basic characteristics of human BMSCs were detected when they grew in vitro.3. Growth curve of fifth-passage BMSCs was drawed and doubling time of cells during logarithmic growth phase was calculated using Patterson formula.4. The fifth generation of BMSCs was harvested using trypsin. The cell cycle and cell surface antigen phenotype were analyzed using flow cytometry with a FACScan (Becton Dickinson, USA). [Results]1. Human BMSCs could be cultured, expanded and maintained their characteristic morphology in vitro in a long period.2. The attached human BMSCs were morphologically similar to fibroblasts and exhibited characteristic spindle shaped fusiform morphology.3. Human BMSCs exhibited a lag phase of growth, followed by an ogarithmic growth phase, and finally resulting in a growth plateau state. Its shape of growth curve is "s" Doubling time of cells during logarithmic growth phase was31h.4. Flow cytometric analysis of the cell cycle demonstrated that a large number of BMSCs were in stationary phase and a small number of BMSCs were in proliferation phase.5. Flow cytometric analysis of the cells from the fifth subculture demonstrated constitutive expression of CD105(SH2) and CD90, but absence of CD45and CD34. These results are consistent with the expression pattern of surface antigens on classical BMSCs.6. Human BMSCs could be cryopreserved and recovery. There were no obvious differences between cultured BMSCs and recovered cells.[Conclusions]The isolation, cultivation and purification of human BMSCs in vitro are feasible. The characteristics of cultured human BMSCs are stable. The cultured human BMSCs in vitro can be used for further research.Section IIHBV infection and replication in human bone-marrow mesenchymal stem cells[Objective]To investigate the infection and replication of HBV in cultures of human BMSCs[Methods] 1. Human BMSCs were incubated with HBV positive serum (HBsAg-positive, HBeAg-positive, HBcAb-positive, and HBV DNA:5.4X108copies/mL).2. The intracellular and extracellular HBV DNA levels were measured using quantitative fluorescence PCR.3. HBV covalently closed circular DNA (cccDNA) in infected human BMSCs was extracted and analyzed using Southern blot analysis.4. The culture medium of infected human BMSCs was collected and assayed for HBsAg and HBeAg using electrochemiluminescence (ECL).5. HBcAg was detected using indirect immunofluorescence in infected human BMSCs.6. The infectious serum source were incubated with mouse antibodies directed against HBsAg (anti-HBsAg) and to infect the human BMSCs, finally assayed for infectivity using ECL.[Results]1. The infected human BMSCs were sustained for10weeks or more and maintained their characteristic phenotype.2. Intracellular HBV DNA was detected at d2after infection and maintained at relatively high levels from d6to d12. The maximal level of intracellular HBV DNA was9.37×105copies/mL. The extracellular HBV DNA was observed from d3to d15, and the levels ranged from3.792×102copies/mL to4.067×105copies/mL.3. Intracellular HBV cccDNA could be detected as early as2days postinfection, and strong signals were obtained with increasing time.4. HBsAg could be detected from d2to d16. The HBsAg levels ranged from1.612to118.100IU/mL (≥1IU/mL was considered positive). HBeAg appeared positive at d5, increased until d10, and decreased thereafter. The HBeAg levels ranged from0.115to3.407s/co (absorbance rate/cut-off ratio)(≥1s/co was considered positive)5. HBcAg expression in human BMSCs was first detected2days after infection. A diffuse but strong green fluorescence was predominantly observed in the cytoplasm of BMSCs that were infected with HBV but was occasionally observed in their nucleus. Approximately7%of the nucleus were stained. Thereafter, the number of nucleus expressing HBcAg increased. Approximately20%of cells expressed HBcAg at9days after infection.6. The anti-HBsAg antibodies reduced the HBV infectivity in human BMSCs. These results suggest that uptake of HBV into human BMSCs follows the authentic entry pathway.[Conclusions]The human BMSCs can maintain HBV infection in vitro and support the replication, expression, and secretion of HBV DNA. Human BMSCs offers a new opportunity for basic research of the HBV life cycle and the mechanism that mediates the early stages of virus-cell interactions.