Hepatitis B virus infection and replication in human bone marrow mesenchymal stem cells

Abstract

Background: Hepatitis B virus (HBV) infection is a blood borne infectious disease that affects the liver. Human bone marrow mesenchymal stem cells (BMSCs) may serve as a cell source for adult stem cell transplantation in liver repair. However, the susceptibility of human BMSCs to HBV infection is poorly understood. The aim of this study was to investigate the infection and replication of HBV in cultures of human BMSCs.

Results: Human BMSCs were confirmed using flow cytometry. Intracellular HBV DNA was detected at d 2 after infection and maintained at relatively high levels from d 6 to d 12. The maximal level of intracellular HBV DNA was 9.37 × 105 copies/mL. The extracellular HBV DNA was observed from d 3 to d 15, and the levels ranged from 3.792 × 102 copies/mL to 4.067 × 105 copies/mL. HBsAg in the culture medium was detected from d 2 to d 16. HBeAg secretion was positive from d 5 to d 13. HBcAg constantly showed positive signals in approximately 7%-20% of BMSCs from 2 days after exposure. Intracellular HBV covalently closed circular DNA (cccDNA) could be detected as early as 2 days postinfection, and strong signals were obtained with increasing time.

Conclusion: HBV can infect and replicate in human BMSCs. Human BMSCs may be a useful tool for investigating HBV life-cycle and the mechanism of initial virus-cell interactions.

Figure 1

Characterization of human BMSCs. (A) Morphology of the third generation of human BMSCs under light microscope. (B) Morphology of the eighth generation of infected human BMSCs under light microscope. (C) Morphology of the eighth generation of uninfected human BMSCs under light microscope. (D) Analysis by flow cytometry showed that human BMSCs at passage 5 were negative for the expression of CD34 and CD45, but positive for the expression of CD90 and CD105.

Figure 2

Analysis of HBV DNA by FQ-PCR. (A) Intracellular HBV DNA was extracted from infected human BMSCs at various times and not detected until 2 days. (B) Extracellular HBV DNA was extracted from cell supernatants at various times and not detected until 3 days. No HBV DNA was detected in the cells or supernatants at time zero (data not shown).

Figure 3

Detection of HBsAg and HBeAg using ECL. (A) HBsAg levels in supernatant (≥1 IU/mL was considered positive). (B) HBeAg levels in supernatant [≥1 s/co (absorbance rate/cut-off ratio) was considered positive].

Figure 4

Detection of HBcAg using indirect immunofluorescence (×200). (A) HBcAg in infected human BMSCs shown by FITC staining (green color). (B) The cellular nuclei in infected human BMSCs were stained with DAPI (blue color). (C) Overlaid image of images from panels A and B. (D) HBcAg in HepG2.2.15 cells (positive control) shown by FITC staining. (E) DAPI staining for nuclei of the same field as in panel D. (F) Overlaid image of images from panels D and E. (G) HBcAg in uninfected human BMSCs (negative control) shown by FITC staining. (H) DAPI staining for nuclei of the same field as in panel G. (I) Overlaid image of images from panels G and H.

Figure 5

Southern blot analysis of HBV cccDNA in infected human BMSCs. (A) HBV cccDNA was extracted from human BMSCs at 2, 4, 6, and 8 days after infection and subjected to Southern blot hybridization. DIG-labeled whole HBV DNA probe served as a positive control (lane M). Uninfected BMSCs served as a negative control (lane U). DNA sizes are indicated on the left (kb). (B) The cccDNA cleaved by EcoRI was analyzed using Southern blot analysis.