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Volume 24 Issue 6
December 2009
    Citation: Ling LI, Bin WANG, Peng LI, Zhi-qiang BAI, Hai-tao WANG, Hai-yan LIU, Dong-meng QIAN, Xiu-xia SONG, Shou-yi DING, Zhi-yong YAN, Guang-yu JIANG. Human Cytomegalovirus Infection Inhibits the Differentiation of Human Hippocampus Neural Precursor Cells into Astrocytes .VIROLOGICA SINICA, 2009, 24(6) : 552-558.  http://dx.doi.org/10.1007/s12250-009-3019-2
    shu

    Human Cytomegalovirus Infection Inhibits the Differentiation of Human Hippocampus Neural Precursor Cells into Astrocytes

    cstr: 32224.14.s12250-009-3019-2
    • 1.

      Department of Histology and Embryology Qingdao University Medical College, Qingdao 266071, China

    • 2.

      Department of Microbiology, Qingdao University Medical College, and Key Laboratory of Medicine and Biotechnology of Qingdao, Qingdao University Medical College, Qingdao 266071, China

    • Corresponding author: Bin WANG, wangbin31@yahoo.com
    • Received Date: 23 December 2008
      Accepted Date: 26 May 2009
      Available online: 01 December 2009

      Fund Project: Qingdao Technology Project 08-1-3-30-jchNational Natural Science Foundation of China 30770105

    • HCMV is a major cause of congenital brain disease in humans, and its neuropathogenesis is not yet fully understood. The objective of the present study is to investigate the effect of human cytomegalovirus (HCMV) infection on human hippocampus neural precursor cell (NPCs) differentiation in vitro. Fetal hippocampus tissue was dissociated mechanically and then cultured in proliferation medium with EGF and bFGF. The identification and purity of the NPCs were confirmed by using immunofluorescence to detect the expression of the NPCs marker-Nestin. To drive NPCs differentiation, bFGF and EGF were withdrawn from the medium and replaced with FBS (10%). HCMV AD169 (MOI=5) was added into the differentiation medium at the onset of the differentiation. After 7 days of differentiation, in order to confirm whether NPCs are permissive for HCMV infection, immunofluorescence was used to stain for the presence of immediate early (IE) and late (pp65) HCMV proteins in the infected cells. The effects of HCMV infection on NPCs’ differentiation was observed by detecting the ratio of nestin and GFAP positive cells with confocal microscopy and immunofluorescence. The data showed that 95%±8% of the cells (passage 4-8) cultured were Nestin positive which suggested that majority of the cells were NPCs. On day 7 postinfection, most of the infected cells were IE and PP65 positive. The percentage of Nestin-positive cells were 93%±10% and 50%±19% (t=6.03, p<0.01) and those of GFAP-positive cells were 55±17% and 81%±11% (t=3.77, p<0.01) in HCMV treated and control groups respectively. These findings indicate that NPCs are HCMV permissive cells and HCMV (AD 169) infection suppresses the differentiation of Hippocampus-genetic human NPCs into astrocytes. These effects may provide part of the explanation for the abnormalities in brain development associated with congenital HCMV infection.
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          doi: 10.1093/ilar.47.1.49

      2. Bazan E, Alonso F J, Redondo C, et al. 2004. In vitro and in vivo characterization of neural stem cells. Histol Histopathol, 19 (4) : 1261-1275.

      3. Gage F H. 2000. Mammalian neural stem cells. Science, 87 (5457): 1433-1438.

      4. Kawasaki H, Kosugi I, Arai Y, et al. 2002. The amount of immature glial cells in organotypic brain slices determines the susceptibility to murine cytomegalovirus infection. Lab Invest, 82: 1347-1358.
          doi: 10.1097/01.LAB.0000032376.58688.D4

      5. Kosugi I, Shinmura Y, Kawasaki H, et al. 2000. Cytomegalovirus infection of the central nervous system stem cells from mouse embryo: a model for developmental brain disorders induced by cytomegalovirus. Lab Invest, 80: 1373-1383.
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      6. Lange C, Mix E, Rateitschak K, et al. 2006. Wnt signal pathways and neural stem cell differentiation. Neurode-gener Dis, 3 (1-2): 76-86.
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      7. Leung A K, Sauve R S, Davies H D. 2003. Congenital cytomegalovirus infection. J Natl Med Assoc, 95: 213-218.

      8. Natali A, Valcavi P, Medici M C, et al. 1997. Cytomega-lovirus infection in an Italian population: antibody prevalence, virus excretion and maternal transmission. New Microbiol, 20: 123-133.

      9. Odeberg J, Wolmer N, Falci S, et al. 2007. Late human cytomegalovirus (HCMV) proteins inhibit differentiation of human neural precursor cells into astrocytes. J Neurosci Res, 85: 583-593.
          doi: 10.1002/(ISSN)1097-4547

      10. Odeberg J, Wolmer N, Westgren M, et al. 2006. Human cytomegalovirus (HCMV) inhibits neuronal differentiation and induces apoptosis in human neural precursor cells. J Virol, 80: 8929-8939.
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      11. Pass R F, Fowler K B, Boppana S B, et al. 2006. Congenital cytomegalovirus infection following first trimester maternal infection: symptoms at birth and outcome. J Clin Virol., 35 (2): 216-220.
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      12. Perlman J M, Argyle C. 1992. Lethal cytomegalovirus infection in preterm infants: clinical, radiological, and neuropathological findings. Ann Neurol, 31: 64-68.
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      13. Rho J Y, Yu K, Han J S, et al. 2006. Transcriptional profiling of the developmentally important signalling pathways in human embryonic stem cells. Hum Reprod, 21 (2): 405-412.

      14. Stagno S, Pass R F, Cloud G, et al. 1986. Primary cytomegalovirus infection in pregnancy. Incidence, transmission to fetus, and clinical outcome. JAMA, 256: 1904-1908.

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      Human Cytomegalovirus Infection Inhibits the Differentiation of Human Hippocampus Neural Precursor Cells into Astrocytes

        Corresponding author: Bin WANG, wangbin31@yahoo.com
      • 1. Department of Histology and Embryology Qingdao University Medical College, Qingdao 266071, China
      • 2. Department of Microbiology, Qingdao University Medical College, and Key Laboratory of Medicine and Biotechnology of Qingdao, Qingdao University Medical College, Qingdao 266071, China
      • Received Date: 23 December 2008
      • Accepted Date: 26 May 2009
      Fund Project:  Qingdao Technology Project 08-1-3-30-jchNational Natural Science Foundation of China 30770105

      Abstract: HCMV is a major cause of congenital brain disease in humans, and its neuropathogenesis is not yet fully understood. The objective of the present study is to investigate the effect of human cytomegalovirus (HCMV) infection on human hippocampus neural precursor cell (NPCs) differentiation in vitro. Fetal hippocampus tissue was dissociated mechanically and then cultured in proliferation medium with EGF and bFGF. The identification and purity of the NPCs were confirmed by using immunofluorescence to detect the expression of the NPCs marker-Nestin. To drive NPCs differentiation, bFGF and EGF were withdrawn from the medium and replaced with FBS (10%). HCMV AD169 (MOI=5) was added into the differentiation medium at the onset of the differentiation. After 7 days of differentiation, in order to confirm whether NPCs are permissive for HCMV infection, immunofluorescence was used to stain for the presence of immediate early (IE) and late (pp65) HCMV proteins in the infected cells. The effects of HCMV infection on NPCs’ differentiation was observed by detecting the ratio of nestin and GFAP positive cells with confocal microscopy and immunofluorescence. The data showed that 95%±8% of the cells (passage 4-8) cultured were Nestin positive which suggested that majority of the cells were NPCs. On day 7 postinfection, most of the infected cells were IE and PP65 positive. The percentage of Nestin-positive cells were 93%±10% and 50%±19% (t=6.03, p<0.01) and those of GFAP-positive cells were 55±17% and 81%±11% (t=3.77, p<0.01) in HCMV treated and control groups respectively. These findings indicate that NPCs are HCMV permissive cells and HCMV (AD 169) infection suppresses the differentiation of Hippocampus-genetic human NPCs into astrocytes. These effects may provide part of the explanation for the abnormalities in brain development associated with congenital HCMV infection.

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