Skip to main content

Advertisement

Labeling and tracking human amniotic epithelial cells with green fluorescent protein in an adeno-associated virus vector

Article metrics

Abstract

Human amniotic epithelial cells (hAECs) are a recently identified type of stem cell. Thanks to their ready availability and the lower risk of teratoma formation, hAECs have been studied and tested for a variety of human disease treatments and tissue reconstruction efforts. This aim of this study was to establish a stable tracking system to further monitor hAECs in vivo after transplantation. hAECs were isolated from the placentas of patients who visited the Hunan Province Maternity and Child Care Hospitals between Jan 2008 and Jan 2009. Using the classic transfection/infection technique, we successfully introduced green fluorescent protein (GFP) into cultured hAECs with an adeno-associated virus (AAV) vector. The initial preparation of the AAV-GFP virus stock was titrated using HT1081 cells, and further used for the infection of hAECs. GFP+ hAECs preserve the capacity of differentiation into hepatocytelike cells with the expression of cytokeratin-18 (CK18) and albumin (ALB). AAV-GFP virus-infected hAECs were transplanted through the spleen into severe combined immune deficiency (SCID) mice via hepatectomy. Four weeks later, the GFP and human albumin expressions were examined in multiple organs through immunoflourence staining. In culture, over 50% of the hAECs were GFP-positive 3 days after infection. Following transplantation, AAV-GFPinfected hAECs survived and continued to express GFP in the host for up to 4 weeks. These cells were primarily found in the spleen and liver, expressing human albumin. This study provides a feasible and stable system to track hAECs. It may prove useful to further identify their biological characteristics after transplantation and to elucidate their beneficial roles for therapeutic purposes.

Abbreviations

AAV:

adeno-associated virus

ALB:

albumin

CK18:

CK18 cytokeratin

Dex:

dexamethasone

FBS:

fetal bovine serum

GFP:

green fluorescent protein

hAEC:

human amniotic epithelial cell

hESC:

human embryonic stem cell

HGF:

hepatocyte growth factor

IGF:

insulin-like growth factor

References

  1. 1.

    Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S. and Jones, J.M. Embryonic stem cell lines derived from human blastocysts. Science 282 (1998) 1145–1147.

  2. 2.

    Ilancheran, S,, Michalska, A., Peh, G., Wallace, E.M., Pera, M. and Manuelpillai, U. Stem cells derived from human fetal membranes display multilineage differentiation potential. Biol. Reprod. 77 (2007) 577–588.

  3. 3.

    Miki, T., Lehmann, T., Cai, H., Stolz, D.B. and Strom, S.C. Stem cell characteristics of amniotic epithelial cells. Stem Cells 23 (2005) 1549–1559.

  4. 4.

    Tseng, S.C., Prabhasawat, P., Barton, K., Gray, T. and Meller, D. Amniotic membrane transplantation with or without limbal allografts for corneal surface reconstruction in patients with limbal stem cell deficiency. Arch. Ophthalmol. 116 (1998) 431–441.

  5. 5.

    Connon, C.J., Doutch, J., Chen, B., Hopkinson, A., Mehta, J.S., Nakamura, T., Kinoshita, S. and Meek, K.M. The variation in transparency of amniotic membrane used in ocular surface regeneration. Br. J. Ophthalmol. 94 (2010) 1057–1061.

  6. 6.

    Meller, D., Pires, R.T., Mack, R.J., Figueiredo, F., Heiligenhaus, A., Park, W.C., Prabhasawat, P., John, T., McLeod, S.D., Steuhl, K.P. and Tseng, S.C. Amniotic membrane transplantation for acute chemical or thermal burns. Ophthalmology 107 (2000) 980–989, discussion 990.

  7. 7.

    Terada, S., Matsuura, K., Enosawa, S., Miki, M., Hoshika, A., Suzuki, S. and Sakuragawa, N. Inducing proliferation of human amniotic epithelial (HAE) cells for cell therapy. Cell Transplant. 9 (2000) 701–704.

  8. 8.

    Sakuragawa, N., Enosawa, S., Ishii, T., Thangavel, R., Tashiro, T., Okuyama, T. and Suzuki, S. Human amniotic epithelial cells are promising transgene carriers for allogeneic cell transplantation into liver. J. Hum. Genet. 45 (2000) 171–176.

  9. 9.

    Kosuga, M., Takahashi, S., Sasaki, K., Enosawa, S., Li, X.K., Okuyama, S., Fujino, M., Suzuki, S., Yamada, M., Matsuo, N., Sakuragawa, N. and Okuyama, T. Phenotype correction in murine mucopolysaccharidosis type VII by transplantation of human amniotic epithelial cells after adenovirus-mediated gene transfer. Cell Transplant. 9 (2000) 687–692.

  10. 10.

    Wei, J.P., Zhang, T.S., Kawa, S., Aizawa, T., Ota, M., Akaike, T., Kato, K., Konishi, I. and Nikaido, T. Human amnion-isolated cells normalize blood glucose in streptozotocin-induced diabetic mice. Cell Transplant. 12 (2003) 545–552.

  11. 11.

    Elwan, M. and Sakuragawa, N. Evidence for synthesis and release of catecholamines by human amniotic epithelial cells. Neuroreport 8 (1997) 3435–3438.

  12. 12.

    Sakuragawa, N., Misawa, H., Ohsugi, K., Kakishita, K., Ishii, T., Thangavel, R., Tohyama, J., Elwan, M., Yokoyama, Y., Okuda, O,, Arai, H., Ogino, I. and Sato, K. Evidence for active acetylcholine metabolism in human amniotic epithelial cells: applicable to intracerebral allografting for neurologic disease. Neurosci. Lett. 232 (1997) 53–56.

  13. 13.

    Kakishita K., Elwan, M.A., Nakao, N., Itakura, T. and Sakuragawa, N. Human amniotic epithelial cells produce dopamine and survive after implantation into the striatum of a rat model of Parkinson’s disease: a potential source of donor for transplantation therapy. Exp. Neurol. 165 (2000) 27–34.

  14. 14.

    Sankar, V. and Muthusamy, R. Role of human amniotic epithelial cell transplantation in spinal cord injury repair research. Neuroscience 118 (2003) 11–17.

  15. 15.

    Bregman, B.S., Broude, E., McAtee, M. and Kelley, M.S. Transplants and neurotrophic factors prevent atrophy of mature CNS neurons after spinal cord injury. Exp. Neurol. 149 (1998) 13–27.

  16. 16.

    Ilancheran, S., Moodley, Y. and Manuelpillai, U. Human fetal membranes: a source of stem cells for tissue regeneration and repair? Placenta 30 (2009) 2–10.

  17. 17.

    Sakuragawa, N., Yoshikawa, H. and Sasaki, M. Amniotic tissue transplantation: clinical and biochemical evaluations for some lysosomal storage diseases. Brain Dev. 14 (1992) 7–11.

  18. 18.

    Hester, M.E., Foust, K.D., Kaspar, R.W. and Kaspar, B.K. AAV as a gene transfer vector for the treatment of neurological disorders: novel treatment thoughts for ALS. Curr. Gene Ther. 9 (2009) 428–433.

  19. 19.

    Lo, W.D., Qu, G., Sferra, T.J., Clark, R., Chen, R. and Johnson, P.R.. Adeno-associated virus-mediated gene transfer to the brain: duration and modulation of expression. Hum. Gene Ther. 10 (1999) 201–213.

  20. 20.

    Cai, J., Ito, M., Nagata, H., Westerman, K.A., Lafleur, D., Chowdhury, J.R., Leboulch, P. and Fox, I.J. Treatment of liver failure in rats with end-stage cirrhosis by transplantation of immortalized hepatocytes. Hepatology 36 (2002) 386–394.

  21. 21.

    Yovchev, M.I., Grozdanov, P.N., Zhou, H., Racherla, H., Guha, C. and Dabeva, M.D. Identification of adult hepatic progenitor cells capable of repopulating injured rat liver. Hepatology 47 (2008) 636–647.

Download references

Author information

Correspondence to Hongwu Luo.

Additional information

These Authors equally contributed to this paper

Rights and permissions

Reprints and Permissions

About this article

Key words

  • Human amniotic epithelial cells
  • Adeno-associated virus
  • Green fluorescent protein
  • Transplantation
  • Tracking system