Isolation of Amniotic Epithelial Stem Cells

Toshio Miki1, Fabio Marongiu1, Kenneth Dorko1, Ewa C.S. Ellis1, Stephen C. Strom1

1 University of Pittsburgh, Pittsburgh, Pennsylvania
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1E.3
DOI:  10.1002/9780470151808.sc01e03s12
Online Posting Date:  January, 2010
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Abstract

Many of the cell types that can be isolated from placental tissues retain phenotypic plasticity that makes them an interesting source of cells for regenerative medicine. Several procedures for the isolation of stem cells from different parts of the placenta have been reported. This unit describes a detailed and simple protocol for the selective isolation of amniotic epithelial cells from human term placenta without disturbing the mesenchymal layer. We also introduce a simple density separation technique for the enrichment of the population for SSEA‐4 positive cells. Curr. Protoc. Stem Cell Biol. 12:1E.3.1‐1E.3.10. © 2010 by John Wiley & Sons, Inc.

Keywords: placenta; amnion; epithelial stem cells

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Human Amniotic Epithelial Cells
  • Alternate Protocol 1: Enrichment of Stem Cell Marker–Positive Cells by Density Separation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation of Human Amniotic Epithelial Cells

  Materials
  • Hanks' balanced salt solution (HBSS; calcium‐ and magnesium‐free; CMF‐HBSS; Lonza, cat. no. 04‐315Q), sterile
  • Term placenta, freshly delivered, in sterile transportation medium (see recipe)
  • 70% ethanol
  • 0.05% (w/v) trypsin/EDTA (Invitrogen, cat. no. 25300‐054)
  • Pre‐digestion buffer (see recipe), sterile
  • Standard culture medium (see recipe), sterile
  • Laminar flow cabinet (BSL‐2) equipped with the following:
    • Absorbent bench paper
    • Sterile field, 16 × 29 in.
    • Sterile scalpel
    • 500‐ml glass beakers (4)
    • Sterile scissors (2) and forceps (2),
    • Sterile gloves and sleeves
    • 100‐µm nylon cell strainers (4)
    • 50‐ml conical polypropylene (e.g., Falcon) centrifuge tubes (8)
  • Refrigerated centrifuge
  • Additional reagents and equipment for cell counting (unit 1.3)

Alternate Protocol 1: Enrichment of Stem Cell Marker–Positive Cells by Density Separation

  • Isolated amniotic epithelial cells ( protocol 1, steps 1 to 16)
  • 24% (v/v) Percoll solution (see recipe), sterile
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Figures

Videos

Literature Cited

Literature Cited
   Elwan, M.A. and Sakuragawa, N. 2007. Uptake and decarboxylation of L‐3,4‐dihydroxyphenylalanine in cultured monkey placenta amniotic epithelial cells. Placenta 28:245‐248.
   Elwan, M.A., Ishii, T., and Sakuragawa, N. 2003a. Characterization of dopamine D2 receptor gene expression and binding sites in human placenta amniotic epithelial cells. Placenta 24:658‐663.
   Elwan, M.A., Ishii, T., and Sakuragawa, N. 2003b. Evidence of dopamine D1 receptor mRNA and binding sites in cultured human amniotic epithelial cells. Neurosci. Lett. 344:157‐160.
   Kakishita, K., Elwan, M.A., Nakao, N., Itakura, T., and Sakuragawa, N. 2000. 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. 165:27‐34.
   Kakishita, K., Nakao, N., Sakuragawa, N., and Itakura, T. 2003. Implantation of human amniotic epithelial cells prevents the degeneration of nigral dopamine neurons in rats with 6‐hydroxydopamine lesions. Brain Res. 980:48‐56.
   Miki, T. and Strom, S.C. 2006. Amnion‐derived pluripotent/multipotent stem cells. Stem Cell Rev. 2:133‐142.
   Miki, T., Lehmann, T., Cai, H., Stolz, D.B., and Strom, S.C. 2005. Stem cell characteristics of amniotic epithelial cells. Stem Cells 23:1549‐1559.
   Sakuragawa, N., Thangavel, R., Mizuguchi, M., Hirasawa, M., and Kamo, I. 1996. Expression of markers for both neuronal and glial cells in human amniotic epithelial cells. Neurosci. Lett. 209:9‐12.
   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. 1997. Evidence for active acetylcholine metabolism in human amniotic epithelial cells: Applicable to intracerebral allografting for neurologic disease. Neurosci. Lett. 232:53‐56.
   Takashima, S., Ise, H., Zhao, P., Akaike, T., and Nikaido, T. 2004. Human amniotic epithelial cells possess hepatocyte‐like characteristics and functions. Cell Struct. Funct. 29:73‐84.
   Wei, J.P., Zhang, T.S., Kawa, S., Aizawa, T., Ota, M., Akaike, T., Kato, K., Konishi, I., and Nikaido, T. 2003. Human amnion‐isolated cells normalize blood glucose in streptozotocin‐ induced diabetic mice. Cell Transplant. 12:545‐552.
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