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Isolation of Amniotic Epithelial Stem Cells

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

1University 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: 
March, 2010
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Learn more about the author(s): 
Fabio Marongiu

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: Isolation of Human Amniotic Epithelial Cells
  • Alternate Protocol: Enrichment of Stem Cell Marker–Positive Cells by Density Separation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Topics
    • Stem Cells
    • Cell Culture
  • Other Versions
     
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Materials

Basic Protocol: 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 1C.3)

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

 Additional Materials (also see Basic Protocol)
  • Isolated amniotic epithelial cells (Basic Protocol, steps 1 to 16)
  • 24% (v/v) Percoll solution (see recipe), sterile
Looking for materials?  Suppliers Guide
     
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Figures

  • Figure 1E.3.1
    Cross-section of human amnion.

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  • Figure 1E.3.2
    Sterile equipment for the isolation of hAECs.

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  • Figure 1E.3.3
    Placenta with amnion membrane facing up.

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  • Figure 1E.3.4
    Placenta with an X-shaped incision.

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  • Figure 1E.3.5
    Peeling the amnion membrane.

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  • Figure 1E.3.6
    Washing the amniotic membrane in HBSS.

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  • Figure 1E.3.7
    Density separation for stem cell enrichment.

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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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Anonymous (not verified)
Posted on March 17, 2010 - 6:56am

This is Batool, PhD medical biotechnology student in UPM-Malaysia, I would like research on stem cells of hAECs at my thesis work. I would be appriciated if you guide me in this area about Isolation protocol to obtain pure hAECs and charactrisation of Amniotic Epithelial Stem Cells and its markers. Certainly, I have to tell you I want to differentiate them to cardiomyocyte.

Thank you very much for your valuable guide  

Dea Dale
Posted on January 29, 2010 - 6:21am

What an amazing procedure! Science continues to break all boundaries.

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