Isolation of Amniotic Mesenchymal Stem Cells

Fabio Marongiu1, Roberto Gramignoli1, Qian Sun1, Veysel Tahan1, Toshio Miki1, Kenneth Dorko1, Ewa Ellis1, Stephen C. Strom1

1 University of Pittsburgh, Pittsburgh, Pennsylvania
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1E.5
DOI:  10.1002/9780470151808.sc01e05s12
Online Posting Date:  March, 2010
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Abstract

Mesenchymal stem cells (MSCs) have the ability to differentiate into osteocytes, chondrocytes, and adipocytes and possess immunomodulatory properties. Amniotic membrane from human term placenta is a potential source of multipotent MSCs that could be useful for regenerative medicine. This unit describes a detailed and simple protocol for the isolation of amniotic mesenchymal cells. We also introduce a simple density separation technique for the purification of this cell type from possible contamination with amniotic epithelial cells. Curr. Protoc. Stem Cell Biol. 12:1E.5.1‐1E.5.11. © 2010 by John Wiley & Sons, Inc.

Keywords: placenta; amnion; mesenchymal stem cells; MSC

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Human Amniotic Mesenchymal Stem Cells
  • Alternate Protocol 1: Purification of Amniotic Mesenchymal Cells from Amniotic Epithelial Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Human Amniotic Mesenchymal Stem Cells

  Materials
  • Hanks' balanced salt solution (HBSS, calcium‐ and magnesium‐free; Lonza, cat. no. 04‐315Q)
  • 70% (w/v) ethanol
  • 0.05% (w/v) trypsin/EDTA (Invitrogen, cat. no. 25300‐054)
  • Term placenta, freshly delivered
  • Digestion Solution (see recipe)
  • Standard culture medium (see recipe)
  • Laminar flow cabinet equipped with the following:
    • Absorbent bench paper
    • Sterile field 16 × 29–in.
    • Sterile scalpel
    • 500‐ml beakers (2)
    • Sterile Scissors (2) and forceps (2)
    • Sterile gloves and sleeves
    • 100‐µm nylon cell strainers (4)
    • 50‐ml Falcon centrifuge tubes (8)
    • Centrifuge
    • 37°C incubator
    • 37°C water bath
  • Additional reagents and equipment for cell counting (unit 1.3)

Alternate Protocol 1: Purification of Amniotic Mesenchymal Cells from Amniotic Epithelial Cells

  • Isolated amniotic mesenchymal cells in culture (see the protocol 1)
  • 0.25% (w/v) trypsin/EDTA (Invitrogen, cat. no. 25200‐056)
  • Percoll (Sigma, cat. no. P4937)
  • 10× HBSS (Invitrogen, cat. no. 14065‐056)
  • Hanks' balanced salt solution, calcium‐ and magnesium‐free (HBSS‐CMF; Lonza, cat. no. 04‐3150)
  • 15‐ml conical tubes
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Figures

Videos

Literature Cited

Literature Cited
   Kobayashi, M., Yakuwa, T., Sasaki, K., Sato, K., Kikuchi, K., Kamo, I., Yokoyama, Y., and Sakuragawa, N. 2008. Multilineage potential of side population cells from human amnion mesenchymal layer. Cell Transplant. 17:291‐301.
   Magatti, M., De Murani, S., Vertua, E., Gibelli, L., Wengler, G.S., and Parolini, O. 2008. Human amnion mesenchyme harbors cells with allogeneic T‐cell suppression and stimulation capabilities. Stem Cells 26:182‐192.
   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., Stotz, D.B., and Strom, S.C. 2005. Stem cell characteristics of amniotic epithelial cells. Stem Cells 23:1549‐1559.
   Parolini, O., Alviano, F., Bagnara, G.P., Bilic, G., Bühring, H.J., Evangelista, M., Hennerbichier, S., Liu, B., Magatti, M., Mao, N., Miki, T., Marongiu, F., Nakajima, H., Nikaido, T., Portmann‐Lanz, C.B., Sankar, V., Soncini, M., Stadler, G., Surbek, D., Takahashi, T.A., Redi, H., Sakuragawa, N., Wolbank, S., Zeisberger, S., Zisch, A., and Strom, S.C. 2008. Concise review: Isolation and characterization of cells from human term placenta: Outcome of the first International Workshop on Placenta Derived Stem Cells. Stem Cells 26:300‐311.
   Soncini, M., Vertua, E., Gibelli, L., Zorzi, F., Denegri, M., Albertini, A., Wengler, G.S., and Parolini, O. 2007. Isolation and characterization of mesenchymal cells from human fetal membranes. J. Tissue Eng. Regen. Med. 1:296‐305.
   Wei, J.P., Nawata, M., Wakitani, S., Kametani, K., Ota, M., Toda, A., Konishi, I., Ebara, S., and Nikaido, T. 2009. Human amniotic mesenchymal cells differentiate into chondrocytes. Cloning Stem Cells 11:19‐26.
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