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Isolation of Amniotic Mesenchymal Stem Cells

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

¹University 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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Fabio Marongiu

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

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Materials

Basic Protocol: 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 1C.3)

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

Additional Materials (also see Basic Protocol)

Isolated amniotic mesenchymal cells in culture (see the Basic Protocol)
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

Figure 1E.5.1

Cross-section of human amnion.

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Figure 1E.5.2

Sterile equipment for the isolation of hAECs.

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Figure 1E.5.3

Placenta with amnion membrane facing up.

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Figure 1E.5.4

Placenta with an X-shaped incision.

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Figure 1E.5.5

Peeling the amnion membrane.

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Figure 1E.5.6

Washing the amniotic membrane in HBSS.

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Figure 1E.5.7

Schematic representation of density separation with Percoll gradients before (A) and after (B) centrifugation.

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Figure 1E.5.8

Morphology of hAMSCs and hAECs cultured for 24 hr after Percoll density separation. (A) Fraction 1: hAMSC monolayer; (B) fraction 2: islands of hAECs (red arrows) on a hAMSC monolayer; (C) fraction 3: islands of hAMSCs (yellow arrows) on a hAEC monolayer; (D) fraction 4: hAEC monolayer.

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Figure 1E.5.9

FACS analysis of MSC marker expression on hAMSCs immediately after isolation. The percentage of the positive fraction is relative to isotype controls.

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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.