Isolation of Human Placenta‐Derived Multipotent Cells and In Vitro Differentiation into Hepatocyte‐Like Cells

Hsing‐I Huang1

1 Cathay General Hospital, Taipei
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1E.1
DOI:  10.1002/9780470151808.sc01e01s1
Online Posting Date:  June, 2007
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Abstract

Several types of progenitor cells can be isolated from various human adult tissues such as bone marrow, adipose tissues, and umbilical cord. Placental tissue collected after labor and delivery can provide a valuable source for adult stem cells. These progenitor cells, termed placenta‐derived multipotent cells (PDMCs), are fibroblast‐like cells which can attach on the bottom of culture vessels. PDMCs are capable of differentiating into various cells such as adipocytes, osteoblasts, chondrocytes, and neurons. Recently, we showed that PDMCs also possess the ability to differentiate into hepatocyte‐like cells. This unit describes the protocols for isolation of PDMCs from human term placental tissue and for setting up in vitro differentiation of PDMCs toward hepatocyte‐like cells. These cells not only express the characteristics of human liver cells, but also demonstrate several functions of typical hepatocytes. Curr. Protoc. Stem Cell Biol. 1:1E.1.1‐1E.1.9. © 2007 by John Wiley & Sons, Inc.

Keywords: placenta; hepatocytes; differentiation; isolation; multipotent progenitors

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of PDMCs from Human Placenta
  • Basic Protocol 2: Hepatic Differentiation of PDMCs in Vitro
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation of PDMCs from Human Placenta

  Materials
  • Donor for term placenta
  • Expansion medium (see recipe), prewarmed before use
  • Dulbecco's phosphate‐buffered saline without calcium or magnesium (CMF‐DPBS; Invitrogen, cat. no. 21600)
  • 70% (v/v) ethanol
  • Trypsin/EDTA solution: 0.5% (w/v) trypsin/0.5 mM EDTA (Invitrogen, cat. no. 15400)
  • 100‐mm culture dishes
  • 15‐ml polypropylene centrifuge tubes
  • Tweezers, sterile
  • Scissors, sterile
  • Centrifuge
  • 25‐cm2 tissue culture flasks
  • Inverted microscope
  • Additional reagents and equipment for counting cells (Phelan, )

Basic Protocol 2: Hepatic Differentiation of PDMCs in Vitro

  Materials
  • 0.01% (w/v) poly‐L‐lysine solution (mol. wt. 70,000 to 150,000; Sigma‐Aldrich), filter sterilized
  • Dulbecco's phosphate‐buffered saline without calcium or magnesium (CMF‐DPBS; Invitrogen, cat. no. 21600)
  • Culture of PDMCs, 80% to 90% confluent ( protocol 1)
  • Trypsin/EDTA solution: 0.5% trypsin/0.5% mM EDTA (Invitrogen, cat. no. 15400)
  • Expansion medium (see recipe)
  • Medium A (see recipe)
  • Medium B (see recipe)
  • 6‐well tissue culture plates or 35‐mm tissue culture dishes
  • 15‐ml centrifuge tubes
  • Centrifuge
  • Additional reagents and equipment for counting viable cell (Phelan, )
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Figures

Videos

Literature Cited

Literature Cited
   Chien, C.C., Yen, B.L., Lee, F.K., Lai, T.H., Chen, Y.C., Chan, S.H., and Huang, H.I. 2006. In vitro differentiation of human placenta‐derived multipotent cells into hepatocyte‐like cells. Stem Cells 24:1759‐1768.
   Evarts, R.P., Nagy, P., Marsden, E., and Thorgeirsson, S.S. 1987. A precursor‐product relationship exists between oval cells and hepatocytes in rat liver. Carcinogenesis 8:1737‐1740.
   In't Anker, P.S., Scherjon, S.A., Kleijburg‐van der Keur, C., de Groot‐Swings, G.M., Claas, F.H., Fibbe, W.E., and Kanhai, H.H. 2004. Isolation of mesenchymal stem cells of fetal or maternal origin from human placenta. Stem Cells 22:1338‐1345.
   Lee, K.D., Kuo, T.K., Whang‐Peng, J., Chung, Y.F., Lin, C.T., Chou, S.H., Chen, J.R., Chen, Y.P., and Lee, O.K. 2004. In vitro hepatic differentiation of human mesenchymal stem cells. Hepatology 40:1275‐1284.
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   Kubota, H., Storms, R.W., and Reid, L.M. 2002. Variant forms of alpha‐fetoprotein transcripts expressed in human hematopoietic progenitors: Implications for their developmental potential towards endoderm. J. Biol. Chem. 277:27629‐27635.
   Petersen, B.E., Bowen, W.C., Patrene, K.D., Mars, W.M., Sullivan, A.K., Murase, N., Boggs, S.S., Greenberger, J.S., and Goff, J.P. 1999. Bone marrow as a potential source of hepatic oval cells. Science 284:1168‐1170.
   Phelan, M.C. 2006. Techniques for mammalian cell tissue culture. Curr. Protoc. Mol. Biol. 74:A.3F.1‐A.3F.8.
   Schwartz, R.E., Reyes, M., Koodie, L., Jiang, Y., Blackstad, M., Lund, T., Lenvik, T., Johnson, S., Hu, W.S., and Verfaille, C.M. 2002. Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte‐like cells. J. Clin. Invest. 109:1291‐1302.
   Seo, M.J., Suh, S.Y., Bae, Y.C., and Jung, J.S. 2005. Differentiation of human adipose stromal cells into hepatic lineage in vitro and in vivo. Biochem. Biophys. Res. Commun. 328:258‐264.
   Suzuki, A., Zheng, Y.W., Fukao, K., Nakauchi, H., and Taniguchi, H. 2004. Liver repopulation by c‐Met positive stem/progenitor cells isolated from the developing rat liver. Hepatogastroenterology 51:423‐426.
   von Mach, M.A., Hengstler, J.G., Brulport, M., Eberhardt, M., Schormann, W., Hermes, M., Prawitt, D., Zabel, B., Grosche, J., Reichenbach, A., Muller, B., Weilemann, L.S., and Zulewski, H. 2004. In vitro cultured islet‐derived progenitor cells of human origin express human albumin in severe combined immunodeficiency mouse liver in vivo. Stem Cells 22:1134‐1141.
   Yen, B.L., Huang, H.I., Chien, C.C., Jui, H.Y., Ko, B.S., Yao, M., Shun, C.T., Yen, M.L., Lee, M.C., and Chen, Y.C. 2005. Isolation of multipotent cells from human term placenta. Stem Cells 23:3‐9.
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