Preparation of Hematopoietic Stem and Progenitor Cells from the Human Placenta

Catherine Robin1, Elaine Dzierzak1

1 Erasmus MC Stem Cell Institute, Department of Cell Biology, Rotterdam, The Netherlands
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2A.9
DOI:  10.1002/9780470151808.sc02a09s14
Online Posting Date:  August, 2010
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This unit describes a protocol to isolate hematopoietic progenitors and stem cells from human placentae isolated at different time points in development and at the full‐term gestational stage. The placenta is extensively washed to eliminate blood contamination on its surface and inside the villi (the vascular compartments of the placenta). The placenta is then mechanically minced into pieces, which are subsequently digested with an enzyme cocktail. After dissociation and filtration, placental cells are available for further phenotypic and functional analyses. Curr. Protoc. Stem Cell Biol. 14:2A.9.1‐2A.9.8. © 2010 by John Wiley & Sons, Inc.

Keywords: human placenta; enzymatic treatment; hematopoietic stem and progenitor cell isolation

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Table of Contents

  • Introduction
  • Basic Protocol 1: Mechanical Dissociation of Human Placenta
  • Basic Protocol 2: Enzymatic Dissociation of Human Placenta
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Mechanical Dissociation of Human Placenta

  • Human placenta
  • PBS supplemented with EDTA (PBS/EDTA; see recipe)
  • PBS supplemented with 10% (v/v) fetal bovine serum (PBS/FBS; see recipe)
  • Ficoll
  • Collagenase (use at 0.125% (w/v) after dilution 1/20 in PBS/FBS; see recipe)
  • 50‐ml collection tubes
  • 10‐ml plastic pipet
  • Absorbent paper
  • Large stainless steel trays (to hold the placenta and fluids during wash procedure)
  • 50‐ml syringe
  • 18‐G needles
  • Clamp
  • Large glass or plastic petri dishes (20‐cm diameter)
  • Cutting board
  • Carving knives
  • Forceps

Basic Protocol 2: Enzymatic Dissociation of Human Placenta

  • Placenta pieces ( protocol 1)
  • Enzyme cocktail (see Table 2.9.1
  • PBS/FBS (see recipe)
  • Ficoll
  • 50‐ml tubes
  • Parafilm
  • 37°C water bath with shaking
  • 10‐ml plastic pipet
  • Sterile cotton gauze placed in a stainless steel soup strainer on top of a sterile glass beaker (500 ml)
  • Cell strainer (40‐µm Nylon)
    Table 2.0.1   MaterialsPreparation of the Enzymatic Cocktail for the Digestion of Placenta Pieces

    Volume of each reagent (ml) a
    Tissue (g) Collagenase Pancreatin Dispase DNase I PBS/FBS Final volume
    50 10 24 13.3 2 150.7 200

     aSee the Reagents and Solutions section for the solution recipes.
NOTE: Keep all cells at 4°C and carry out all procedures except Ficoll separation at 4°C.
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Literature Cited

Literature Cited
   Alvarez‐Silva, M., Belo‐Diabangouaya, P., Salaun, J., and Dieterlen‐Lievre, F. 2003. Mouse placenta is a major hematopoietic organ. Development 130:5437‐5444.
   Barcena, A., Kapidzic, M., Muench, M.O., Gormley, M., Scott, M.A., Weier, J.F., Ferlatte, C., and Fisher, S.J. 2009a. The human placenta is a hematopoietic organ during the embryonic and fetal periods of development. Dev. Biol. 327:24‐33.
   Barcena, A., Muench, M.O., Kapidzic, M., and Fisher, S.J. 2009b. A new role for the human placenta as a hematopoietic site throughout gestation. Reprod. Sci. 16:178‐187.
   Challier, J.C., Dubernard., G., Galtier, M., Bintein, T., Vervelle, C., Raison, D., Espié, M.J., and Uzan, S. 2005. Immunocytological evidence for hematopoiesis in the early human placenta. Placenta 26:282‐288.
   Corbel, C., Salaun, J., Belo‐Diabangouaya, P., and Dieterlen‐Lievre, F. 2007. Hematopoietic potential of the pre‐fusion allantois. Devel. Biol. 301:478‐488.
   Gekas, C., Dieterlen‐Lievre, F., Orkin, S.H., and Mikkola, H.K. 2005. The placenta is a niche for hematopoietic stem cells. Dev. Cell 8:365‐375.
   Gude, N.M., Roberts, C.T., Kalionis, B., and King, R.G. 2004. Growth and function of the normal human placenta. Thromb. Res. 114:397‐407.
   Kim, S.J., Song, J.H., Sung, H.J., Yoo, Y.D., Geum, D.H., Park, S.H., Yoo, J.H., Oh, J.H., Shin, H.J., Kim, S.H., Kim, J.S., and Kim, B.S. 2007. Human placenta‐derived feeders support prolonged undifferentiated propagation of a human embryonic stem cell line, SNUhES3: Comparison with human bone marrow‐derived feeders. Stem Cells Dev. 16:421‐428.
   Miyamoto, K., Hayashi, K., Suzuki, T., Ichihara, S., Yamada, T., Kao, Y., Yamabe, T., and Ito, Y. 2004. Human placenta feeder layers support undifferentiated growth of primate embryonic stem cells. Stem Cells 22:433‐440.
   Ottersbach, K. and Dzierzak, E. 2005. The murine placenta contains hematopoietic stem cells within the vascular labyrinth region. Dev. Cell 8:377‐387.
   Rhodes, K.E., Gekas, C., Wang, Y., Lux, C.T., Francis, C.S., Chan, D.N., Conway, S., Orkin, S.H., Yoder, M.C., and Mikkola, H.K. 2008. The emergence of hematopoietic stem cells is initiated in the placental vasculature in the absence of circulation. Cell Stem Cell 2:252‐263.
   Robin, C., Bollerot, K., Mendes, S., Haak, E., Crisan, M., Cerisoli, F., Lauw, I., Kaimakis, P., Jorna, R., Vermeulin, M., Kayser, M., van der Linden, R., Imanirad, V., Verstegen, M., Nawaz‐Jousef, H., Papazian, N., Steegers, E., Cupedo, T., and Dzierzak, E. 2009. Human placenta is a potent hematopoietic niche containing hematopoietic stem and progenitor cells throughout development. Cell Stem Cell 5:385‐395.
   Tavian, M. and Peault, B. 2005. Embryonic development of the human hematopoietic system. Int. J. Devel. Biol. 49:243‐250.
   Tavian, M., Robin, C., Coulombel, L., and Peault, B. 2001. The human embryo, but not its yolk sac, generates lympho‐myeloid stem cells: mapping multipotent hematopoietic cell fate in intraembryonic mesoderm. Immunity 15:487‐495.
   Zeigler, B.M., Sugiyama, D., Chen, M., Guo, Y., Downs, K.M., and Speck, N.A. 2006. The allantois and chorion, when isolated before circulation or chorio‐allantoic fusion, have hematopoietic potential. Development 133:4183‐4192.
   Zhang, Y., Li, C., Jiang, X., Zhang, S., Wu, Y., Liu, B., Tang, P., and Mao, N. 2004. Human placenta‐derived mesenchymal progenitor cells support culture expansion of long‐term culture‐initiating cells from cord blood CD34+ cells. Exp. Hematol. 32:657‐664.
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