Isolation of Human Placental Fibroblasts

Dusko Ilic1, Mirhan Kapidzic2, Olga Genbacev2

1 StemLifeLine, San Carlos, California, 2 University of California, San Francisco, California
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1C.6
DOI:  10.1002/9780470151808.sc01c06s5
Online Posting Date:  June, 2008
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Abstract

The first human embryonic stem cell lines (hESCs) were derived using mouse embryonic fibroblasts as feeder cells. In attempts to replace mouse embryonic fibroblasts with feeders of human origin, irradiated human placental fibroblasts were successfully used as feeder cells for the derivation and propagation of hESCs. Here we describe a protocol for the isolation and expansion of fibroblasts from placental villous stroma. We include a description of placental architecture to provide the background for a stepwise tissue digestion that leads to the isolation of villous stroma. Villous stroma from the first trimester tissue is different from term placenta and contains mesenchymal, fibroblast‐like cells, only a few blood vessels, and a network of matrix fibers. The fibroblasts isolated from a single placenta of 6‐ to 8‐weeks gestation proliferate rapidly and retain the ability to support hESC growth between passage doubling (PD) 8 and PD 12. Curr. Protoc. Stem Cell Biol. 5:1C.6.1‐1C.6.17. © 2008 by John Wiley & Sons, Inc.

Keywords: placenta; fibroblasts; human feeders; human embryonic stem cells

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Collection of Placental Tissue and Isolation of Placental Fibroblasts
  • Support Protocol 1: Dressing for Collection and Isolation of Placental Fibroblasts
  • Basic Protocol 2: Expanding and Freezing Placental Fibroblasts
  • Support Protocol 2: Counting Cells Using a Coulter Counter
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Collection of Placental Tissue and Isolation of Placental Fibroblasts

  Materials
  • Placental tissue
  • Phosphate‐buffered saline (PBS), Ca2+‐, Mg2+‐free (CMF‐PBS; Invitrogen, cat. no. 14190‐144)
  • Cytowash medium (see recipe)
  • Ice bucket or Styrofoam box filled with ice
  • 70% ethanol (Fisher Scientific, cat. no. 254670–32)
  • Collagenase solution (see recipe)
  • Trypsin solution (see recipe)
  • Fetal bovine serum (Hyclone, cat. no. SH30071.03)
  • Fibroblast culture medium (see recipe)
  • Full height sieves, stainless steel wire and frame, Tyler (VWR Lab Shop, cat. no. 57324‐181), 1‐mm mesh opening
  • 15‐cm petri dish
  • White Light Transilluminator (Fisher Scientific; Fisher Biotech, cat. no. FB‐WLT‐1417)
  • Sterile surgical instruments:
    • Forceps (Fisher, cat. no. 10275)
    • Dumont tweezers, high precision, 120 mm, stainless steel (Ted Pella, cat. no. 5617)
    • General laboratory scissors, curved with sharp points, 110 mm, stainless steel (Ted Pella, cat. no. 1332)
    • Vannas‐type Micro Scissors, straight, 80 mm, stainless steel (Ted Pella, cat. no. 1346)
  • 50‐ml polypropylene conical tube (BD Falcon, cat. no. 352098)
  • 37°C shaking water bath
  • Centrifuge
  • 145 × 20–mm cell culture dish (USA Scientific, cat. no. 5663‐9160)
  • 100‐ml heat‐resistant glass bottle (Corning, cat. no. X1395), sterile
  • Medical sterile gauze sponges, 2 × 2–in. (Kendall Curity Gauze Sponge, cat. no. 1806)
  • 100‐µm cell strainer (Fisher Scientific, cat. no. 08‐771‐19), sterile
  • 25‐cm2 flasks
  • Additional reagents and equipment for dressing for collection and isolation of placental fibroblasts ( protocol 2) and counting cells ( protocol 4)
NOTE: One set of sterile instruments and labware listed above is required for each placenta processed. Catalog numbers for the surgical instruments and labware are given just as examples—instruments and labware of the equivalent quality can be purchased from other manufacturers without altering the outcome of the procedure.

Support Protocol 1: Dressing for Collection and Isolation of Placental Fibroblasts

  Materials
  • Antibacterial soap
  • Sterile surgical gown (VWR, cat. no. 10845–016)
  • Shoe covers (Fisher Scientific, cat. no. 19–098–921)
  • Hair cover (Fisher Scientific, cat. no. 17–981–43A)
  • Protective glasses (Fisher Scientific, cat. no. 19–130–2088)
  • Surgical face‐mask (Fisher Scientific, cat. no. 18–096B)
  • Sterile surgical gloves (Fisher Scientific, cat. no. 18–999–2668)
NOTE: Catalog numbers for the protective clothing are given just as examples—protective clothing of the equivalent quality can be purchased from other manufacturers without altering the outcome of the procedure.

Basic Protocol 2: Expanding and Freezing Placental Fibroblasts

  Materials
  • Cultures of human placental fibroblasts in 25‐ or 75‐cm2 cell culture flasks ( protocol 1)
  • Phosphate‐buffered saline (PBS), Ca2+‐, Mg2+‐free (CMF‐PBS; Invitrogen, cat. no. 14190‐144)
  • 0.05% Trypsin/0.53 mM EDTA (Invitrogen, cat. no. 25300‐054)
  • Fibroblast culture medium (see recipe)
  • Fibroblast freezing medium (see recipe)
  • Liquid nitrogen
  • Inverted microscope
  • 5‐ml pipet
  • 25‐cm2 and 75‐cm2 cell culture flasks
  • Cryovials
  • Additional reagents and equipment for cell counting ( protocol 4) and mycoplasma testing (Harlin and Gajewski, )

Support Protocol 2: Counting Cells Using a Coulter Counter

  Materials
  • Diluent (Beckman Coulter)
  • Cleaning agent (e.g., Clenz; Beckman)
  • Cells to be counted (Basic Protocols protocol 11 and protocol 32)
  • Z1 Particle Counter (Beckman Coulter)
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Figures

Videos

Literature Cited

Literature Cited
   Amenta, P.S., Gay, S., Vaheri, A., and Martinez‐Hernandez, A. 1986. The extracellular matrix is an integrated unit: Ultrastructural localization of collagen types I, III, IV, V, VI, fibronectin, and laminin in human term placenta. Coll. Relat. Res. 6: 125‐152.
   Amit, M., Margulets, V., Segev, H., Shariki, K., Laevsky, I., Coleman, R., and Itskovitz‐Eldor, J. 2003. Human feeder layers for human embryonic stem cells. Biol. Reprod. 68: 2150‐2156.
   Autio‐Harmainen, H., Sandberg, M., Pihlajaniemi, T., and Vuorio, E. 1991. Synthesis of laminin and type IV collagen by trophoblastic cells and fibroblastic stromal cells in the early human placenta. Lab. Invest. 64: 483‐491.
   Boyd, J.D. and Hamilton, W.J. 1967. Development and structure of the human placenta from the end of the 3rd month of gestation. J. Obstet. Gynaecol. Br. Commonw. 74: 161‐226.
   Brosens, I. and Dixon, H.G. 1966. The anatomy of the maternal side of the placenta. J. Obstet. Gynaecol. Br. Commonw. 73: 357‐363.
   Castellucci, M. and Kaufmann, P. 1982a. A three‐dimensional study of the normal human placental villous core: II. Stromal architecture. Placenta 3: 269‐285.
   Castellucci, M. and Kaufmann, P. 1982b. Evolution of the stroma in human chorionic villi throughout pregnancy. Bibl Anat. 22: 40‐45.
   Castellucci, M., Zaccheo, D., and Pescetto, G. 1980. A three‐dimensional study of the normal human placental villous core. I. The Hofbauer cells. Cell Tissue Res. 210: 235‐247.
   Castellucci, M., Schweikhart, G., Kaufmann, P., and Zaccheo, D. 1984. The stromal architecture of the immature intermediate villus of the human placenta. Functional and clinical implications. Gynecol. Obstet. Invest. 18: 95‐99.
   Castellucci, M., Classen‐Linke, I., Muhlhauser, J., Kaufmann, P., Zardi, L., and Chiquet‐Ehrismann, R. 1991. The human placenta: A model for tenascin expression. Histochemistry 95: 449‐458.
   Cheng, L., Hammond, H., Ye, Z., Zhan, X., and Dravid, G. 2003. Human adult marrow cells support prolonged expansion of human embryonic stem cells in culture. Stem Cells 21: 131‐42.
   Cross, J.C., Werb, Z., and Fisher, S.J. 1994. Implantation and the placenta: Key pieces of the development puzzle. Science 266: 1508‐1518.
   Demir, R., Kosanke, G., Kohnen, G., Kertschanska, S., and Kaufmann, P. 1997. Classification of human placental stem villi: Review of structural and functional aspects. Microsc. Res. Tech. 38: 29‐41.
   Earl, U., Estlin, C., and Bulmer, J.N. 1990. Fibronectin and laminin in the early human placenta. Placenta 11: 223‐231.
   Frank, H.G., Malekzadeh, F., Kertschanska, S., Crescimanno, C., Castellucci, M., Lang, I., Desoye, G., and Kaufmann, P. 1994. Immunohistochemistry of two different types of placental fibrinoid. Acta Anat. (Basel) 150: 55‐68.
   Genbacev, O., Krtolica, A., Zdravkovic, T., Brunette, E., Powell, S., Nath, A., Caceres, E., McMaster, M., McDonagh, S., Li, Y., Mandalam, R., Lebkowski, J., and Fisher, S.J. 2005. Serum‐free derivation of human embryonic stem cell lines on human placental fibroblast feeders. Fertil. Steril. 83: 1517‐1529.
   Harlin, H. and Gajewski, T.F. 2005. Diagnosis and treatment of mycoplasma‐contaminated cell cultures. Curr. Protoc. Microbiol. 0: A.3B.1‐A.3B.6.
   Hovatta, O., Mikkola, M., Gertow, K., Stromberg, A.M., Inzunza, J., Hreinsson, J., Rozell, B., Blennow, E., Andang, M., and Ahrlund‐Richter, L. 2003. A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum. Reprod. 18: 1404‐1409.
   Kaufmann, P., Stark, J., and Stegner, H.E. 1977. The villous stroma of the human placenta. I. The ultrastructure of fixed connective tissue cells. Cell Tissue Res. 177: 105‐121.
   King, B.F. 1987. Ultrastructural differentiation of stromal and vascular components in early macaque placental villi. Am. J. Anat. 178: 30‐44.
   Kohnen, G., Kertschanska, S., Demir, R., and Kaufmann, P. 1996. Placental villous stroma as a model system for myofibroblast differentiation. Histochem. Cell Biol. 105: 415‐429.
   Kohnen, G., Mackenzie, F., Collett, G.F., Campbell, S., Davenport, A.P., Cameron, A.D., and Cameron. I.T. 1997. Differential distribution of endothelin receptor subtypes in placentae from normal and growth‐restricted pregnancies. Placenta 18: 173‐180.
   Lee, J.B., Lee, J.E., Park, J.H., Kim, S.J., Kim, M/K., Roh, S.I., and Yoon, H.S. 2005. Establishment and maintenance of human embryonic stem cell lines on human feeder cells derived from uterine endometrium under serum‐free condition. Biol. Reprod. 72: 42‐49.
   Martinoli, C., Castellucci, M., Zaccheo, D., and Kaufmann, P. 1984. Scanning electron microscopy of stromal cells of human placental villi throughout pregnancy. Cell Tissue Res. 235: 647‐655.
   Muhlhauser, J., Marzioni, D., Morroni, M., Vuckovic, M., Crescimanno, C., and Castellucci, M. 1996. Codistribution of basic fibroblast growth factor and heparan sulfate proteoglycan in the growth zones of the human placenta. Cell Tissue Res. 285: 101‐107.
   Nanaev, A.K., Rukosuev, V.S., Shirinsky, V.P., Milovanov, A.P., Domogatsky, S.P., Duance, V.C., Bradbury, F.M., Yarrow, P., Gardiner, L., d'Lacey, C., and et al. 1991. Confocal and conventional immunofluorescent and immunogold electron microscopic localization of collagen types III and IV in human placenta. Placenta 12: 573‐595.
   Pfister, C., Scheuner, G., Bahn, H., and Stiller, D. 1988. Immunohistochemical demonstration of fibronectin in the human placenta. Acta Histochem. 84: 83‐91.
   Reubinoff, B.E., Pera, M.F., Fong, C.Y., Trounson, A., and Bongso, A. 2000. Embryonic stem cell lines from human blastocysts: Somatic differentiation in vitro. Nat. Biotechnol. 18: 399‐404.
   Richards, M., Fong, C.Y., Chan, W.K., Wong, P.C., and Bongso, A. 2002. Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat. Biotechnol. 20: 933‐936.
   Richards, M., Tan, S., Fong, C.Y., Biswas, A., Chan, W.K., and Bongso, A. 2003. Comparative evaluation of various human feeders for prolonged undifferentiated growth of human embryonic stem cells. Stem Cells 21: 546‐556.
   Rukosuev, V.S. 1992. Immunofluorescent localization of collagen types I, III, IV, V, fibronectin, laminin, entactin, and heparan sulphate proteoglycan in human immature placenta. Experientia 48: 285‐287.
   Simon, C., Escobedo, C., Valbuena, D., Genbacev, O., Galan, A., Krtolica, A., Asensi, A., Sanchez, E., Esplugues, J., Fisher, S., and Pellicer, A. 2005. First derivation in Spain of human embryonic stem cell lines: Use of long‐term cryopreserved embryos and animal‐free conditions. Fertil. Steril. 83: 246‐249.
   Stojkovic, P., Lako, M., Stewart, R., Przyborski, S., Armstrong, L., Evans, J., Murdoch, A., Strachan, T., and Stojkovic, M. 2005. An autogeneic feeder cell system that efficiently supports growth of undifferentiated human embryonic stem cells. Stem Cells 23: 306‐314.
   Thomson, J.A., Itskovitz‐Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., and Jones, J.M. 1998. Embryonic stem cell lines derived from human blastocysts. Science 282: 1145‐1147.
   Virtanen, I., Laitinen, L., and Vartio, T. 1988. Differential expression of the extra domain‐containing form of cellular fibronectin in human placentas at different stages of maturation. Histochemistry 90: 25‐30.
   Wang, Q., Fang, Z.F., Jin, F., Lu, Y., Gai, H., and Sheng, H.Z. 2005. Derivation and growing human embryonic stem cells on feeders derived from themselves. Stem Cells 23: 1221‐1227.
   Xu, C., Jiang, J., Sottile, V., McWhir, J., Lebkowski, J., and Carpenter, M.K. 2004. Immortalized fibroblast‐like cells derived from human embryonic stem cells support undifferentiated cell growth. Stem Cells 22: 972‐980.
   Yamada, T., Isemura, M., Yamaguchi, Y., Munakata, H., Hayashi, N., and Kyogoku, M. 1987. Immunohistochemical localization of fibronectin in the human placentas at their different stages of maturation. Histochemistry 86: 579‐584.
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