Preparation of Autogenic Human Feeder Cells for Growth of Human Embryonic Stem Cells

Rodolfo Gonzalez1, Jeanne F. Loring2, Evan Y. Snyder1

1 Burnham Institute for Medical Research, La Jolla, California, 2 Scripps Research Institute, La Jolla, California
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1C.5
DOI:  10.1002/9780470151808.sc01c05s4
Online Posting Date:  March, 2008
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Abstract

Human embryonic stem cells (hESCs) that are currently distributed under NIH guidelines, as well as many of those that are not on the NIH registry, have been derived and maintained in coculture with growth‐arrested mouse embryonic fibroblasts (MEFs). Using this mouse support system may compromise the therapeutic potential of these hESCs because of the risk of transmitting xenopathogens. Alternatively, to reduce this risk, methods to culture undifferentiated hESCs on autologous hESC‐derived human feeder layers have now been developed. This feeder cell system derived from hESCs successfully prolongs growth of undifferentiated hESCs and eliminates risk factors and concerns about using xenogeneic or unknown allogeneic feeders. In this unit, we provide the necessary protocols for an autogeneic human feeder system that efficiently supports hESC growth and maintenance of pluripotency. Curr. Protoc. Stem Cell Biol. 4:1C.5.1‐1C.5.15. © 2008 by John Wiley & Sons, Inc.

Keywords: human embryonic stem cells; fibroblast‐like; autologous; feeders

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Stock hESC‐Derived Feeder Cells
  • Support Protocol 1: Passaging hESCs onto hESC‐Derived Feeders by Mechanical Dissociation
  • Support Protocol 2: Passaging hESCs onto hESC‐Derived Feeders by Enzymatic Dissociation
  • Support Protocol 3: Growth of hESCs under Feeder‐Free MEF‐CM Culture Conditions
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Stock hESC‐Derived Feeder Cells

  Materials
  • hESC cultures grown under feeder‐free conditions ( protocol 4) in 6‐well plates
  • MEF‐conditioned medium ( protocol 4)
  • Calcium‐ and magnesium‐free Dulbecco's phosphate‐buffered saline (CMF‐DPBS; e.g., Invitrogen or Sigma)
  • Trypsin/EDTA: 0.05% (w/v) trypsin/0.53 mM EDTA (Invitrogen)
  • Feeder medium (see recipe)
  • hESC medium (see recipe)
  • Mitomycin C medium (see recipe)
  • Freezing medium (90 % v/v FBS/10% v/v DMSO), ice cold
  • Liquid nitrogen
  • Low‐power dissecting microscope
  • Pipettor with 20‐µl sterile filter tip, or 23‐G needle
  • 15‐ml conical tubes
  • Centrifuge
  • Gelatin‐coated 75‐cm2 culture flasks (and other appropriately sized vessels as needed): add 0.1% (w/v) gelatin to dishes/flasks at 1 ml/cm2 and leave 1 hr to overnight at room temperature; just before plating the cells remove gelatin and rinse with CMF‐DPBS
  • γ irradiator (Nordion Gammacell 40 Exactor, or equivalent low‐dose 137Cs irradiator for both cells and whole animals with central dose rate of ∼1.10 Gy/min (∼110 rad/min).
  • 1‐ml cryovials
  • Isopropanol freezing container
  • Additional reagents and equipment for growing hESCs under feeder‐free growth conditions ( protocol 4)
NOTE: Volumes are described for one well of a 6‐well plate or 35‐mm tissue culture dish. They should be adjusted for other sizes of tissue culture vessels based on the relative surface area.

Support Protocol 1: Passaging hESCs onto hESC‐Derived Feeders by Mechanical Dissociation

  Materials
  • hESC cultures
  • hESC medium (see recipe)
  • Stock hESC‐derived feeder cells, irradiated or mitomycin C‐treated ( protocol 1)
  • Microscope: inverted phase‐contrast with 4×, 10×, 20× objectives
  • 15‐ml conical centrifuge tubes
  • Pipettor with 20‐µl sterile filter tip, or 23‐G needle

Support Protocol 2: Passaging hESCs onto hESC‐Derived Feeders by Enzymatic Dissociation

  Materials
  • hESC cultures
  • Calcium and magnesium‐free Dulbecco's phosphate‐buffered saline (CMF‐DPBS; e.g., Sigma or Invitrogen)
  • 200 U/ml collagenase IV (see recipe)
  • hESC medium (see recipe)
  • Stock hESC‐derived feeder cells, irradiated or mitomycin C‐treated ( protocol 1)
  • 15‐ml conical tubes

Support Protocol 3: Growth of hESCs under Feeder‐Free MEF‐CM Culture Conditions

  Materials
  • MEFs (see above), inactivated by γ irradiation or mitomycin C treatment
  • Feeder medium (see recipe)
  • hESC medium (see recipe) containing 4 ng/ml human FGF2
  • 10 µg/ml human FGF2 (see recipe)
  • Matrigel (BD Biosciences)
  • KnockOUT DMEM medium (Invitrogen)
  • 10‐cm culture dishes (∼55 cm2 area)
  • 0.2‐µm low‐protein‐binding filters
  • 1‐, 10‐, 15‐, 25‐, and 50‐ml tubes
  • 6‐well tissue culture plates, prechilled
  • Inverted phase‐contrast microscope
  • Low‐power dissecting microscope
  • Pipettor with 20‐µl sterile filter tip, or 23‐G needle
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Figures

Videos

Literature Cited

Literature Cited
   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.
   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: 131‐142.
   Choo, A.B., Padmanabhan, J., Chin, A.C., and Oh, S.K. 2004. Expansion of pluripotent human embryonic stem cells on human feeders. Biotechnol. Bioeng. 88: 321‐331.
   Genbacev, O., Krtolica, A., Zdravkovic, T., Brunette, E., Powell, S., Nath, A., Caceres, E., McMaster, M., McDonagh, S., Li, Y., Mandalam, R., Liebowski, 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.
   Lee, J.B., Song, J.M., Lee, J.E., Park, J.H., Kim, S.J., Kang, S.M., Kwon, J.N., Kim, M.K., Roh, S.I., and Yoon, H.S. 2004. Available human feeder cells for the maintenance of human embryonic stem cells. Reproduction 128: 727‐735.
   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.
   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.
   Stewart, M.H., Bosse, M., Chadwick, K., Menendez, P., Bendall, S.C., and Bhatia, M. 2006. Clonal isolation of hESCs reveals heterogeneity within the pluripotent stem cell compartment. Nat. Methods 3: 807‐815.
   Stojkovic, P., Lako, M., Przyborski, S., Stewart, R., Armstrong, L., Evans, J., Zhang, X., and Stojkovic, M. 2005. Human‐serum matrix supports undifferentiated growth of human embryonic stem cells. Stem Cells 23: 895‐902.
   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.
   Yoo, S.J., Yoon, B.S., Kim, J.M., Song, J.M., Roh, S., You, S., and Yoon, H.S. 2005. Efficient culture system for human embryonic stem cells using autologous human embryonic stem cell‐derived feeder cells. Exp. Mol. Med. 37: 399‐407.
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