Mouse Embryonic Stem Cell Derivation, and Mouse and Human Embryonic Stem Cell Culture and Differentiation as Embryoid Bodies

Brock J. Conley1, Mark Denham1, Lerna Gulluyan1, Fredrik Olsson1, Timothy J. Cole1, Richard Mollard1

1 Monash University, Melbourne
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 23.2
DOI:  10.1002/0471143030.cb2302s28
Online Posting Date:  October, 2005
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Abstract

Embryonic stem (ES) cells are pluripotent cells derived from developing mouse blastocysts in vitro that maintain long‐term self renewal and the capacity to give rise to all cell types in the adult body (including some extraembryonic cell types) when subjected to the appropriate conditions. It is envisaged that the development of methods enabling controlled differentiation of mouse ES cell counterparts from human blastocysts would enable the provision of an unlimited supply of tissue for cell and tissue transplantation therapies for the repair and replacement of diseased, injured, and senescent tissue. Furthermore, derivation of mouse ES cells has allowed for the generation of thousands of gene‐targeted mouse mutants. Culture of mouse ES cells as embryoid bodies (EBs) has provided a convenient system for studying early mouse developmental processes, including several aspects of extraembryonic lineage and axis formation associated with the pre‐ and peri‐gastrulating mouse embryo. Relatively little is known regarding the corresponding development of the early human embryo due to limitations associated with the acquisition of relevant tissue material for study. The transfer of methods such as EB formation to human systems should, by association, facilitate a more advanced understanding of similar processes associated with early human development. This unit describes protocols for isolating mouse embryonic stem cells and methods for propagating, freezing, and producing EBs from both mouse and human embryonic stem cells.

Keywords: Human embryonic stem cells; mouse embryonic stem cells; mouse embryonic fibroblast feeder cells; tissue culture; embryoid body formation; vitrification

     
 
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Table of Contents

  • Basic Protocol 1: Deriving, Culturing, and Freezing Mouse Embryonic Stem Cells
  • Alternate Protocol 1: MOUSE ES Cell Culture in the Absence of Feeder Cells
  • Support Protocol 1: Preparation of Mouse Embryonic Fibroblasts
  • Support Protocol 2: Passaging and Mitotic Inactivation of MEFs by Mitomycin C
  • Support Protocol 3: Passaging and Mitotic Inactivation of MEFs by γ‐Irradiation
  • Support Protocol 4: Freezing MEFs
  • Basic Protocol 2: Mouse Embryoid Body Formation (Hanging Drop Culture)
  • Basic Protocol 3: Human Embryonic Stem Cell Propagation
  • Alternate Protocol 2: Preparation of Conditioned Medium Feeder‐Layer‐Free Culture of Human ES Cells
  • Alternate Protocol 3: Enzymatic Passage of Human ES Cells for Culture on Feeder Layers and on Matrigel
  • Basic Protocol 4: Producing Human ES Cell Embryoid Bodies by Mechanical Dissociation
  • Alternate Protocol 4: Producing Human ES Cell Embryoid Bodies by Enzymatic Digestion
  • Basic Protocol 5: Vitrification/Thawing of Human ES Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Deriving, Culturing, and Freezing Mouse Embryonic Stem Cells

  Materials
  • 3.5 days post coitum (dpc) pregnant strain 129sv mouse
  • M2 medium (Sigma), sterile
  • M16 medium (Sigma), sterile
  • Cell culture–grade distilled water (JRH Biosciences), sterile
  • Organ culture dishes treated with 0.1% (w/v) gelatin and coated with mitotically inactivated MEFs (see protocol 4 or protocol 53)
  • mES cell medium (see recipe), sterile
  • PBS, calcium‐ and magnesium‐free (CMF‐PBS; Invitrogen), sterile
  • 0.025% trypsin/0.04% EDTA (see recipe), sterile
  • Cell culture–grade distilled water (JRH Biosciences), sterile
  • 1‐ml syringe and 26‐G needle
  • Petri dishes
  • Organ culture dish (Becton Dickinson)
  • Finely drawn glass capillary pipets, 1 mm i.d.
  • Microscope
  • 25‐cm2 tissue culture flask (Becton Dickinson), optional

Alternate Protocol 1: MOUSE ES Cell Culture in the Absence of Feeder Cells

  Materials
  • 12.5 to 13.5 days post coitum (dpc) pregnant strain 129sv females
  • PBS, calcium‐ and magnesium‐free (CMF‐PBS; Invitrogen), sterile
  • 0.025% (w/v) trypsin/EDTA (see recipe), sterile
  • MEF medium (see recipe), sterile
  • 90‐mm bacteriological petri dish
  • Scalpel, sterile
  • 20‐G needle (Becton Dickinson)
  • 75‐cm2 tissue culture flasks (Becton Dickinson/Falcon)
  • Platform shaker
  • 15‐ml tubes

Support Protocol 1: Preparation of Mouse Embryonic Fibroblasts

  Materials
  • MEF cultures in 75‐cm2 culture flasks (see protocol 3)
  • MEF medium (see recipe)
  • Mitomycin C (see recipe)
  • 0.1% (w/v) gelatin (see recipe)
  • PBS, calcium‐ and magnesium‐free (CMF‐PBS; Invitrogen)
  • 0.025% (w/v) trypsin/EDTA (see recipe)
  • Cell culture–grade water (JRH Biosciences), sterile
  • Organ culture dishes
  • 15‐ml centrifuge tubes
  • Additional reagents and equipment for counting cells (unit 1.1)

Support Protocol 2: Passaging and Mitotic Inactivation of MEFs by Mitomycin C

  Materials
  • MEF cultures (for fresh MEF, see protocol 3 or for frozen MEF, see protocol 6)
  • 0.025% (w/v) trypsin/EDTA (see recipe)
  • MEF medium (see recipe)
  • Human ES (hES) cell culture medium (see recipe)
  • 75‐cm2 culture flasks
  • 50‐ml centrifuge tube
  • γ irradiator (Gammacell 1000, Nordion)
  • 150‐ and 100‐mm gelatin‐coated tissue culture dishes (optional)
  • Organ culture dishes

Support Protocol 3: Passaging and Mitotic Inactivation of MEFs by γ‐Irradiation

  Materials
  • Freezing solution (see recipe)
  • MEF cultures in 75‐cm2 tissue culture flasks (see protocol 3)
  • PBS, calcium‐ and magnesium‐free (CMF‐PBS; Invitrogen)
  • 0.05% (w/v) trypsin/EDTA (Invitrogen)
  • MEF medium (see recipe)
  • Liquid nitrogen
  • 1‐ml cryovial

Support Protocol 4: Freezing MEFs

  Materials
  • mES cell culture in the absence of a feeder layer (see protocol 2) in a 25‐cm2 tissue culture flask
  • PBS, calcium‐ and magnesium‐free (CMF‐PBS; Invitrogen)
  • 0.025% (w/v) trypsin/EDTA (see recipe)
  • mES medium (see recipe) without LIF
  • Cell culture–grade distilled water (JRH Biosciences), sterile
  • 15‐ml centrifuge tubes
  • 90‐mm bacterial culture plate
  • Additional reagents and equipment for counting cells (unit 1.1)

Basic Protocol 2: Mouse Embryoid Body Formation (Hanging Drop Culture)

  Materials
  • Mitomycin C–treated MEFs in organ culture dish (see protocol 4)
  • Human ES cell medium (see recipe)
  • Cultures of human ES cells
  • Finely drawn glass capillaries, 1.0‐mm o.d. (Clark Electromagnetic Industries)

Basic Protocol 3: Human Embryonic Stem Cell Propagation

  Materials
  • Mitotically inactivated MEFs (see protocol 4 or protocol 53)
  • MEF medium (see recipe)
  • Serum‐free medium (see recipe)
  • 0.1 mg/ml human recombinant basic fibroblast growth factor stock (hbFGF; Invitrogen): reconstitute 10 µg in 100 µl of 10 mM Tris⋅Cl, pH 7.6 ( appendix 2A), store at −20°C
  • hES cells
  • 25‐cm2 tissue culture flasks
  • 0.22‐µm filter

Alternate Protocol 2: Preparation of Conditioned Medium Feeder‐Layer‐Free Culture of Human ES Cells

  Materials
  • hES cell cultures in Matrigel‐coated 4‐well plates (see recipe for plates)
  • 1 mg/ml collagenase IV (see recipe)
  • PBS, calcium‐ and magnesium‐free (CMF‐PBS; Invitrogen)
  • Serum‐free medium (see recipe)
  • Conditioned medium (CM, see protocol 9)
  • Matrigel‐coated 4‐well plates (see recipe)
  • 15‐ml centrifuge tubes

Alternate Protocol 3: Enzymatic Passage of Human ES Cells for Culture on Feeder Layers and on Matrigel

  Materials
  • Cultures of human ES cell colonies in organ culture dishes on MEF feeder layer, 6 to 9 colonies/dish
  • Human ES cell medium (see recipe)
  • Finely drawn glass capillary (1.0‐mm o.d.)
  • 90‐mm bacteriological petri dish

Basic Protocol 4: Producing Human ES Cell Embryoid Bodies by Mechanical Dissociation

  Materials
  • Human ES cell colonies
  • Serum‐free medium (see recipe)
  • 1 mg/ml collagenase/dispase stock (see recipe)
  • Certified fetal bovine serum (Invitrogen)
  • Human ES cell medium (see recipe)
  • Finely drawn glass capillary (1.0‐mm o.d.)
  • 15‐ml centrifuge tube
  • 90‐mm bacteriological petri dish

Alternate Protocol 4: Producing Human ES Cell Embryoid Bodies by Enzymatic Digestion

  Materials
  • Bench medium (BM; see recipe)
  • Vitrification solution 1 (VS1; see recipe)
  • Vitrification solution 2 (VS2; see recipe)
  • Liquid nitrogen
  • hES cell culture
  • Warming solution 1 (WS1; see recipe)
  • Warming solution 2 (WS2; see recipe)
  • Organ culture dish seeded with mitotically inactivated MEFs (see protocol 4 or protocol 53)
  • Human ES cell medium (see recipe)
  • 4‐well tissue culture plates (NUNC)
  • 5‐ml cryovials (NUNC)
  • 18‐G needle
  • Canes for liquid nitrogen tank
  • 20‐µl micropipettor
  • Vitrification straws (LEC Instruments)
  • Forceps
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Figures

Videos

Literature Cited

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Key References
   Joyner, A.L. ed. 1993. Gene Targeting: A Practical Approach. (D. Rickwood and B.D. Hames, eds.), IRL Press, Oxford, UK.
  Protocols for gene targeting.
   Nagy, A., Gertsenstein, M., Vintersten, K., and Behringer, R. 2002. Manipulating the Mouse Embryo: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory Press, New York.
  Important work describing manipulation of mouse embryo at various stages, and culture conditions required.
   Tymms, M.J. and Kola, I. 2001. Gene Knockout Protocols. Methods in Molecular Biology, Vol. 158 Humana Press, Totowa, NJ, USA.
  Protocol for gene knockout.
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