Isolation and Manipulation of Mouse Trophoblast Stem Cells

Koji Hayakawa1, Emi Himeno1, Satoshi Tanaka1, Tilo Kunath2

1 Laboratory of Cellular Biochemistry, Animal Resource Sciences/Veterinary Medical Sciences, The University of Tokyo, Tokyo, 2 MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, University of Edinburgh, Edinburgh
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1E.4
DOI:  10.1002/9780470151808.sc01e04s32
Online Posting Date:  February, 2015
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Abstract

The isolation of stable trophoblast stem (TS) cell lines from early mouse embryos has provided a useful cell culture model to study trophoblast development. TS cells are derived from pre‐implantation blastocysts or from the extraembryonic ectoderm of early post‐implantation embryos. The derivation and maintenance of mouse TS cells is dependent upon continuous fibroblast growth factor (FGF) signaling. Gene expression analysis, differentiation in culture, and chimera formation show that TS cells accurately model the mouse trophoblast lineage. This unit describes how to derive, maintain, and manipulate TS cells, including DNA transfection and chimera formation. © 2015 by John Wiley & Sons, Inc.

Keywords: trophoblast stem cells; TS cells; extraembryonic ectoderm; trophectoderm; trophoblast; FGF4

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Isolation of Trophoblast Stem Cells from Blastocysts
  • Alternate Protocol 1: Isolation of Trophoblast Stem Cells from Post‐Implantation Embryos
  • Support Protocol 1: Mitomycin C Treatment of Mouse Embryonic Fibroblasts
  • Support Protocol 2: Preparation of MEF‐Conditioned Medium
  • Basic Protocol 2: Maintenance of TS Cells
  • Support Protocol 3: Removing MMC‐MEFs from TS Cell Cultures
  • Support Protocol 4: Freezing TS Cells
  • Support Protocol 5: Thawing TS Cells
  • Genetic Manipulation of TS Cells
  • Basic Protocol 3: DNA Transfection with jetPRIME
  • Alternate Protocol 2: DNA Transfection with Lipofectamine
  • Alternate Protocol 3: Transfection Using the NEON Transfection System
  • Alternate Protocol 4: Nucleofection of TS Cells
  • Alternate Protocol 5: Bio‐Rad Electroporation of TS Cells
  • Support Protocol 6: Establishing Stable TS Lines
  • Basic Protocol 4: Generation of TS Cell Chimeras
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Trophoblast Stem Cells from Blastocysts

  Materials
  • Mitomycin C–treated MEFs (MMC‐MEFs; see protocol 3)
  • TS medium (see recipe)
  • TS + F4H medium (see recipe)
  • 3.5 days post‐coitum (dpc) mice
  • M2 medium (Sigma, cat. no. M7167)
  • KSOM medium (optional)
  • Acid Tyrode's (optional)
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS; Invitrogen)
  • 0.1% (w/v) trypsin/1 mM EDTA (see recipe)
  • 70CM + 1.5× F4H medium (see recipe and protocol 4)
  • TS + 1.5× F4H medium (see recipe)
  • 4‐well and 6‐well tissue culture plates
  • 100‐mm petri dishes
  • 1‐ml syringes and 26‐G needles
  • Dissecting microscope
  • Finely‐drawn mouth (Pasteur) pipets with tubing and mouthpiece
  • Inverted microscope
  • Organ culture dish (optional)
  • 20‐μl and 200‐μl adjustable pipets
  • U‐bottom 96‐well nontissue culture plates
  • 35‐mm tissue culture dishes (optional)
  • Additional reagents and equipment for euthanasia of mice (Donovan and Brown, )

Alternate Protocol 1: Isolation of Trophoblast Stem Cells from Post‐Implantation Embryos

  Materials
  • Mitomycin C‐treated MEFs (MMC‐MEFs; see protocol 3)
  • 0.5% (w/v) trypsin/2.5% (w/v) pancreatin (see recipe)
  • Ice
  • 6.5 dpc mice
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS; Invitrogen)
  • CMF‐PBS/10% (v/v) FBS
  • 0.1% (w/v) trypsin/1 mM EDTA (see recipe)
  • TS + F4H medium (see recipe)
  • 60‐mm Center‐well Organ Culture Dish (BD Falcon, cat. no. 353037)
  • 100‐mm petri dishes
  • 200‐μl pipet tips
  • Fine forceps
  • Dissecting microscope
  • Glass needles or 29‐G needles
  • Finely drawn mouth (Pasteur) pipets with tubing and mouthpiece
  • U‐bottom 96‐well nontissue culture plates
  • Inverted microscope

Support Protocol 1: Mitomycin C Treatment of Mouse Embryonic Fibroblasts

  Materials
  • Mouse embryonic fibroblasts (MEFs), frozen
  • DMEM/10% FBS (see recipe)
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS; Invitrogen)
  • 0.05% (w/v) trypsin/1 mM EDTA (see recipe)
  • Mitomycin C (MMC; see recipe)
  • 2× freezing medium for MEFs (see recipe)
  • 37°C water bath
  • 50‐ml centrifuge tubes
  • 150‐mm tissue culture dishes
  • Freezing vials
  • Cell‐freezing container (e.g., 5100 Cryo 1°C Freezing Container, Nalgene)
  • Liquid nitrogen tank
  • Additional reagents and equipment for performing a viable cell count (Michalska, ; unit )

Support Protocol 2: Preparation of MEF‐Conditioned Medium

  Materials
  • Mitomycin C–treated MEFs (MMC‐MEFs; see protocol 3)
  • DMEM/10% FBS (see recipe)
  • TS medium (see recipe)
  • 37°C water bath
  • 50‐ml centrifuge tubes
  • 100‐mm dishes or 150‐mm dishes
  • 0.22‐μm filter unit for a glass bottle (Millipore)
  • Glass fiber prefilter (Millipore)
  • 500‐ml glass bottles, autoclaved

Basic Protocol 2: Maintenance of TS Cells

  Materials
  • TS cells in culture
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS; Invitrogen)
  • 0.05% (w/v) trypsin/1 mM EDTA (see recipe)
  • TS medium (see recipe)
  • TS medium + F4H (see recipe)
  • 70CM + F4H medium (optional; see recipe)
  • 50‐ml centrifuge tubes
  • Cell culture dishes (see Table 1.4.1 for sizes)

Support Protocol 3: Removing MMC‐MEFs from TS Cell Cultures

  Materials
  • Cultures of TS cells
  • Mitomycin C–treated MEFs (MMC‐MEFs; see protocol 3)
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS; Invitrogen)
  • 0.05% (w/v) trypsin/1 mM EDTA (see recipe)
  • TS medium (see recipe)
  • 70CM + F4H medium (see recipe)
  • TS + F4H medium (see recipe)
  • 100‐mm cell culture dishes
  • 50‐ml centrifuge tubes
  • Additional reagents and equipment for performing a viable cell count (Michalska, ; unit )

Support Protocol 4: Freezing TS Cells

  Materials
  • 2× freezing medium for TS cells (see recipe)
  • TS cell cultures
  • TS medium (see recipe)
  • 1‐ml cryovials
  • Cell‐freezing container (e.g., 5100 Cryo 1°C Freezing Container, Nalgene)
  • –80°C freezer
  • Liquid nitrogen tank
  • Additional reagents and equipment for trypsinization and pelleting of cells ( protocol 5)

Support Protocol 5: Thawing TS Cells

  Materials
  • Mitomycin C–treated MEFs (MMC‐MEFs; see protocol 3)
  • Vials of frozen TS cells
  • TS medium (see recipe)
  • TS + F4H medium (see recipe)
  • 37°C water bath
  • 50‐ml centrifuge tubes
  • 100‐mm cell culture dishes

Basic Protocol 3: DNA Transfection with jetPRIME

  Materials
  • TS cells
  • 70CM + F4H medium (see recipe)
  • Plasmid DNA (1 μg/μl) in sterile H 2O (linearize plasmid for stable lines)
  • jetPRIME reagent (Polyplus transfection, cat no. 114‐15)
  • jetPRIME buffer (included in jetPRIME package)
  • 35‐mm culture dishes
  • 1.5‐ml microcentrifuge tubes
  • Vortex mixer
  • Centrifuge
  • 37°C, 5% CO 2/95% air incubator

Alternate Protocol 2: DNA Transfection with Lipofectamine

  Materials
  • TS cells
  • Mitomycin C–treated MEFs (MMC‐MEFs; protocol 3)
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS; Invitrogen)
  • 0.05% (w/v) trypsin/1 mM EDTA (see recipe)
  • TS medium (see recipe)
  • TS medium + 1.5× F4H (see recipe)
  • 70CM + 1.5× F4H medium (see recipe)
  • Plasmid DNA (4 μg) in 10 to 20 μl sterile H 2O (linearize plasmid for stable lines)
  • Opti‐MEM (Invitrogen)
  • Lipofectamine 2000 (Invitrogen)
  • 1 mM EDTA/CMF‐PBS (see recipe)
  • 50‐ml tubes
  • 1.5‐ml microcentrifuge tubes
  • 35‐mm petri dishes or 6‐well non‐tissue culture plates
  • 100‐mm culture dishes
  • Additional reagents and equipment for counting viable cells (Michalska, ; unit )

Alternate Protocol 3: Transfection Using the NEON Transfection System

  Materials
  • TS cells
  • Antibiotics‐free 70CM + F4H medium (see recipe)
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS)
  • 0.05% (w/v) trypsin/1 mM EDTA (see recipe)
  • TS medium (see recipe)
  • Neon Transfection System 10 μl kit (Life technologies, cat no. MPK1025) containing:
    • 10‐μl Neon tips
    • Resuspension buffer R
    • Electrolytic buffer E
    • Electroporation tubes
  • 1 μg/μl plasmid DNA in sterile H 2O
  • 35‐mm culture dishes
  • 50‐ml tubes
  • 2‐ml microcentrifuge tubes
  • Neon Transfection System (Life technologies, cat no. MPK5000)
  • Additional reagents and equipment for counting viable cells (Michalska, ; unit )

Alternate Protocol 4: Nucleofection of TS Cells

  Materials
  • Mouse ES Cell Nucleofector Kit (Amaxa, cat no. VPH‐1001) containing:
    • Supplement
    • Mouse ES Cell Nucleofector Solution
  • TS cells
  • 70CM + F4H medium (see recipe)
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS; Invitrogen)
  • 0.05% (w/v) trypsin/1 mM EDTA (see recipe)
  • TS medium (see recipe)
  • Plasmid DNA (5 μg) in 1 to 5 μl sterile H 2O
  • 50‐ml tubes
  • 15‐ml tubes (optional)
  • Amaxa‐certified cuvette (included in the Mouse ES Nucleofector kit)
  • Nucleofector II Device (Amaxa, cat no. AAD‐1001)
  • 100‐mm culture dishes
  • Additional reagents and equipment for counting viable cells (Michalska, ; unit )

Alternate Protocol 5: Bio‐Rad Electroporation of TS Cells

  Materials
  • Appropriate restriction enzyme
  • Linear plasmid DNA (with mammalian antibiotic resistance gene)
  • 3 M sodium acetate (see recipe)
  • 70% and 100% ethanol
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS; Invitrogen)
  • TS cells
  • 70CM + F4H medium (see recipe)
  • 0.05% (w/v) trypsin/1mM EDTA (see recipe)
  • TS medium (see recipe)
  • Microcentrifuge at 4°C
  • Tissue culture hood
  • 50‐ml tubes
  • 15‐ml tubes (optional)
  • Gene Pulser cuvette, 0.4 cm (Bio‐Rad, cat no. 1652088)
  • Gene Pulser electroporation device (Bio‐Rad)
  • Capacitance Extender (Bio‐Rad)
  • 100‐mm culture dish
  • Additional reagents and equipment for counting viable cells (Michalska, ; unit )

Support Protocol 6: Establishing Stable TS Lines

  Materials
  • 70CM + 1.5× F4H medium (see recipe)
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS; Invitrogen)
  • Antibiotic (e.g., neomycin, puromycin, and zeocin)
  • Culture of transfected TS cells ( protocol 9, protocol 10, or protocol 10)
  • 0.05% (w/v) trypsin/1 mM EDTA (see recipe)
  • 4‐well plates
  • 96‐well plate
  • 20‐μl adjustable pipet with appropriate tips
  • Dissecting microscope
  • Multichannel pipettor
  • 1.5‐ml microcentrifuge tubes
  • 30‐mm dish or 4‐well plate
  • 100‐mm dish

Basic Protocol 4: Generation of TS Cell Chimeras

  Materials
  • Genetically labeled TS cells (e.g., GFP, LacZ)
  • 70CM + F4H medium (see recipe)
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS; Invitrogen)
  • 0.05% (w/v) trypsin/1 mM EDTA (see recipe)
  • TS medium (see recipe)
  • Blastocysts (E3.5)
  • Pseudo‐pregnant females (E2.5)
  • 14‐ml round‐bottom tubes (BD Falcon)
  • Microinjection facility with operator
  • Dissecting microscope with UV fluorescence
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Figures

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Literature Cited

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