Generation of Human Embryonic Stem Cells

Teija Peura1, Julia Schaft1, Biljana Dumevska1, Tomas Stojanov1

1 Sydney IVF, Sydney, New South Wales, Australia
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1A.5
DOI:  10.1002/9780470151808.sc01a05s16
Online Posting Date:  March, 2011
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Abstract

This unit describes generation of human embryonic stem cell lines from early human embryos. The focus is on actual handling of embryos and early embryonic outgrowths, omitting steps required for actual generation, freezing, and thawing of embryos, as well as further culture and characterization of newly derived stem cells. Hence, the initial culture of embryos to a blastocyst stage is described, followed by removal of the protective zona pellucida layer, isolation of the inner cell mass (ICM), subsequent plating of ICM or whole embryo and, finally, the first few passages of an early embryonic outgrowth. A few alternative procedures for some steps such as zona removal and inner cell mass isolation are described, to allow procedures to be modified according to circumstances. Curr. Protoc. Stem Cell Biol. 16:1A.5.1‐1A.5.19. © 2011 by John Wiley & Sons, Inc.

Keywords: human; embryonic stem cell; derivation; inner cell mass; hESC

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Embryo Culture
  • Basic Protocol 2: Zona Pellucida Removal, Isolation of Inner Cell Mass, and Plating of Embryonic Cells
  • Basic Protocol 3: Passaging of the Embryonic Outgrowth
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Embryo Culture

  Materials
  • Human embryos at any stage of development
  • Culture oil (Cook IVF, cat no. G32717 or G26708)
  • Media:
    • Embryo Cleavage Medium (Cook IVF, cat no. G20720, G20721, or G19018)
    • Blastocyst Medium (Cook IVF, cat no. G20722 or G20929)
  • 4‐well plates (Nunc, cat no. 179830)
  • K‐MINC‐1000 mini‐incubator (Cook IVF, cat no. K‐MINC‐1000‐US)
  • Dissecting microscope (if working inside a Class II Biological Safety Cabinet, microscope must be fitted with extended eyepieces, e.g., Olympus SZX7 with extended eyepieces)
  • Heated stage for the dissecting microscope (e.g., LEC instruments, cat no. LEC944) and warm stage controller (e.g., LEC Instruments, cat no. LEC916)
  • Inverted microscope with phase‐contrast optics and 40× to 100× magnification

Basic Protocol 2: Zona Pellucida Removal, Isolation of Inner Cell Mass, and Plating of Embryonic Cells

  Materials
  • Embryos ( protocol 1)
  • 4 mg/ml pronase (see recipe)
  • Culture oil (Cook IVF, cat no. G32717 or G26708)
  • Gamete Buffer (Cook IVF, cat. nos. G48258, G48259, or G48260)
  • Acidic Tyrode's solution (Sigma, cat. no. T1788); thaw and divide into 1‐ to 5‐ml aliquots in sterile disposable tubes and refreeze; store at −20°C for ≥1 year (avoid additional thawing and re‐freezing)
  • KSR‐based stem cell medium with and without bFGF (see recipe; alternatively, use FBS‐based stem cell medium, see recipe); note that while the use of other hESC culture media (e.g., fully defined or xeno‐free media) is possible and sometimes desirable, it may be necessary to adapt the derivation protocol from the one described here
  • 1% 7X cleaning solution (MP Biomedicals, cat. no. 76670)
  • 70% ethanol
  • Anti‐human whole serum (see recipe)
  • Guinea pig complement (see recipe)
  • Mitotically inactivated (irradiated or mitomycin‐C treated) feeder cell plates (also see Michalska, , and annotation to step 14, below)
  • Basic fibroblast growth factor (bFGF)
  • 35‐mm culture dishes
  • Dissecting microscope (if working inside a Class II Biological Safety Cabinet, microscope needs to have been fitted with extended eyepieces, e.g., Olympus SZX7 with extended eyepieces)
  • Heated stage for the dissecting microscope (e.g., LEC instruments, cat no. LEC944) and warm stage controller (e.g., LEC Instruments, cat no. LEC916)
  • Embryo transfer pipets, e.g., glass Pasteur pipets (Sigma, cat no. P 1736); treat first by soaking overnight in fresh 3% Analar HCl (1260 ml HCl in 14 liters Milli‐Q water), rinse thoroughly in Milli‐Q water, followed by another overnight soak, and rinse in fresh Milli‐Q water; sonicate at 60°C with Milli‐Q water for 10 min, rinse once more with fresh running Milli‐Q water, and dry in oven; flame‐pull and fire‐polish the pipets just before using to create a suitable‐size narrow glass pipet
  • Disposable sterile embryo transfer pipet (0.29 to 0.31 mm, Swemed Int., cat no. H‐290‐310; http://www.vitrolife.com/) can be used as an alternative to flame‐pulled glass pipets
  • Indirect mouth hose (Tek‐Event, IVF connection‐set II mouth tubing, cat no. HPF6237) to use with flame‐pulled Pasteur pipet: has a thin membrane inside a bell‐shaped attachment to the tubing, mediating the aspirating and pulling pressure without exposing the embryo or the operator to a direct aerosol contact; the membrane prevents open airway contact between the solution and the mouth, ensuring protection both for the operator as well as for the cellular material
  • Ultra Sharp splitting blades (Bioniche Animal Health, cat. no. ESE020; http://bionicheanimalhealth.com/)
  • Pipettor with narrow (P‐20) tip
  • Additional reagents for preparation and plating of feeder cells (e.g., Michalska, )

Basic Protocol 3: Passaging of the Embryonic Outgrowth

  Materials
  • Embryos, plated ( protocol 2)
  • Mitotically inactivated (irradiated or mitomycin‐C treated) feeder cell plates (also see Michalska, , and protocol 2)
  • KSR‐based medium (see recipe) freshly supplemented with 4 ng/ml basic fibroblast growth factor (bFGF)
  • Ultra Sharp splitting blades (Bioniche Animal Health, cat. no. ESE020; http://bionicheanimalhealth.com/)
  • Inverted microscope with phase‐contrast optics and 40× to 100× magnification
  • Dissecting microscope (if working inside a Class II Biological Safety Cabinet, microscope needs to have been fitted with extended eyepieces, e.g., Olympus SZX7 with extended eyepieces)
  • Heated stage for the dissecting microscope (e.g., LEC instruments, cat no. LEC944) and warm stage controller (e.g., LEC Instruments, cat no. LEC916)
  • Embryo transfer pipettes, e.g., glass Pasteur pipets (Sigma, cat no. P 1736); treat first by soaking overnight in fresh 3% Analar HCl (1260 ml HCl in 14 liters Milli‐Q water), rinse thoroughly in Milli‐Q water, followed by another overnight soak and rinse in fresh Milli‐Q water; sonicate at 60°C with Milli‐Q water for 10 min, rinse once more with fresh running Milli‐Q water, and dry in oven; flame‐pull and fire‐polish the pipets just before using to create a suitable‐size narrow glass pipet
  • Disposable sterile embryo transfer pipet (0.29 to 0.31 mm, Swemed Int., cat no. H‐290‐310; http://www.vitrolife.com/) can be used as an alternative to flame‐pulled glass pipets
  • Indirect mouth hose (Tek‐Event, IVF connection‐set II mouth tubing, cat no. HPF6237) to use with flame‐pulled Pasteur pipet: has a thin membrane inside a bell‐shaped attachment to the tubing, mediating the aspirating and pulling pressure without exposing the embryo or the operator to a direct aerosol contact; the membrane prevents open airway contact between the solution and the mouth, ensuring protection both for the operator as well as for the cellular material
  • P‐20 pipettor (2.0 to 20 µl) and 20‐µl tips
  • Additional reagents for preparation and plating of feeder cells (for example, as Michalska, )
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Figures

Videos

Literature Cited

Literature Cited
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