Derivation of hESC from Intact Blastocysts

Dusko Ilic1, Olga Genbacev1, Ana Krtolica2

1 University of California, San Francisco, California, 2 Lawrence Berkeley National Laboratory, Berkeley, California
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1A.2
DOI:  10.1002/9780470151808.sc01a02s1
Online Posting Date:  June, 2007
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit describes protocols for culturing human embryos and deriving human embryonic stem cells from the intact blastocyst. Description of the culturing begins with methods for obtaining human blastocysts using pronuclear or cleavage stage embryos left over after in vitro fertilization. Then there is a description of methods that can be used to derive human embryonic stem cell lines from the blastocyst without trophectoderm removal. Also included is a discussion of the critical steps and parameters such as zona pellucida removal, embryo quality assessment, feeder selection, when and how to transfer early embryonic outgrowths. In addition, there are protocols for embryo thawing, seeding of feeder cells, gelatin coating of plates, cleavage and blastocyst stage embryo grading, preparation and storage of reagents and solutions. Finally, there is a discussion of alternative derivation approaches as well as the timeline for the procedures. Curr. Protoc. Stem Cell Biol. 1:1A.2.1‐1A.2.18. © 2007 by John Wiley & Sons, Inc.

Keywords: human embryonic stem cells (hESC); inner cell mass (ICM); trophectoderm (TE); zona pellucida removal; feeders

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Human Embryonic Stem Cell (hESC) Derivation
  • Support Protocol 1: In Vitro Development of Blastocysts
  • Support Protocol 2: Removal of the Zona Pellucida with Acidified Tyrode's Solution
  • Support Protocol 3: Removal of the Zona Pellucida with Pronase
  • Support Protocol 4: Thawing Embryos
  • Support Protocol 5: Plating of Feeder Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Human Embryonic Stem Cell (hESC) Derivation

  Materials
  • KSR embryo culture medium supplemented with 25 ng/ml bFGF (see recipe)
  • Zona pellucida–free blastocyst‐stage embryos (Support Protocols protocol 32 and protocol 43)
  • 26‐G needle, sterile
  • The Stripper micropipettor (MidAtlantic Diagnostics MXL3‐STR) and 600‐µm polycarbonate tips (MidAtlantic Diagnostics MXL3‐600)
  • 1.8‐ml cryovials
  • Additional reagents and equipment for preparing feeder cells in 4‐ or 6‐well tissue culture plates ( protocol 6)

Support Protocol 1: In Vitro Development of Blastocysts

  Materials
  • Appropriate cell culture medium: G‐1 v3 Plus medium (Vitrolife) for the 1‐ to 8‐cell stage (day 1 pronuclear to day 3 cleavage); G‐2 v3 Plus blastocyst medium (Vitrolife) for the 8‐cell (day 3 cleavage) to blastocyst (day 5 or 6) stage
  • Oil for embryo culture (sterile light mineral oil; Irvine Scientific)
  • Pronuclear or cleaving embryos from IVF, fresh or frozen (see protocol 5 for thawing directions)
  • 6‐cm tissue culture–treated plastic dish (e.g., Falcon 3046)
  • The Stripper micropipettor (MidAtlantic Diagnostics MXL3‐STR) with 135‐µm and 600‐µm polycarbonate tips (MidAtlantic Diagnostics MXL3‐135 and MXL3‐600)

Support Protocol 2: Removal of the Zona Pellucida with Acidified Tyrode's Solution

  Materials
  • KSR embryo culture medium with and without 25 ng/ml bFGF (see recipe)
  • Acidified Tyrode's solution (Irvine Scientific)
  • Embryos in culture ( protocol 2)
  • 4‐well tissue culture plate with feeder cells ( protocol 6)
  • G‐2 v3 Plus blastocyst medium (Vitrolife)
  • 6‐cm tissue culture dish with cell culture–treated surface (e.g., Falcon 3046)
  • The Stripper micropipettor (MidAtlantic Diagnostics MXL3‐STR) with 600‐µm (MidAtlantic Diagnostics MXL3‐600) and other appropriate size tips
  • Microscope with camera

Support Protocol 3: Removal of the Zona Pellucida with Pronase

  Materials
  • KSR embryo culture medium with and without 25 ng/ml bFGF (see recipe)
  • 0.5% (w/v) pronase E (Sigma) in KSR embryo culture medium (see recipe)
  • Embryos in culture ( protocol 2)
  • 4‐well tissue culture plate with feeder cells ( protocol 6)
  • 6‐cm tissue culture dishes with cell culture–treated surface
  • The Stripper micropipettor (MidAtlantic Diagnostics MXL3‐STR) and 600‐µm tips

Support Protocol 4: Thawing Embryos

  Materials
  • Embryos frozen in straws under liquid nitrogen (from IVF center)
  • Embryo Thaw Media Kit containing solutions T1, T2, and T3 (Irvine Scientific)
  • 100 mg/ml human serum albumin solution (HSA; Irvine Scientific)
  • Modified human tubal fluid medium (mHTF; Irvine Scientific)
  • 6‐cm tissue culture dish (e.g., Falcon, 3046)
  • The Stripper micropipettor (MidAtlantic Diagnostics MXL3‐STR) and appropriate size tips

Support Protocol 5: Plating of Feeder Cells

  Materials
  • 0.5% (w/v) gelatin (see recipe)
  • Phosphate‐buffered saline (PBS), calcium and magnesium free (Gibco/Invitrogen)
  • Fibroblasts: irradiated and frozen mouse or human cells (see Conner, ; Nagy, )
  • Fibroblast feeder medium (see recipe), prewarmed to 37°C
  • 15‐ml centrifuge tube, sterile
  • 6‐well, tissue culture–treated plates (e.g., Corning) or
  • 4‐well, tissue culture–treated plated (e.g., Nunc)
  • Additional reagents and equipment for counting cells (Phelan, )
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Amit, M., Carpenter, M.K., Inokuma, M.S., Chiu, C.P., Harris, C.P., Waknitz, M.A., Itskovitz‐Eldor, J., and Thomson, J.A. 2000. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev. Biol. 227:271‐278.
   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.
   Carpenter, M.K., Rosler, E., and Rao, M.S. 2003. Characterization and differentiation of human embryonic stem cells. Cloning Stem Cells 5:79‐88.
   Conner, D.A. 2000. Mouse embryo fibroblast (MEF) feeder cell preparation. Curr. Protoc. Mol. Biol. 51:23.2.1‐23.2.7.
   Draper, J.S. and Fox, V. 2003. Human embryonic stem cells: Multilineage differentiation and mechanisms of self‐renewal. Arch. Med. Res. 34:558‐564.
   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.
   Hovatta, O., Mikkola, M., Gertow, K., Stromberg, A.M., Inzunza, J., Hreinsson, J., Rozell, B., Blennow, E., Andang, M., and Arhlund‐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.
   Ilic, D. 2006. Culture of human embryonic stem cells and extracellular matrix microenvironment. Regener. Med. 1:95‐101.
   Johnson, M.H. and McConnell, J.M. 2004. Lineage allocation and cell polarity during mouse embryogenesis. Semin. Cell Dev. Biol. 15:583‐597.
   Krtolica, A. and Campisi, J. 2002. Cancer and aging: A model for the cancer promoting effects of the aging stroma. Int. J. Biochem. Cell Biol. 34:1401‐1414.
   Krtolica, A., and Genbacev, O. 2007 Cell polarity, pluripotency and differentiation. In Stem Cells in Human Reproduction (C. Simon and A. Pellicer, eds.) pp. 183‐188. Informa Healthcare, London.
   Martin, M.J., Muotri, A., Gage, F., and Varki, A. 2005. Human embryonic stem cells express immunogenic nonhuman sialic acid. Nat. Med. 11:228‐232
   Nagy, A., Gertsenstein, M., and Vintersten, K. 2003. Manipulating the Mouse Embryo: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Noda, Y., Goto, Y., Umaoka, Y., Shiotani, M., Nakayama, T., and Mori, T. 1994. Culture of human embryos in alpha modification of Eagle's medium under low oxygen tension and low illumination. Fertil. Steril. 62:1022‐1027.
   Phelan, M.C., 2006. Techniques for mammalian cell tissue culture. Curr. Protoc. Mol. Biol. 74:A.3F.1‐A.3F.8.
   Rasmussen, T.P. 2003. Embryonic stem cell differentiation: A chromatin perspective. Reprod. Biol. Endocrinol. 1:100.
   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.
   Verfaillie, C.M., Pera, M.F., and Lansdorp, P.M. 2002. Stem cells: Hype and reality. Hematology Am. Soc. Hematol. Educ. Program 369‐391.
   Xu, R.H., Chen, X., Li, D.S., Li, R., Addicks, G.C., Glennon, C., Zwaka, T.P., and Thomson, J.A. 2002. BMP4 initiates human embryonic stem cell differentiation to trophoblast. Nat. Biotechnol. 20:1261‐1264.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library