Skip to main content
SpringerLink
Log in
Book cover

Human Embryonic Stem Cells Handbook pp 69–80Cite as

Derivation and Maintenance of Undifferentiated Human Embryonic Stem Cells

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 873))

Abstract

Human embryonic stem cells (hESCs) are self-renewing, pluripotent cells derived from the inner cell mass of blastocysts, early-stage embryos, or blastomeres. hESCs can be propagated indefinitely in an undifferentiated state in vitro and have the ability to differentiate into all cell types of the body. Therefore, these cells can potentially provide an unlimited source of cells and hold promise for transplantation therapy, regenerative medicine, drug screening and discovery, and basic scientific research. Surplus human embryos donated for hESC derivation are extremely valuable, and inefficient derivation of hESCs would be a terrible waste of human embryos. Here, we describe a method for isolating hESC lines from human blastocysts with high efficiency. We also describe the methods for excising differentiated areas from partially differentiated hESC colonies and re-isolating undifferentiated hESCs from extremely differentiated hESC colonies.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   59.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   79.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM (1998) Embryonic stem cell lines derived from human blastocysts. Science 282:1145–1147

    Article  PubMed  CAS  Google Scholar 

  2. Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A (2000) Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 18:399–404

    Article  PubMed  CAS  Google Scholar 

  3. Strelchenko N, Verlinsky O, Kukharenko V, Verlinsky Y (2004) Morula-derived human embryonic stem cells. Reprod Biomed Online 9:623–629

    Article  PubMed  Google Scholar 

  4. Klimanskaya I, Chung Y, Becker S, Lu SJ, Lanza R (2006) Human embryonic stem cell lines derived from single blastomeres. Nature 444:481–485

    Article  PubMed  CAS  Google Scholar 

  5. Park JH, Kim SJ, Oh EJ, Moon SY, Roh SI, Kim CG, Yoon HS (2003) Establishment and maintenance of human embryonic stem cells on STO, a permanently growing cell line. Biol Reprod 69:2007–2014

    Article  PubMed  CAS  Google Scholar 

  6. Findikli N, Kahraman S, Akcin O, Sertyel S, Candan Z (2005) Establishment and characterization of new human embryonic stem cell lines. Reprod Biomed Online 10:617–627

    Article  PubMed  Google Scholar 

  7. Chen HF, Kuo HC, Chien CL, Shun CT, Yao YL, Ip PL (2007) Derivation, characterization and differentiation of human embryonic stem cells: comparing serum-containing versus serum-free media and evidence of germ cell differentiation. Hum Reprod 22:567–577

    Article  PubMed  Google Scholar 

  8. Suss-Toby E, Gerecht-Nir S, Amit M, Manor D, Itskovitz-Eldor J (2004) Derivation of a diploid human embryonic stem cell line from a mononuclear zygote. Hum Reprod 19:670–675

    Article  PubMed  Google Scholar 

  9. Simon C, Escobedo C, Valbuena D, Genbacev O, Galan A, Krtolica A, Asensi A, Sánchez E, Esplugues J, Fisher S, Pellicer A (2005) First derivation in Spain of human embryonic stem cell lines: use of long-term cryopreserved embryos and animal-free conditions. Fertil Steril 83:246–249

    Article  PubMed  Google Scholar 

  10. Strom S, Inzunza J, Grinnemo KH, Holmberg K, Matilainen E, Stromberg AM, Blennow E, Hovatta O (2007) Mechanical isolation of the inner cell mass is effective in derivation of new human embryonic stem cell lines. Hum Reprod 22:3051–3058

    Article  PubMed  Google Scholar 

  11. Meng G, Liu S, Li X, Krawetz R, Rancourt DE (2010) Derivation of human embryonic stem cell lines after blastocyst microsurgery. Biochem Cell Biol 88:479–490

    Article  PubMed  CAS  Google Scholar 

  12. Pakzad M, Totonchi M, Taei A, Seifinejad A, Hassani SN, Baharvand H (2010) Presence of a ROCK inhibitor in extracellular matrix supports more undifferentiated growth of feeder-free human embryonic and induced pluripotent stem cells upon passaging. Stem Cell Rev 6:96–107

    Article  PubMed  CAS  Google Scholar 

  13. Li X, Meng G, Krawetz R, Liu S, Rancourt DE (2008) The ROCK inhibitor Y-27632 enhances the survival rate of human embryonic stem cells following cryopreservation. Stem Cells Dev 17:1079–1085

    Article  PubMed  CAS  Google Scholar 

  14. Watanabe K, Ueno M, Kamiya D, Nishiyama A, Matsumura M, Wataya T, Takahashi JB, Nishikawa S, Nishikawa S, Muguruma K, Sasai Y (2007) AROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol 25:681–686

    Article  PubMed  CAS  Google Scholar 

  15. Gauthaman K, Fong CY, Bongso A (2010) Effect of ROCK inhibitor Y-27632 on normal and variant human embryonic stem cells (hESCs) in vitro: its benefits in hESC expansion. Stem Cell Rev 6:86–95

    Article  PubMed  CAS  Google Scholar 

  16. Meng G, Liu S, Rancourt DE (2011) Rapid isolation of undifferentiated human pluripotent stem cells from extremely differentiated colonies. Stem Cells Dev 20(4): 583–591

    Article  PubMed  Google Scholar 

  17. BresaGen, Inc. (2004) Human embryonic stem cell protocols. P5–P8

    Google Scholar 

  18. Meng GL, Zur Nieden NI, Liu SY, Cormier JT, Kallos MS, Rancourt DE (2008) Properties of murine embryonic stem cells maintained on human foreskin fibroblasts without LIF. Mol Reprod Dev 75:614–622

    Article  PubMed  CAS  Google Scholar 

  19. Heins N, Englund MC, Sjöblom C, Dahl U, Tonning A, Bergh C, Lindahl A, Hanson C, Semb H (2004) Derivation, characterization, and differentiation of human embryonic stem cells. Stem Cells 22:367–376

    Article  PubMed  Google Scholar 

  20. Hoffman LM, Carpenter MK (2005) Characterization and culture of human embryonic stem cells. Nat Biotechnol 23:699–708

    Article  PubMed  CAS  Google Scholar 

  21. Werbowetski-Ogilvie TE, Bossé M, Stewart M, Schnerch A, Ramos-Mejia V, Rouleau A, Wynder T, Smith MJ, Dingwall S, Carter T, Williams C, Harris C, Dolling J, Wynder C, Boreham D, Bhatia M (2009) Characterization of human embryonic stem cells with features of neoplastic progression. Nat Biotechnol 27:91–97

    Article  PubMed  CAS  Google Scholar 

  22. Meng G, Liu S, Krawetz R, Chan M, Chernos J, Rancourt DE (2008) A novel method for generating xeno-free human feeder cells for human embryonic stem cell culture. Stem Cells Dev 17:413–422

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. University of Calgary, Calgary, AB, Canada

    Guoliang Meng

  2. Departments of Oncology and Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada

    Derrick E. Rancourt

Authors
  1. Guoliang Meng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Derrick E. Rancourt
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Derrick E. Rancourt .

Editor information

Editors and Affiliations

  1. Sprott Centre for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital Research Institute, Smyth Road 501, Ottawa, K1Y 8L6, Canada

    Kursad Turksen

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Meng, G., Rancourt, D.E. (2012). Derivation and Maintenance of Undifferentiated Human Embryonic Stem Cells. In: Turksen, K. (eds) Human Embryonic Stem Cells Handbook. Methods in Molecular Biology, vol 873. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-794-1_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-794-1_5

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-793-4

  • Online ISBN: 978-1-61779-794-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation