Skip to main content
SpringerLink
Log in

Human Embryonic Stem Cells

Isolation, Maintenance, and Differentiation

  • Protocol
Human Embryonic Stem Cell Protocols

Part of the book series: Methods In Molecular Biology ((MIMB,volume 331))

Abstract

The isolation of pluripotent human embryonic stem (hES) cells having the capacity to differentiate in vitro to numerous cell types generated much excitement and promise in the field of regenerative medicine. However, along with great enthusiasm came hot contro-versy for stem cell research and researchers alike because available hES cell lines were isolated from “excess” embryos from in vitro fertilization clinics. Despite ethical and political debates, the methods and protocols to study diverse lineages are developing. Furthermore, strategies using specific growth factor combinations, cell-cell and cell-extracellular matrix induction systems are being explored for directed differentiation along a desired lineage. However, there is a great need to characterize the mechanisms that control self-renewal and differentiation and a necessity to improve methodologies and develop new purification protocols for the potential future clinical application of hES cells. After the scientific and political obstacles are overcome, it is anticipated that the hES cell field will make a tremendous difference in conditions, such as burn traumas and diabetic foot ulcers, as well a number of degenerative diseases such as Parkinson’s disease, type 1 diabetes, rheumatoid arthritis, and myocardial infarction. In this introductory chapter, we will summarize and review recent progress in the field of hES cell differenti-ation protocols and discuss some of the current issues surrounding hES cell research.

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

Access this chapter

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 109.00
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Smith A. G. (2001) Embryo-derived stem cells: of mice and men. Annu. Rev. Cell Dev. Biol. 17, 435–462.

    Article  PubMed  CAS  Google Scholar 

  2. Hall P. A. and Watt F. M. (1989) Stem cells: the generation and maintenance of cellular diversity. Development 106, 619–633.

    PubMed  CAS  Google Scholar 

  3. Watt F. M. and Hogan B. L. (2000) Out of Eden: stem cells and their niches. Science 287, 1427–1430.

    Article  PubMed  CAS  Google Scholar 

  4. Evans M. J. and Kaufman M. H. (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156.

    Article  PubMed  CAS  Google Scholar 

  5. Martin G. R. (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl. Acad. Sci. USA 78, 7634–7638.

    Article  PubMed  CAS  Google Scholar 

  6. Cazpecchi M. R. (2005) Gene targeting in mice: functional analysis of the mam-malian genome for the twenty-first century. Nat. Rev. Genet. 6, 507–512.

    Article  CAS  Google Scholar 

  7. Mansour S. L., Thomas K. R., and Capecchi M. R. (1988) Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature 336, 348–352.

    Article  PubMed  CAS  Google Scholar 

  8. Downing G. J. and Battey J. F., Jr. (2004) Technical assessment of the first 20 years of research using mouse embryonic stem cell lines. Stem Cells 22, 1168–1180.

    Article  PubMed  Google Scholar 

  9. Coraux C., Nawrocki-Raby B., Hinnrasky J., et al. (2005) Embryonic stem cells generate airway epithelial tissue. Am. J. Respir. Cell. Mol. Biol. 32, 87–92.

    Article  PubMed  CAS  Google Scholar 

  10. Kawaguchi J., Mee P. J., and Smith A. G. (2005) Osteogenic and chondrogenic differentiation of embryonic stem cells in response to specific growth factors. Bone 36, 758–769.

    Article  PubMed  CAS  Google Scholar 

  11. Troy T. C. and Turksen K. (2005) Commitment of embryonic stem cells to an epidermal cell fate and differentiation in vitro. Dev. Dyn. 232, 293–300.

    Article  PubMed  CAS  Google Scholar 

  12. Turksen K. (2002) Embryonic Stem Cells: Methods and Protocols. Methods in Molecular Biology Series Vol. 185. Humana, Totowa NJ.

    Google Scholar 

  13. Watanabe K., Kamiya D., Nishiyama A., et al. (2005) Directed differentiation of telencephalic precursors from embryonic stem cells. Nat. Neurosci. 8, 288–296.

    Article  PubMed  CAS  Google Scholar 

  14. Yuasa S., Itabashi Y., Koshimizu U., et al. (2005) Transient inhibition of BMP signaling by Noggin induces cardiomyocyte differentiation of mouse embryonic stem cells. Nat. Biotechnol. 23, 607–611.

    Article  PubMed  CAS  Google Scholar 

  15. Thomson J. A., Itskovitz-Eldor J., Shapiro S. S., et al. (1998) Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147.

    Article  PubMed  CAS  Google Scholar 

  16. Pera M. F., Reubinoff B., and Trounson A. (2000) Human embryonic stem cells. J. Cell. Sci. 113, 5–10.

    PubMed  CAS  Google Scholar 

  17. Pera M. F. and Trounson A. O. (2004) Human embryonic stem cells: prospects for development. Development 131, 5515–5525.

    Article  PubMed  CAS  Google Scholar 

  18. Reubinoff B. E., Pera M. F., Fong C. Y., Trounson A., and Bongso A. (2000) Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat. Biotechnol. 18, 399–404.

    Article  PubMed  CAS  Google Scholar 

  19. Stojkovic M., Lako M., Stojkovic P., et al. (2004) Derivation of human embryonic stem cells from day-8 blastocysts recovered after three-step in vitro culture. Stem Cells 22, 790–797.

    Article  PubMed  Google Scholar 

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

    Article  PubMed  Google Scholar 

  21. Verlinsky Y., Strelchenko N., Kukharenko V., et al. (2005) Human embryonic stem cell lines with genetic disorders. Reprod. Biomed. Online 10, 105–110.

    Article  PubMed  CAS  Google Scholar 

  22. Hwang W. S., Roh S. I., Lee B. C., et al. (2005) Patient-specific embryonic stem cells derived from human SCNT blastocysts. Science 308, 1777–1783.

    Article  PubMed  CAS  Google Scholar 

  23. Amit M., Carpenter M. K., Inokuma M. S., et al. (2000) Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev. Biol. 227, 271–278.

    Article  PubMed  CAS  Google Scholar 

  24. Oh S. K., Kim H. S., Park Y. B., et al. (2005) Methods for expansion of human embryonic stem cells. Stem Cells 23, 605–609.

    Article  PubMed  CAS  Google Scholar 

  25. Schatten G., Smith J., Navara C., Park J. H., and Pedersen R. (2005) Culture of human embryonic stem cells. Nat. Methods 2, 455–463.

    Article  PubMed  CAS  Google Scholar 

  26. Miura T., Luo Y., Khrebtukova I., et al. (2004) Monitoring early differentiation events in human embryonic stem cells by massively parallel signature sequencing and expressed sequence tag scan. Stem Cells Dev. 13, 694–715.

    Article  PubMed  CAS  Google Scholar 

  27. Amit M., Winkler M. E., Menke S., et al. (2005) No evidence for infection of human embryonic stem cells by feeder cell-derived murine leukemia viruses. Stem Cells 23, 761–771.

    Article  PubMed  CAS  Google Scholar 

  28. Martin M. J., Muotri A., Gage F., and Varki A. (2005) Human embryonic stem cells express an immunogenic nonhuman sialic acid. Nat. Med. 11, 228–232.

    Article  PubMed  CAS  Google Scholar 

  29. Cheng L., Hammond H., Ye Z., Zhan X., and Dravid G. (2003) Human adult marrow cells support prolonged expansion of human embryonic stem cells in culture. Stem Cells 21, 131–142.

    Article  PubMed  CAS  Google Scholar 

  30. 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.

    Article  PubMed  CAS  Google Scholar 

  31. Stojkovic P., Lako M., Stewart R., et al. (2005) An autogeneic feeder cell system that efficiently supports growth of undifferentiated human embryonic stem cells. Stem Cells 23, 306–314.

    Article  PubMed  CAS  Google Scholar 

  32. Prowse A. B., McQuade L. R., Bryant K. J., Van Dyk D. D., Tuch B. E., and Gray P. P. (2005) Aproteome analysis of conditioned media from human neona-tal fibroblasts used in the maintenance of human embryonic stem cells. Proteomics 5, 978–989.

    Article  PubMed  CAS  Google Scholar 

  33. Stojkovic P., Lako M., Przyborski S., et al. (2005) Human-serum matrix sup-ports undifferentiated growth of human embryonic stem cells. Stem Cells 2005 Aug; 23(7), 895–902.

    Article  PubMed  CAS  Google Scholar 

  34. Wang L., Li L., Menendez P., Cerdan C., and Bhatia M. (2005) Human embry-onic stem cells maintained in the absence of mouse embryonic fibroblasts or conditioned media are capable of hematopoietic development. Blood 105, 4598–4603.

    Article  PubMed  CAS  Google Scholar 

  35. Genbacev O., Krtolica A., Zdravkovic T., et al. (2005) Serum-free derivation of human embryonic stem cell lines on human placental fibroblast feeders. Fertil. Steril. 83, 1517–1529.

    Article  PubMed  Google Scholar 

  36. Inzunza J., Gertow K., Stromberg M. A., et al. (2005) Derivation of human embryonic stem cell lines in serum replacement medium using postnatal human fibroblasts as feeder cells. Stem Cells 23, 544–549.

    Article  PubMed  CAS  Google Scholar 

  37. Wang G., Zhang H., Zhao Y., et al. (2005) Noggin and bFGF cooperate to maintain the pluripotency of human embryonic stem cells in the absence of feed-er layers. Biochem. Biophys. Res. Commun. 330, 934–942.

    Article  PubMed  CAS  Google Scholar 

  38. Xu C., Rosler E., Jiang J., et al. (2005) Basic fibroblast growth factor supports undifferentiated human embryonic stem cell growth without conditioned medium. Stem Cells 23, 315–323.

    Article  PubMed  CAS  Google Scholar 

  39. Xu R. H., Peck R. M., Li D. S., Feng X., Ludwig T., and Thomson J. A. (2005) Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells. Nat. Methods 2, 185–190.

    Article  PubMed  CAS  Google Scholar 

  40. Beattie G. M., Lopez A. D., Bucay N., et al. (2005) Activin A maintains pluri-potency of human embryonic stem cells in the absence of feeder layers. Stem Cells 23, 489–495.

    Article  PubMed  CAS  Google Scholar 

  41. Zaehres H., Lensch M. W., Daheron L., Stewart S. A., Itskovitz-Eldor J., and Daley G. Q. (2005) High-efficiency RNA interference in human embryonic stem cells. Stem Cells 23, 299–305.

    Article  PubMed  CAS  Google Scholar 

  42. Rao M. (2004) Conserved and divergent paths that regulate self-renewal in mouse and human embryonic stem cells. Dev. Biol. 275, 269–286.

    Article  PubMed  CAS  Google Scholar 

  43. Wei C. L., Miura T., Robson P., et al. (2005) Transcriptome profiling of human and murine ESCs identifies divergent paths required to maintain the stem cell state. Stem Cells 23, 166–185.

    Article  PubMed  CAS  Google Scholar 

  44. Itskovitz-Eldor J., Schuldiner M., Karsenti D., et al. (2000) Differentiation of human embryonic stem cells into embryoid bodies compromising the three embry-onic germ layers. Mol. Med. 6, 88–95.

    PubMed  CAS  Google Scholar 

  45. Schuldiner M., Yanuka O., Itskovitz-Eldor J., Melton D. A., and Benvenisty N. (2000) Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc. Natl. Acad. Sci. USA 97, 11,307-11,312.

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Development Program, Ottawa Health Research Institute, Ottawa, Ontario, Canada

    Kursad Turksen & Tammy-Claire Troy

Authors
  1. Kursad Turksen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tammy-Claire Troy
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Ottawa Health Research Institute, Ottawa, Ontario, Canada

    Kursad Turksen

Rights and permissions

Reprints and permissions

Copyright information

© 2006 Humana Press Inc.

About this protocol

Cite this protocol

Turksen, K., Troy, TC. (2006). Human Embryonic Stem Cells. In: Turksen, K. (eds) Human Embryonic Stem Cell Protocols. Methods In Molecular Biology, vol 331. Humana Press. https://doi.org/10.1385/1-59745-046-4:1

Download citation

  • DOI: https://doi.org/10.1385/1-59745-046-4:1

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-497-5

  • Online ISBN: 978-1-59745-046-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation