Skip to main content

Advertisement

SpringerLink
Log in

Derivation, characterization, differentiation, and registration of seven human embryonic stem cell lines (VAL-3, -4, -5, -6M, -7, -8, and -9) on human feeder

  • Published:
In Vitro Cellular & Developmental Biology - Animal Aims and scope Submit manuscript

Abstract

Derivation of human embryonic stem cell lines has been a remarkable scientific achievement during the last decade. Human embryonic stem cells are regarded as an unlimited cell source for replacement therapy in regenerative medicine. Clearly, the scientific community requires proper derivation, characterization, and registration with the purpose of making them available for research and future medical applications worldwide. In this paper, we report our derivation work as the Valencian Node of the Spanish Stem Cell Bank in the generation, characterization, and registration of VAL-3, -4, -5, -6M, -7, -8, and 9 (www.isciii/htdocs/terapia/terapia_bancocelular.jsp). The derivation process was performed on microbiologically tested and irradiated human foreskin fibroblasts and designed to minimize contact with xeno-components in knockout Dulbecco’s modified Eagle’s medium supplemented with knockout serum replacement and basic fibroblast growth factor. Fingerprinting of the cell lines was performed to allow their identification and traceability. All lines were expressed at the mRNA and specific protein markers for undifferentiation and were found to be negative for classical differentiation markers such as neurofilament heavy chain (ectoderm), renin (mesoderm), and amylase (endoderm). All lines displayed high levels of telomerase activity and were shown to successfully overcome cryopreservation and thawing. Finally, we demonstrated the potential to differentiate in vitro (embryoid body formation) and in vivo (teratoma formation) into cell types from all three germ layers. Teratoma derived from all human embryonic stem cell lines present similar morphological features except VAL-8 that display more aggressive tumor behavior with a larger proportion of solid tissues, as opposed to cyst formation in the other cell lines.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.

Similar content being viewed by others

References

  • Bjorklund A.; Lindvall O. Cell replacement therapies for central nervous system disorders. Nat. Neurosci. 3: 537–544; 2000.

    Article  CAS  PubMed  Google Scholar 

  • Guhr A.; Kurtz A.; Friedgen K.; Löser P. Current state of human embryonic stem cell research: an overview of cell lines and their use in experimental work. Stem Cells 24: 2187–2191; 2006.

    Article  PubMed  Google Scholar 

  • Hoffman L.; Carpenter M. Characterization and culture of human embryonic stem cells. Nat. Biotechnol. 23: 699–708; 2005.

    Article  CAS  PubMed  Google Scholar 

  • Hovatta O.; Mikkola M.; Gertow K.; Strömberg A. M.; Inzunza J.; Hreinsson J.; Rozell B.; Blennow E.; Andäng M.; Ahrlund-Richter L. A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum. Reprod. 18: 1404–1409; 2003.

    Article  PubMed  Google Scholar 

  • Hudson T. J.; Stein L. D.; Gerety S. S.; Ma J.; Castle A. B.; Silva J.; Slonim D. K.; Baptista R.; Kruglyak L.; Xu S. H.; Hu X.; Colbert A. M.; Rosenberg C.; Reeve-Daly M. P.; Rozen S.; Hui L.; Wu X.; Vestergaard C.; Wilson K. M.; Bae J. S.; Maitra S.; Ganiatsas S.; Evans C. A.; DeAngelis M. M.; Ingalls K. A.; Nahf R. W.; Horton Jr. L. T.; Anderson M. O.; Collymore A. J.; Ye W.; Kouyoumjian V.; Zemsteva I. S.; Tam J.; Devine R.; Courtney D. F.; Renaud M. T.; Nguyen H.; O’Connor T. J.; Fizames C.; Fauré S.; Gyapay G.; Dib C.; Morissette J.; Orlin J. B.; Birren B. W.; Goodman N.; Weissenbach J.; Hawkins T. L.; Foote S.; Page D. C.; Lander E. S. An STS-based map of the human genome. Science 270: 1945–1954; 1995.

    Article  CAS  PubMed  Google Scholar 

  • Itskovitz-Eldor J.; Schuldiner M.; Karsenti D.; Eden A.; Yanuka O.; Amit M.; Soreq H.; Benvenisty N. Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol. Med. 6: 88–95; 2000.

    CAS  PubMed  Google Scholar 

  • Kimpton C.; Walton A.; Gill P. A further tetranucleotide repeat polymorphism in the vWF gene. Hum. Mol. Genet. 1: 287; 1981.

    Article  Google Scholar 

  • Li H.; Schmidt L.; Wei M. H.; Hustad T.; Lerman M. I.; Zbar B.; Tory K. Three tetranucleotide polymorphisms for loci: D3S1352; D3S1358; D3S1359. Hum. Mol. Genet. 2: 1327; 1993.

    Article  CAS  PubMed  Google Scholar 

  • Martin M.; Muotri A.; Gage F.; Varki A. Human embryonic stem cells express an immunogenic nonhuman sialic acid. Nat. Med. 11: 228–232; 2005.

    Article  CAS  PubMed  Google Scholar 

  • Mills K.; Even D.; Murray J. Tetranucleotide repeat polymorphism at the human alpha fibrinogen locus (FGA). Hum. Mol. Genet. 1: 779; 1992.

    Article  CAS  PubMed  Google Scholar 

  • Oldroyd N.; Urquhart A.; Kimpton C.; Millican E. S.; Watson S. K.; Downes T.; Gill P. D. A highly discriminating octoplex short tandem repeat polymerase chain reaction system suitable for human individual identification. Electrophoresis 16: 334–337; 1995.

    Article  CAS  PubMed  Google Scholar 

  • Pickering S.; Braude P.; Patel M.; Burns C. J.; Trussler J.; Bolton V.; Minger S. Preimplantation genetic diagnosis as a novel source of embryos for stem cell research. Reprod. Biomed. Online 7: 353–364; 2003.

    Article  PubMed  Google Scholar 

  • Reubinoff B.; Pera M.; Fong C.; Trounson A.; Bongso A. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat. Biotechnol. 18: 399–404; 2000.

    Article  CAS  PubMed  Google Scholar 

  • Rodriguez C.; Galan A.; Valvuena D.; Simon C. Derivation of clinical-grade human embryonic stem cells. Reprod. Biomed. Online 12: 112–118; 2006.

    Article  PubMed  Google Scholar 

  • Sharma V.; Litt M. Tetranucleotide repeat polymorphism at the D21S11 locus. Hum. Mol. Genet. 1: 67; 1992.

    Article  CAS  PubMed  Google Scholar 

  • Simon C.; Escobedo C.; Valbuena D.; Genbacev O.; Galan A.; Krtolica A.; Asensi A.; Sánchez E.; Esplugues J.; Fisher S.; Pellicer A. 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; 2005.

    Article  PubMed  Google Scholar 

  • Sullivan K.; Mannucci A.; Kimpton C.; Gill P. A rapid and quantitative DNA sex test: fluorescence-based PCR analysis of X-Y homologous gene amelogenin. Biotechniques 15: 636–641; 1993.

    CAS  PubMed  Google Scholar 

  • The international stem cell initiative. Characteristics of human embryonic stem cell lines: results from the international Stem Cell Initiative. Nat. Biotechnol. 25: 803–816; 2007.

    Article  CAS  Google Scholar 

  • Thomson J.; Itskovitz-Eldor J.; Shapiro S.; Waknitz M.; Swiergiel J.; Marshall V.; Jones J. Embryonic stem cell lines derived from human blastocysts. Science 282: 1145–1147; 1998.

    Article  CAS  PubMed  Google Scholar 

  • Urquhart A.; Oldrovd N.; Kimpton C. Highly discriminating heptaplex short tandem repeat PCR system for forensic identification. Biotechniques 18(116–118): 120–121; 1995.

    Google Scholar 

  • Valbuena D.; Galán A.; Sánchez E.; Poo M. E.; Gómez E.; Sánchez-Luengo S.; Melguizo D.; García A.; Ruiz V.; Moreno R.; Pellicer A.; Simón C. Derivation and characterization of three new Spanish human embryonic stem cell lines (VAL -3–4 -5) on human feeder and in serum-free conditions. Reprod. Biomed. Online 13: 875–886; 2006.

    Article  PubMed  Google Scholar 

  • Valbuena D.; Sánchez-Luengo S.; Galán A.; Sánchez E.; Gómez E.; Poo M. E.; Ruiz V.; Genbacev O.; Krtolica A.; Pellicer A.; Moreno R.; Simón C. Efficient method for slow cryopreservation of human embryonic stem cells in xeno-free conditions. Reprod. Biomed. Online 17: 127–135; 2008.

    CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was founded by the Regenerative Medicine Program, Conselleria de Sanidad, Generalitat Valenciana, Institute of Health Carlos III (Ministry of Health), Spanish Government, PI Carlos Simon and FIS (PI08-1134) PI Diana Valbuena.

Author information

Authors and Affiliations

  1. Valencian Node of the Spanish Stem Cell Bank, Prince Felipe Research Centre (CIPF), Valencia, Spain

    Cristobal Aguilar-Gallardo, Maria Poo, Eva Gomez, Amparo Galan, Eva Sanchez, Ana Marques-Mari, Veronica Ruiz, Jose Medrano, Marcia Riboldi, Diana Valbuena & Carlos Simon

  2. Department of Obstetrics & Gynecology, University of Valencia, Valencia, Spain

    Carlos Simon

  3. Fundación IVI-Instituto Universitario IVI, University of Valencia, Valencia, Spain

    Carlos Simon

  4. Banco Nacional de Líneas Celulares, Nodo de Valencia, Centro de Investigación Príncipe Felipe (CIPF), Avda. Autopista del Saler, 16, 46013, Valencia, Spain

    Carlos Simon

Authors
  1. Cristobal Aguilar-Gallardo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Maria Poo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Eva Gomez
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Amparo Galan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Eva Sanchez
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ana Marques-Mari
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Veronica Ruiz
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jose Medrano
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Marcia Riboldi
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Diana Valbuena
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Carlos Simon
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Carlos Simon.

Additional information

Editor: J. Denry Sato

Rights and permissions

Reprints and permissions

About this article

Cite this article

Aguilar-Gallardo, C., Poo, M., Gomez, E. et al. Derivation, characterization, differentiation, and registration of seven human embryonic stem cell lines (VAL-3, -4, -5, -6M, -7, -8, and -9) on human feeder. In Vitro Cell.Dev.Biol.-Animal 46, 317–326 (2010). https://doi.org/10.1007/s11626-010-9285-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11626-010-9285-3

Keywords

Search

Navigation