Skip to main content
SpringerLink
Log in

Endoplasmic Reticulum Stress Signals in Defined Human Embryonic Stem Cell Lines and Culture Conditions

  • Published:
Stem Cell Reviews and Reports Aims and scope Submit manuscript

An Erratum to this article was published on 03 December 2010

Abstract

Human embryonic stem cells (hESCs) are especially resistant to several cellular stresses, but the existence and induction of Endoplasmic Reticulum (ER) stress by culture conditions are unknown. Using qPCR, here, we investigated the behavior of the principal sensors of ER stress and their relation with the feeder layer, the type of conditioned media used in feeder free systems and the upregulation of several differentiation markers. We observed the preservation of pluripotency, and detected differential expression of differentiation markers in HS181 and SHEF1 hESCs growing on Adipose-derived mesenchymal stem cells (ASCs) and feeder-free system with different conditioned media (HEF-CM and ASC-CM). Taken together, these results demonstrate evidence of ER stress events that cells must resolve to survive and maintenance of markers of pluripotency. The early differentiation status defined could progress into a more differentiated state, and may be influenced by culture conditions.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. Saretzki, G., Walter, T., Atkinson, S., Passos, J. F., Bareth, B., Keith, W. N., et al. (2008). Downregulation of multiple stress defense mechanisms during differentiation of human embryonic stem cells. Stem Cells, 26, 455–464.

    Article  CAS  PubMed  Google Scholar 

  2. Saretzki, G., Armstrong, L., Leake, A., Lako, M., & Von Zglinicki, T. (2004). Stress defense in murine embryonic stem cells is superior to that of various differentiated murine cells. Stem Cells, 22, 962–971.

    Article  CAS  PubMed  Google Scholar 

  3. Prinsloo, E., Setati, M. M., Longshaw, V. M., & Blatch, G. L. (2009). Chaperoning stem cells: a role for heat shock proteins in the modulation of stem cell self-renewal and differentiation? BioEssays, 31, 1–8.

    Article  Google Scholar 

  4. Brodsky, J. L. (2007). The protective and destructive roles played by molecular chaperones during ERAD (endoplasmic-reticulum-associated degradation). Biochemical Journal, 404, 353–363.

    Article  CAS  PubMed  Google Scholar 

  5. Kim, R., Emi, M., Tanabe, K., & Murakami, S. (2006). Role of the unfolded protein response in cell death. Apoptosis, 11, 5–13.

    Article  CAS  PubMed  Google Scholar 

  6. Vembar, S. S., & Brodsky, J. L. (2008). One step at a time: endoplasmic reticulum-associated degradation. Nature Reviews. Molecular Cell Biology, 9, 944–957.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  8. Thomson, J. A., Kalishman, J., Golos, T. G., Durning, M., Harris, C. P., Becker, R. A., et al. (1995). Isolation of a primate embryonic stem cell line. Proceedings of the National Academy of Science, 92, 7844–7848.

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  10. Richards, M., Fong, C. Y., Chan, W. K., Wong, P. C., & Bongso, A. (2002). Human feeders support prolonged undifferentiated growth of human inner cell mass and embryonic stem cells. Nature Biotechnology, 20, 882–883. NormFinder Software http://mdl.dk/publicationsnormfinder.htm.

    Article  Google Scholar 

  11. Xu, C., Inokuma, M. S., Denham, J., Golds, K., Kundu, P., Gold, J. D., et al. (2001). Feeder-free growth of undifferentiated human embryonic stem cells. Nature Biotechnology, 19, 971–974.

    Article  CAS  PubMed  Google Scholar 

  12. Escobedo-Lucea, C., & Stojkovic, M. (2010). Growth of human embryonic stem cells using derivates of human fibroblast. Methods in Molecular Biology, 584, 55–69.

    Article  CAS  PubMed  Google Scholar 

  13. Wang, Q., Fang, Z. F., Jin, F., Lu, Y., Gai, H., & Sheng, H. Z. (2005). Derivation and growing human embryonic stem cells on feeders derived from themselves. Stem Cells, 23, 1221–1227.

    Article  PubMed  Google Scholar 

  14. Choo, A., Ngo, A. S., Ding, V., Oh, S., & Kiang, L. S. (2008). Autogeneic feeders for the culture of undifferentiated human embryonic stem cells in feeder and feeder-free conditions. Methods in Cell Biology, 86, 15–28.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  16. Cortes, J. L., Sanchez, L., Ligero, G., Gutierrez-Aranda, I., Catalina, P., Elosua, C., et al. (2009). Mesenchymal stem cells facilitate the derivation of human embryonic stem cells from cryopreserved poor-quality embryos. Human Reproduction, 24, 1854–1851.

    Article  Google Scholar 

  17. Dubois, S. G., Floyd, E. Z., Zvonic, S., Kilroy, G., Wu, X., Carling, S., et al. (2008). Isolation of human adipose-derived stem cells from biopsies and liposuction specimens. Methods in Molecular Biology, 449, 69–79.

    Article  PubMed  Google Scholar 

  18. Gimble, J. M., Katz, A. J., & Bunnell, B. A. (2007). Adipose-derived stem cells for regenerative medicine. Circulation Research, 100, 1249–1260.

    Article  CAS  PubMed  Google Scholar 

  19. Bunnell, B. A., Flaat, M., Gagliardi, C., Patel, B., & Ripoll, C. (2008). Adipose-derived stem cells: isolation, expansion and differentiation. Methods, 45, 115–120.

    Article  CAS  PubMed  Google Scholar 

  20. Flynn, L., Prestwich, G. D., Semple, J. L., & Woodhouse, K. A. (2007). Adipose tissue engineering with naturally derived scaffolds and adipose-derived stem cells. Biomaterials, 28, 3834–3842.

    Article  CAS  PubMed  Google Scholar 

  21. Flynn, L., Prestwich, G. D., Semple, J. L., & Woodhouse, K. A. (2009). Adipose tissue engineering in vivo with adipose-derived stem cells on naturally derived scaffolds. Journal of Biomedical Materials Research: Part A, 89(4), 929–941.

    Article  CAS  Google Scholar 

  22. Flynn, L., Prestwich, G. D., Semple, J. L., & Woodhouse, K. A. (2008). Proliferation and differentiation of adipose-derived stem cells on naturally derived scaffolds. Biomaterials, 29, 1862–1871.

    Article  CAS  PubMed  Google Scholar 

  23. Assou, S., Le Carrour, T., Tondeur, S., Ström, S., Gabelle, A., Marty, S., et al. (2007). A meta-analysis of human embryonic stem cells transcriptome integrated into a web-based expression atlas. Stem Cells, 25, 961–973.

    Article  CAS  PubMed  Google Scholar 

  24. Kim, J., Chu, J., Shen, X., Wang, J., & Orkin, S. H. (2008). An extended transcriptional network for pluripotency of embryonic stem cells. Cell, 132, 1049–1061.

    Article  CAS  PubMed  Google Scholar 

  25. Bendall, S. C., Stewart, M. H., Menendez, P., George, D., Vijayaragavan, K., Werbowetski-Ogilvie, T., et al. (2007). IGF and FGF cooperatively establish the regulatory stem cell niche of pluripotent human cells in vitro. Nature, 441, 1075–1079.

    Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  27. Rubio, D., Garcia, S., Paz, M., De la Cueva, T., Lopez-Fernandez, L. A., Lloyd, A. C., et al. (2008). Molecular characterization of spontaneous mesenchymal stem cell transformation. PloS One, 3, e1398.

    Article  PubMed  Google Scholar 

  28. Rubio, D., Garcia-Castro, J., Martin, M. C., de la Fuente, R., Cigudosa, J. C., Lloyd, A. C., et al. (2005). Spontaneous human adult stem cell transformation. Cancer Research, 65, 3035–3039.

    CAS  PubMed  Google Scholar 

  29. Cobo, F., Navarro, J. M., Herrera, M. L., Vivo, A., Porcel, D., Hernández, C., et al. (2008). Electron microscopy reveals the presence of viruses in mouse embryonic fibroblasts but neither in human embryonic fibroblasts nor in human mesenchymal cells used for hESC maintenance: toward an implementation of microbiological quality assurance program in stem cell banks. Cloning Stem Cells, 10, 65–74.

    Article  CAS  PubMed  Google Scholar 

  30. Garcia-Castro, J., Trigueros, C., Madreras, J., Pérez-Simón, J. A., Rodriguez, R., & Menendez, P. (2008). Mesenchymal stem cells and their use as cell replacement therapy and disease modelling tool. Journal of Cellular and Molecular Medicine, 12, 1–14.

    Article  Google Scholar 

  31. Andersen, C. L., Ledet-Jensen, J., & Orntoft, T. (2004). Normalization of real-time quantitative RT-PCR data: a model based variance estimation approach to identify genes suited for normalization—applied to bladder- and colon-cancer data-sets. Cancer Research, 64, 5245–5250.

    Article  CAS  PubMed  Google Scholar 

  32. Lowry, W. E., Richter, L., Yachechko, R., Pyle, A. D., Tchieu, J., Sridharan, R., et al. (2008). Generation of human induced pluripotent stem cells from dermal fibroblasts. Proceedings of the National Academy of Sciences of the United States of America, 105, 2883–2888.

    Article  CAS  PubMed  Google Scholar 

  33. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., et al. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 131, 861–872.

    Article  CAS  PubMed  Google Scholar 

  34. Takahashi, K., & Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126, 663–676.

    Article  CAS  PubMed  Google Scholar 

  35. Bustin, S. A., & Nolan, T. (2004). Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. Journal of Biomolecular Techniques, 15, 155–165.

    PubMed  Google Scholar 

  36. Radonic, A., Thulke, S., Mackay, I. M., Landt, O., Siegert, W., & Nitsche, A. (2004). Guideline to reference gene selection for quantitative real-time PCR. Biochemical and Biophysical Research Communications, 313, 856–862.

    Article  CAS  PubMed  Google Scholar 

  37. Fink, T., Lund, P., Pilgaard, L., Rasmussen, J. G., Duroux, M., & Zachar, V. (2008). Instability of standard PCR reference genes in adipose-derived stem cells during propagation, differentiation and hypoxic exposure. BMC Molecular Biology, 31, 98.

    Article  Google Scholar 

  38. Perez, S., Royo, L. J., Astudillo, A., Escudero, D., Alvarez, F., Rodríguez, A., et al. (2007). Identifying the most suitable endogenous control for determining gene expression in hearts from organ donors. BMC Molecular Biology, 8, 114.

    Article  PubMed  Google Scholar 

  39. Piana, C., Wirth, M., Gerbes, S., Viernstein, H., Gabor, F., & Toegel, S. (2008). Validation of reference genes for qPCR studies on Caco-2 cell differentiation. European Journal of Pharmaceutics and Biopharmaceutics, 69, 1187–92.

    Article  CAS  PubMed  Google Scholar 

  40. Pilbrow, A. P., Ellmers, L. J., Black, M. A., Moravec, C. S., Sweet, W. E., Troughton, R. W., et al. (2008). Genomic selection of reference genes for real-time PCR in human myocardium. BMC Medical Genomics, 29, 64.

    Article  Google Scholar 

  41. Puente, L. G., Borris, D. J., Carriere, J. F., Kelly, J. F., & Megeney, L. A. (2006). Identification of candidate regulators of embryonic stem cell differentiation by comparing phosphoprotein affinity profiling. Molecular & Cellular Proteomics, 5, 57–67.

    Article  CAS  Google Scholar 

  42. Li, Y., Kang, X., Guo, K., Li, X., Gao, D., Cui, J., et al. (2009). Proteome alteration of early-stage differentiation of mouse embryonic stem cells into hepatocyte-like cells. Electrophoresis, 30, 1431–1440.

    Article  CAS  PubMed  Google Scholar 

  43. Marcu, M. G., Doyle, M., Bertolotti, A., Ron, D., Hendershot, L., & Neckers, L. (2002). Heat shock protein 90 modulates the unfolded protein response by stabilizing IRE1. Molecular and Cellular Biology, 22, 8506–8513.

    Article  CAS  PubMed  Google Scholar 

  44. Ni, M., & Lee, A. S. (2007). ER chaperones in mammalian development and human diseases. FEBS Letters, 581, 3641–3651.

    Article  CAS  PubMed  Google Scholar 

  45. Schroder, M., & Kaufman, R. (2005). The mammalian unfolded protein response. Annual Review of Biochemistry, 74, 739–789.

    Article  PubMed  Google Scholar 

  46. Kimata, Y., Ishiwata-Kimata, Y., Ito, T., Hirata, A., Suzuki, T., Oikawa, D., et al. (2007). Two regulatory steps of ER-stress sensor Ire1 involving its cluster formation and interaction with unfolded proteins. Journal of Cell Biology, 179, 75–86.

    Article  CAS  PubMed  Google Scholar 

  47. Shen, J., Snapp, E. L., Lippincott-Schwartz, J., & Prywes, R. (2005). Stable binding of ATF6 to BiP in the endoplasmic reticulum stress response. Molecular and Cellular Biology, 25, 921–932.

    Article  PubMed  Google Scholar 

  48. Sommer, T., & Jarosch, E. (2002). BiP binding keeps ATF6 at bay. Developmental Cell, 25, 1–2.

    Article  Google Scholar 

  49. Yamamoto, K., Yoshida, H., Kokame, K., Kaufman, R. J., & Mori, K. (2004). Differential contributions of ATF6 and XBP1 to the activation of endoplasmic reticulum stress-responsive cis-acting elements ERSE, UPRE and ERSE-II. Journal of Biochemistry, 136, 343–350.

    Article  CAS  PubMed  Google Scholar 

  50. Jones, D. L., & Wagers, A. J. (2008). No place like home: anatomy and function of the stem cell niche. Nature Reviews. Molecular Cell Biology, 9, 11–21.

    Article  CAS  PubMed  Google Scholar 

  51. Stewart, M. H., Bendall, S. C., & Bhatia, M. (2008). Deconstructing human embryonic stem cell cultures: niche regulation of self-renewal and pluripotency. Journal of Molecular Medicine, 86, 875–886.

    Article  PubMed  Google Scholar 

  52. Abeyta, M. J., Clark, A. T., Rodriguez, R. T., Bodnar, M. S., Pera, R. A., & Firpo, M. T. (2004). Unique gene expression signatures of independently-derived human embryonic stem cell lines. Human Molecular Genetics, 13, 601–608.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors would like to thank Peter W. Andrews for technical assistance. This work was supported by grants FIS PI051707 and FIS PI080566 from the Fondo de Investigacion Sanitaria (Instituto de Salud Carlos III), Ministry of Science and Innovation (Spain). P.M was funded by Consejería de Innovación (P08-CTS-3678) de la Junta de Andalucía and FIS (PI070026).

Author information

Authors and Affiliations

  1. Histocompatibility and Transplantation Unit, Hospital Universitario Central de Asturias, 33006, Oviedo, Spain

    Miguel Angel Blanco-Gelaz, Beatriz Suarez-Alvarez, Jose Ramon Vidal-Castiñeira & Carlos Lopez-Larrea

  2. Andalusian Stem Cell Bank, Instituto de Investigación Biomédica, University of Granada, Granada, Spain

    Gertrudis Ligero, Laura Sanchez & Pablo Menendez

  3. Department of Molecular Genetics, Hospital Universitario Central de Asturias, Oviedo, Spain

    Eliecer Coto

  4. Centre for Stem Cell Biology, University of Sheffield, Western Bank, Sheffield, UK

    Harry Moore

  5. Red de Investigacion Renal—REDinREN, Instituto de Salud “Carlos III”, Madrid, Spain

    Eliecer Coto & Carlos Lopez-Larrea

  6. Fundacion Renal “Iñigo Alvarez de Toledo”, Madrid, Spain

    Eliecer Coto & Carlos Lopez-Larrea

Authors
  1. Miguel Angel Blanco-Gelaz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Beatriz Suarez-Alvarez
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Gertrudis Ligero
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Laura Sanchez
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jose Ramon Vidal-Castiñeira
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Eliecer Coto
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Harry Moore
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Pablo Menendez
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Carlos Lopez-Larrea
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Carlos Lopez-Larrea.

Additional information

An erratum to this article can be found at http://dx.doi.org/10.1007/s12015-010-9207-5

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplemental Data 1

(XLS 26 kb)

Supplemental Data 2

(XLS 15 kb)

Supplemental Data 3

(XLS 33 kb)

Supplemental Data 4

(XLS 32 kb)

Supplemental Data 5

(XLS 33 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Blanco-Gelaz, M.A., Suarez-Alvarez, B., Ligero, G. et al. Endoplasmic Reticulum Stress Signals in Defined Human Embryonic Stem Cell Lines and Culture Conditions. Stem Cell Rev and Rep 6, 462–472 (2010). https://doi.org/10.1007/s12015-010-9135-4

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12015-010-9135-4

Keywords

Search

Navigation