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Stem Cell Reviews and Reports

, Volume 6, Issue 3, pp 450–461 | Cite as

Selective Removal of Undifferentiated Embryonic Stem Cells from Differentiation Cultures Through HSV1 Thymidine Kinase and Ganciclovir Treatment

  • Ortwin NaujokEmail author
  • Joanna Kaldrack
  • Terbish Taivankhuu
  • Anne Jörns
  • Sigurd Lenzen
Article

Abstract

Pluripotent cell lines such as embryonic stem cells are an attractive source for a potential cell replacement therapy. However, transplantation of differentiated cells harbors the risk of teratoma formation, presenting a serious health risk. To overcome this obstacle, a negative selection system was established that permits selective removal of undifferentiated cells during in vitro differentiation. Use of the HSV1 thymidine kinase and eGFP under the control of the Oct4 promoter allowed the destruction of undifferentiated ES cells by ganciclovir treatment; differentiated cells were unharmed. Clonal ES cells remained pluripotent and showed positive staining for a wide range of embryonic markers. Thus, treatment with ganciclovir during in vitro differentiation effectively removed the population of undifferentiated cells and provided a pure population of completely differentiated cells. This approach may pave the way for a safe application of ES cells in regenerative medicine in the future.

Keywords

Embryonic stem cells Insulin-producing cells Teratoma Ganciclovir treatment 

Notes

Acknowledgements

This work was supported by funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) for the Cluster of Excellence REBIRTH (From Regenerative Biology to Reconstructive Therapy).

Conflicts of Interest

The authors declare no potential conflicts of interest.

Supplementary material

12015_2010_9148_MOESM1_ESM.doc (48 kb)
Supplemental Table 1 Primers used for qPCR. Fw – forward (sense) primer; Rv – reverse (antisense) primer. Amplicons were designed exon-spanning and were in a size ranging from 116–266 bp. (DOC 48 kb)
12015_2010_9148_MOESM2_ESM.doc (184 kb)
Supplemental Fig. 1 Concentration-dependent toxicity against ganciclovir after treatment for 24 h to 72 h in undifferentiated eHSVtk and control cells. Shown is the concentration-dependent toxicity of ganciclovir from 0.01 to 10 μM in eHSVtk cells compared to ES-D3 control cells. (A–C). After 24 h of treatment, toxicity of ganciclovir is low in eHSVtk cells (A) while after 48 h and 72 h the toxicity increases (B, C). Control cells remained unaffected by ganciclovir treatment (A, B, C). Values shown are means + SEM of the viability measured by MTT-assay in 4–5 independent experiments. (DOC 183 kb)
12015_2010_9148_MOESM3_ESM.doc (1.2 mb)
Supplemental Fig. 2 Treatment of eHSVtk after in vitro differentiation with 5 and 10 μM GCV exhibits no additional decrease of eGFP-positive or SSEA-1 positive cells in comparison to treatment with 1 μM GCV. (A–D) Comparison of eGFP-expression in untreated cells (A) and eHSVtk cells treated with 1 μM (B), 5 μM (C) and 10 μM (D) for seven days during the differentiation. (E) SSEA-1 staining of untreated eHSVtk cells and cells treated with 1, 5 and 10 μM ganciclovir. (DOC 1263 kb)
12015_2010_9148_MOESM4_ESM.doc (736 kb)
Supplemental Fig. 3 Decrease of alkaline phosphatase positive cells after 7 days and complete removal of alkaline phosphatase positive cells after 14 days of treatment with ganciclovir. Representive bright field contrast image of eHSVtk cells fixed and stained for alkaline phosphatase (red) after 19 or 26 days of differentiation comprising 7 or 14 days of treatment with 1 μM ganciclovir compared to untreated cells. (DOC 735 kb)

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Ortwin Naujok
    • 1
    Email author
  • Joanna Kaldrack
    • 1
  • Terbish Taivankhuu
    • 1
  • Anne Jörns
    • 1
    • 2
  • Sigurd Lenzen
    • 1
  1. 1.Institute of Clinical BiochemistryHannover Medical SchoolHannoverGermany
  2. 2.Center of AnatomyHannover Medical SchoolHannoverGermany

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