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Laser-Assisted Photoablation of Human Pluripotent Stem Cells from Differentiating Cultures

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Abstract

Due to their pluripotency and their self-renewal capacity, human pluripotent stem cells (hPSC) provide fascinating perspectives for biomedical applications. In the long term, hPSC-derived tissue-specific cells will constitute an important source for cell replacement therapies in non-regenerative organs. These therapeutic approaches, however, will critically depend on the purity of the in vitro differentiated cell populations. In particular, remaining undifferentiated hPSC in a transplant can induce teratoma formation. In order to address this challenge, we have developed a laser-based method for the ablation of hPSC from differentiating cell cultures. Specific antibodies were directed against the hPSC surface markers tumor related antigen (Tra)-1-60 and Tra-1-81. These antibodies, in turn, were targeted with nanogold particles. Subsequent laser exposure resulted in a 98,9 ± 0,9% elimination of hPSCs within undifferentiated cell cultures. In order to study potential side effects of laser ablation on cells negative for Tra-1-60 and Tra-1-81, hPSC were mixed with GFP-positive hPSC-derived neural precursors (hESCNP) prior to ablation. These studies showed efficient elimination of hPSC while co-treated hESCNP maintained their normal proliferation and differentiation potential. In vivo transplantation of treated and untreated mixed hPSC/hESCNP cultures revealed that laser ablation can dramatically reduce the risk of teratoma formation. Laser-assisted photothermolysis thus represents a novel contact-free method for the efficient elimination of hPSC from in vitro differentiated hPSC-derived somatic cell populations.

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Acknowledgements

We are grateful to Michal Amit and Joseph Itskovitz-Eldor for providing the hESC line H9.2. We would like to thank Björn Scheffler (Institute of Reconstructive Neurobiology, Life & Brain Center, University of Bonn) for help with teratoma analysis. This work was supported by the German Bundesministerium für Bildung und Forschung (grant no. 13N8854), the DFG (SFB-TR3 D2), the EU 6FP Project NeuroScreen (LSHB-CT-2007-037766) and the Hertie Foundation.

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Authors and Affiliations

  1. Institute of Reconstructive Neurobiology, Life & Brain Center, University of Bonn and Hertie Foundation, Sigmund-Freud-Str. 25, 53105, Bonn, Germany

    Stefanie Terstegge, Claudia Schwarz, Anke Leinhaas, Simone Haupt, Philipp Koch & Oliver Brüstle

  2. Life & Brain GmbH, Sigmund-Freud-Str. 25, 53127, Bonn, Germany

    Stefanie Terstegge, Franziska Winter, Barbara H. Rath, Iris Laufenberg, Simone Haupt & Oliver Brüstle

  3. Institute of Molecular Medicine, University of Bonn, Sigmund-Freud-Str. 25, 53127, Bonn, Germany

    Florian Levold, Andreas Dolf & Elmar Endl

Authors
  1. Stefanie Terstegge
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  2. Franziska Winter
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  3. Barbara H. Rath
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  4. Iris Laufenberg
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  7. Florian Levold
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  8. Andreas Dolf
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  9. Simone Haupt
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  10. Philipp Koch
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  11. Elmar Endl
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  12. Oliver Brüstle
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Corresponding author

Correspondence to Oliver Brüstle.

Additional information

Stefanie Terstegge and Franziska Winter contributed equally to this work.

This work was supported by the German Bundesministerium für Bildung und Forschung (grant no. 13N8854), the DFG (SFB-TR3 D2), the EU 6FP Project NeuroScreen (LSHB-CT-2007-037766) and the Hertie Foundation.

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Supplemental Fig. S1

Photothermolysis competes well with established cell purification techniques. Neural precursor cells were purified from mixed suspensions of hESCs and hESCNPs (1:1) by photothermolysis, MACS® or FACSort. For MACS, cells were stained with both Tra-1-60 and Tra-1-81 antibodies for 20 min on ice. Subsequently, cells were resuspended in MACS buffer and incubated with rat-α-mouse IgM MicroBeads for 15 min at 4°C and secondary antibody (goat-α-mouse-Cy5) for further 10 min. Cells were separated with an autoMACS Separator using the “depletion s” program and Tra-1-60/Tra-1-81-positive (hESCs) and -negative (hESCNPs, feeder cells) fractions were collected and analysed by flow cytometry. For FACS, cells were stained with both Tra-1-60 and Tra-1-81 antibodies as described in the Material and Methods section and sorted with a BD FACSDiva cell sorter using a 90 µm nozzle. The Sorter was running on 20 psi sheat fluid pressure, 40 kHz drop drive frequency and an analysis rate of 5.000 events per second. Re-analysis was performed by using propidium iodide (PI) staining for dead cells and cell counting. Compared to MACS® and FACSort, photothermolysis resulted in higher yield, i.e. higher recovery of target cells (A) and similar or better survival rates (B). Please note that data for MACS and FACS were generated in a routine setup and might be further optimized for better yield and survival, e.g. by varying magnetic bead concentration or column material for MACS and sorting speed or sort modus for FACS (GIF 222 kb)

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Terstegge, S., Winter, F., Rath, B.H. et al. Laser-Assisted Photoablation of Human Pluripotent Stem Cells from Differentiating Cultures. Stem Cell Rev and Rep 6, 260–269 (2010). https://doi.org/10.1007/s12015-010-9114-9

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