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Molecular Biology Reports

, Volume 46, Issue 6, pp 6675–6683 | Cite as

Detection of small numbers of iPSCs in different heterogeneous cell mixtures with highly sensitive droplet digital PCR

  • A. S. ArtyuhovEmail author
  • E. B. Dashinimaev
  • N. V. Mescheryakova
  • A. A. Ashikhmina
  • E. A. Vorotelyak
  • A. V. Vasiliev
Methods Paper

Abstract

Induced pluripotent stem cells (iPS cells) are a prospective resource for regenerative biomedicine. iPS cells can differentiate into any type of stem, progenitor and somatic cells to help replace structures within damaged organs or tissues. However, iPS cells themselves, can produce malignant tumors if they are injected into the body of an immunocompatible or immunodeficient recipient. Thus, it is necessary to detect any residual iPS cells content in biomedical cell products obtained from iPS cells and destined for transplantation. In this article we describe searches for iPS cells in heterogeneous cell mixtures, using two different methods—quantitative RT-PCR and droplet digital PCR (ddPCR). In experiments with various heterogeneous mixtures, including mixtures with neural stem cells, we found that the OCT4, TDGF1 and LIN28 genes are the best markers for such a search, and droplet digital PCR provides the greatest measurement accuracy, which is 0.002%. Thus, we have confirmed the advantage of using droplet digital PCR in the search for pluripotent stem cells in heterogeneous cell mixtures. We hope that this data can be useful for biosafety control in regenerative biomedicine.

Keywords

iPS cells Induced pluripotent stem cells Droplet digital PCR ddPCR OCT4 TDGF1 LIN28 Neural progenitor cells NPC 

Notes

Acknowledgements

The authors would like to thank M.A. Lagarkova, E.E. Egorov and O.S.Rogovaya for providing the hES-MK05, 1608-hT and PFCH-O cell lines respectively. The authors also would like to thank Anna Smirnova and Anrey Verner for help with ddPCR performance.

Funding

This research was funded by the IDB RAS government program of basic research № 0108-2019-0004. The part of the work on neural stem cells has been financially supported by Russian Science Foundation (Grant No. 17-75-20178). The work of Dashinimaev E.B. was supported by a grant to creating the “Center for Precise Gene Editing and Genetic Technologies for Biomedicine” of the Ministry of Science and Higher Education of the Russian Federation. The funders had no role in study design, data analysis and interpretation or manuscript writing.

Compliance with ethical standards

Conflict of interest

The authors have declared that there is no conflict of interest.

Supplementary material

11033_2019_5100_MOESM1_ESM.docx (1.7 mb)
Supplementary material 1 (DOCX 1776 kb)

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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Pirogov Russian National Research Medical UniversityMoscowRussia
  2. 2.Moscow Institute of Physics and Technology (State University)Moscow RegionRussia
  3. 3.Koltzov Institute of Developmental Biology, Russian Academy of SciencesMoscowRussia
  4. 4.RUDN UniversityMoscowRussia
  5. 5.Lomonosov Moscow State UniversityMoscowRussia

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