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.
This is a preview of subscription content, log in to check access.
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.
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.
Muratore CR et al (2014) Comparison and optimization of hiPS cells forebrain cortical differentiation protocols. PLoS ONE 9(8):e105807CrossRefGoogle Scholar
Chambers SM et al (2009) Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol 27(3):275–280CrossRefGoogle Scholar
Gong J et al (2015) Differentiation of human protein-induced pluripotent stem cells toward a retinal pigment epithelial cell fate. PLoS ONE 10(11):e0143272CrossRefGoogle Scholar
Rowland TJ et al (2013) Differentiation of human pluripotent stem cells to retinal pigmented epithelium in defined conditions using purified extracellular matrix proteins. J Tissue Eng Regen Med 7(8):642–653CrossRefGoogle Scholar
Zhong X et al (2014) Generation of three-dimensional retinal tissue with functional photoreceptors from human iPS cellss. Nat Commun 5:4047CrossRefGoogle Scholar
Leach LL et al (2016) Induced pluripotent stem cell-derived retinal pigmented epithelium: a comparative study between cell lines and differentiation methods. J Ocul PharmacolTher 32(5):317–330CrossRefGoogle Scholar
Kaserman JE, Wilson AA (2017) Protocol for directed differentiation of human induced pluripotent stem cells (iPS cellss) to a hepatic lineage. Methods Mol Biol 1639:151–160CrossRefGoogle Scholar
Roy-Chowdhury N et al (2017) Hepatocyte-like cells derived from induced pluripotent stem cells. Hepatol Int 11(1):54–69CrossRefGoogle Scholar
Ezashi T et al (2009) Derivation of induced pluripotent stem cells from pig somatic cells. Proc Natl Acad Sci USA 106(27):10993–10998CrossRefGoogle Scholar
Takahashi K et al (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131(5):861–872CrossRefGoogle Scholar
Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126(4):663–676CrossRefGoogle Scholar
Egorov EE et al (2003) Telomerization as a method of obtaining immortal human cells preserving normal properties. Ontogenez 34(3):183–192PubMedGoogle Scholar
Dashinimaev EB et al (2017) Neurons derived from induced pluripotent stem cells of patients with down syndrome reproduce early stages of Alzheimer’s disease type pathology in vitro. J Alzheimer’s Dis 56(2):835–847CrossRefGoogle Scholar
Artyukhov AS et al (2017) New genes for accurate normalization of qRT-PCR results in study of iPS and iPS-derived cells. Gene 626:234–240CrossRefGoogle Scholar
Kuroda T et al (2015) Highly sensitive droplet digital PCR method for detection of residual undifferentiated cells in cardiomyocytes derived from human pluripotent stem cells. Regen Ther 2:17–23CrossRefGoogle Scholar
Pillai V, Cibas ES, Dorfman DM (2013) A simplified flow cytometric immunophenotyping procedure for the diagnosis of effusions caused by epithelial malignancies. Am J Clin Pathol 139(5):672–681CrossRefGoogle Scholar
Inocencio J, Frenster JD, Placantonakis DG (2018) Isolation of glioblastoma stem cells with flow cytometry. Methods Mol Biol 1741:71–79CrossRefGoogle Scholar
Jagric T et al (2018) Can flow cytometry reinvent the sentinel lymph node concept in gastric cancer patients? J Surg Res 223:46–57CrossRefGoogle Scholar
Sykes PJ et al (1992) Quantitation of targets for PCR by use of limiting dilution. BioTech 13(3):444–449Google Scholar
Sclafani F et al (2018) KRAS and BRAF mutations in circulating tumour DNA from locally advanced rectal cancer. Sci Rep 8(1):1445CrossRefGoogle Scholar
Hirano M et al (2018) A novel high-sensitivity assay to detect a small fraction of mutant IDH1 using droplet digital PCR. Brain Tumor Pathol 35(2):97–105CrossRefGoogle Scholar
Nystrand CF et al (2018) JAK2 V617F mutation can be reliably detected in serum using droplet digital PCR. Int J Lab Hematol 40(2):181–186CrossRefGoogle Scholar
Pohl G, Shih I-M (2004) Principle and applications of digital PCR. Expert Rev Mol Diagn 4(1):41–47CrossRefGoogle Scholar
Vandesompele J et al (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3(7):RESEARCH0034CrossRefGoogle Scholar
Fagerberg L et al (2014) Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics. Mol Cell Proteom 13(2):397–406CrossRefGoogle Scholar
Kuroda T et al (2012) Highly sensitive in vitro methods for detection of residual undifferentiated cells in retinal pigment epithelial cells derived from human iPS cells. PLoS ONE 7(5):e37342CrossRefGoogle Scholar