In Vitro Detection of Residual Undifferentiated Cells in Retinal Pigment Epithelial Cells Derived from Human Induced Pluripotent Stem Cells

  • Takuya Kuroda
  • Satoshi Yasuda
  • Yoji SatoEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1210)


Human pluripotent stem cells (hPSCs) such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are a leading candidate for regenerative medicine/cell therapies because of their capacity for pluripotency and unlimited self-renewal. However, there are significant obstacles preventing the clinical use of hPSCs. A significant safety issues is the presence of residual undifferentiated cells that have the potential to form tumors in vivo. Here, we describe the highly sensitive qRT-PCR methods for detection of residual undifferentiated cells in retinal pigment epithelial (RPE) cells derived from hiPSCs. qRT-PCR using probes and primers targeting LIN28A (LIN28) transcripts can detect residual undifferentiated cell levels as low as 0.002 % in hiPSC-derived RPE cells. We expect this method to contribute to process validation and quality control of hiPSC-derived cell therapy product.

Key words

Human induced pluripotent stem cells Retinal pigment epithelial cells Tumorigenicity LIN28 qRT-PCR 


  1. 1.
    Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM (1998) Embryonic stem cell lines derived from human blastocysts. Science 282(5391): 1145–1147PubMedCrossRefGoogle Scholar
  2. 2.
    Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131(5): 861–872. doi: 10.1016/j.cell.2007.11.019 PubMedCrossRefGoogle Scholar
  3. 3.
    Lu B, Malcuit C, Wang S, Girman S, Francis P, Lemieux L, Lanza R, Lund R (2009) Long-term safety and function of RPE from human embryonic stem cells in preclinical models of macular degeneration. Stem Cells 27(9):2126–2135. doi: 10.1002/stem.149 PubMedCrossRefGoogle Scholar
  4. 4.
    Ben-David U, Benvenisty N (2011) The tumorigenicity of human embryonic and induced pluripotent stem cells. Nat Rev Cancer 11(4): 268–277. doi: 10.1038/nrc3034 PubMedCrossRefGoogle Scholar
  5. 5.
    Knoepfler PS (2009) Deconstructing stem cell tumorigenicity: a roadmap to safe regenerative medicine. Stem Cells 27(5):1050–1056. doi: 10.1002/stem.37 PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Hentze H, Soong PL, Wang ST, Phillips BW, Putti TC, Dunn NR (2009) Teratoma formation by human embryonic stem cells: evaluation of essential parameters for future safety studies. Stem Cell Res 2(3):198–210. doi: 10.1016/j.scr.2009.02.002 PubMedCrossRefGoogle Scholar
  7. 7.
    Noaksson K, Zoric N, Zeng X, Rao MS, Hyllner J, Semb H, Kubista M, Sartipy P (2005) Monitoring differentiation of human embryonic stem cells using real-time PCR. Stem Cells 23(10):1460–1467. doi: 10.1634/stemcells.2005-0093 PubMedCrossRefGoogle Scholar
  8. 8.
    Wright AJ, Andrews PW (2009) Surface marker antigens in the characterization of human embryonic stem cells. Stem Cell Res 3(1): 3–11. doi: 10.1016/j.scr.2009.04.001 PubMedCrossRefGoogle Scholar
  9. 9.
    Kuroda T, Yasuda S, Kusakawa S, Hirata N, Kanda Y, Suzuki K, Takahashi M, Nishikawa S, Kawamata S, Sato Y (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):e37342. doi: 10.1371/journal.pone. 0037342 PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Osakada F, Jin ZB, Hirami Y, Ikeda H, Danjyo T, Watanabe K, Sasai Y, Takahashi M (2009) In vitro differentiation of retinal cells from human pluripotent stem cells by small-molecule induction. J Cell Sci 122(Pt 17):3169–3179. doi: 10.1242/jcs.050393 PubMedCrossRefGoogle Scholar
  11. 11.
    Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, Ichisaka T, Aoi T, Okita K, Mochiduki Y, Takizawa N, Yamanaka S (2008) Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol 26(1):101–106. doi:  10.1038/nbt1374 PubMedCrossRefGoogle Scholar
  12. 12.
    James NM, Jane CM (2005) Statistics and Chemometrics for Analytical Chemistry Fifth edition. Harlow: Person Education LimitedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Division of Cellular and Gene Therapy ProductsNational Institute of Health SciencesSetagayaJapan

Personalised recommendations