Stem Cell Reviews and Reports

, Volume 11, Issue 2, pp 357–372 | Cite as

Current Methods and Challenges in the Comprehensive Characterization of Human Pluripotent Stem Cells

  • Joanna S. T. Asprer
  • Uma LakshmipathyEmail author


Pluripotent stem cells (PSCs) are powerful tools for basic scientific research and promising agents for drug discovery and regenerative medicine. Technological advances have made it increasingly easy to generate PSCs but the various lines generated may differ in their characteristics based on their origin, derivation, number of passages, and culture conditions. In order to confirm the pluripotency, quality, identity, and safety of pluripotent cell lines as they are derived and maintained, it is critical to perform a panel of characterization assays. Functional pluripotency is determined using tests that rely on the expression of specific markers in the undifferentiated and differentiated states; tests for quality, identity and safety are less specialized. This article provides a comprehensive review of current practices in PSC characterization and explores challenges in the field, from the selection of markers to the development of simple and scalable methods. It also delves into emerging trends like the adoption of alternative assays that could be used to supplement or replace traditional methods, specifically the use of in silico assays for determining pluripotency.


Pluripotent stem cells Embryonic stem cells Induced pluripotent stem cells PSC ESC iPSC Characterization 



We would like to thank J. Fergus, C. MacArthur, R. Quintanilla, M. Sridharan, and K. Sylakowski for the helpful discussions.

Conflict of Interest

Both authors, JA and UL, are employed by Thermo Fisher Scientific and receive financial compensation. This does not alter their adherence to all the policies on sharing data and materials.


  1. 1.
    Fusaki, N., Ban, H., Nishiyama, A., Saeki, K., & Hasegawa, M. (2009). Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proceedings of the Japan Academy. Series B, Physical and Biological Sciences, 85, 348–62.PubMedCentralPubMedGoogle Scholar
  2. 2.
    Yoshioka, N., Gros, E., Li, H. R., et al. (2013). Efficient generation of human iPSCs by a synthetic self-replicative RNA. Cell Stem Cell, 13, 246–54.PubMedGoogle Scholar
  3. 3.
    Yu, J., Hu, K., Smuga-Otto, K., et al. (2009). Human induced pluripotent stem cells free of vector and transgene sequences. Science, 324, 797–801.PubMedCentralPubMedGoogle Scholar
  4. 4.
    Hu, K., Yu, J., Suknuntha, K., et al. Efficient generation of transgene-free induced pluripotent stem cells from normal and neoplastic bone marrow and cord blood mononuclear cells. Blood;117:e109-19Google Scholar
  5. 5.
    Okita, K., Yamakawa, T., Matsumura, Y., et al An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells. Stem Cells;31:458–66.Google Scholar
  6. 6.
    Abeyta, M. J., Clark, A. T., Rodriguez, R. T., Bodnar, M. S., Pera, R. A., & Firpo, M. T. (2004). Unique gene expression signatures of independently-derived human embryonic stem cell lines. Human Molecular Genetics, 13, 601–8.PubMedGoogle Scholar
  7. 7.
    Bock, C., Kiskinis, E., Verstappen, G., et al. (2011). Reference Maps of human ES and iPS cell variation enable high-throughput characterization of pluripotent cell lines. Cell, 144, 439–52.PubMedCentralPubMedGoogle Scholar
  8. 8.
    Cheng, L., Hansen, N. F., Zhao, L., et al. (2012). Low incidence of DNA sequence variation in human induced pluripotent stem cells generated by nonintegrating plasmid expression. Cell Stem Cell, 10, 337–44.PubMedCentralPubMedGoogle Scholar
  9. 9.
    Draper, J. S., Smith, K., Gokhale, P., et al. (2004). Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells. Nature Biotechnology, 22, 53–4.PubMedGoogle Scholar
  10. 10.
    Enver, T., Soneji, S., Joshi, C., et al. (2005). Cellular differentiation hierarchies in normal and culture-adapted human embryonic stem cells. Human Molecular Genetics, 14, 3129–40.PubMedGoogle Scholar
  11. 11.
    Vitale, A. M., Matigian, N. A., Ravishankar, S., et al. (2012). Variability in the generation of induced pluripotent stem cells: importance for disease modeling. Stem Cells Translational Medicine, 1, 641–50.PubMedCentralPubMedGoogle Scholar
  12. 12.
    Marti, M., Mulero, L., Pardo, C., et al. (2013). Characterization of pluripotent stem cells. Nature Protocols, 8, 223–53.PubMedGoogle Scholar
  13. 13.
    Meissner, A., Wernig, M., & Jaenisch, R. (2007). Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells. Nature Biotechnology, 25, 1177–81.PubMedGoogle Scholar
  14. 14.
    Singh, U., Quintanilla, R. H., Grecian, S., Gee, K. R., Rao, M. S., & Lakshmipathy, U. (2012). Novel live alkaline phosphatase substrate for identification of pluripotent stem cells. Stem Cell Reviews, 8, 1021–9.PubMedCentralPubMedGoogle Scholar
  15. 15.
    Takahashi, K., Tanabe, K., Ohnuki, M., et al. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 131, 861–72.PubMedGoogle Scholar
  16. 16.
    Adewumi, O., Aflatoonian, B., Ahrlund-Richter, L., et al. (2007). Characterization of human embryonic stem cell lines by the International Stem Cell Initiative. Nature Biotechnology, 25, 803–16.PubMedGoogle Scholar
  17. 17.
    ISCBI. (2009). Consensus guidance for banking and supply of human embryonic stem cell lines for research purposes. Stem Cell Reviews, 5, 301–14.Google Scholar
  18. 18.
    Itskovitz-Eldor, J., Schuldiner, M., Karsenti, D., et al. (2000). Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Molecular Medicine, 6, 88–95.PubMedCentralPubMedGoogle Scholar
  19. 19.
    Muller, F. J., Goldmann, J., Loser, P., & Loring, J. F. (2010). A call to standardize teratoma assays used to define human pluripotent cell lines. Cell Stem Cell, 6, 412–4.PubMedGoogle Scholar
  20. 20.
    Gore, A., Li, Z., Fung, H. L., et al. (2011). Somatic coding mutations in human induced pluripotent stem cells. Nature, 471, 63–7.PubMedCentralPubMedGoogle Scholar
  21. 21.
    Hussein, S. M., Batada, N. N., Vuoristo, S., et al. (2011). Copy number variation and selection during reprogramming to pluripotency. Nature, 471, 58–62.PubMedGoogle Scholar
  22. 22.
    Laurent, L. C., Ulitsky, I., Slavin, I., et al. (2011). Dynamic changes in the copy number of pluripotency and cell proliferation genes in human ESCs and iPSCs during reprogramming and time in culture. Cell Stem Cell, 8, 106–18.PubMedCentralPubMedGoogle Scholar
  23. 23.
    Martins-Taylor, K., Nisler, B. S., Taapken, S. M., et al. (2011). Recurrent copy number variations in human induced pluripotent stem cells. Nature Biotechnology, 29, 488–91.PubMedGoogle Scholar
  24. 24.
    Mayshar, Y., Ben-David, U., Lavon, N., et al. (2010). Identification and classification of chromosomal aberrations in human induced pluripotent stem cells. Cell Stem Cell, 7, 521–31.PubMedGoogle Scholar
  25. 25.
    Stem Cell Registry. International Stem Cell Forum, 2010. (Accessed Feb 11, 2014, at
  26. 26.
    Brimble, S. N., Zeng, X., Weiler, D. A., et al. (2004). Karyotypic stability, genotyping, differentiation, feeder-free maintenance, and gene expression sampling in three human embryonic stem cell lines derived prior to August 9, 2001. Stem Cells and Development, 13, 585–97.PubMedGoogle Scholar
  27. 27.
    Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., et al. (1998). Embryonic stem cell lines derived from human blastocysts. Science, 282, 1145–7.PubMedGoogle Scholar
  28. 28.
    Wakao, S., Kitada, M., Kuroda, Y., et al. (2012). Morphologic and gene expression criteria for identifying human induced pluripotent stem cells. PLoS One, 7, e48677.PubMedCentralPubMedGoogle Scholar
  29. 29.
    Ludwig, T. E., Bergendahl, V., Levenstein, M. E., Yu, J., Probasco, M. D., & Thomson, J. A. (2006). Feeder-independent culture of human embryonic stem cells. Nature Methods, 3, 637–46.PubMedGoogle Scholar
  30. 30.
    Muller, F.J., Brandl, B., Loring J.F., (2008). Assessment of human pluripotent stem cells with PluriTest. In: StemBook. Cambridge (MA).Google Scholar
  31. 31.
    Wang, L., Schulz, T. C., Sherrer, E. S., et al. (2007). Self-renewal of human embryonic stem cells requires insulin-like growth factor-1 receptor and ERBB2 receptor signaling. Blood, 110, 4111–9.PubMedCentralPubMedGoogle Scholar
  32. 32.
    Dvash, T., Mayshar, Y., Darr, H., et al. (2004). Temporal gene expression during differentiation of human embryonic stem cells and embryoid bodies. Human Reproduction, 19, 2875–83.PubMedGoogle Scholar
  33. 33.
    Sato, N., Sanjuan, I. M., Heke, M., Uchida, M., Naef, F., & Brivanlou, A. H. (2003). Molecular signature of human embryonic stem cells and its comparison with the mouse. Developmental Biology, 260, 404–13.PubMedGoogle Scholar
  34. 34.
    Bhattacharya, B., Miura, T., Brandenberger, R., et al. (2004). Gene expression in human embryonic stem cell lines: unique molecular signature. Blood, 103, 2956–64.PubMedGoogle Scholar
  35. 35.
    Zeng, X., Miura, T., Luo, Y., et al. (2004). Properties of pluripotent human embryonic stem cells BG01 and BG02. Stem Cells, 22, 292–312.PubMedGoogle Scholar
  36. 36.
    Richards, M., Tan, S. P., Tan, J. H., Chan, W. K., & Bongso, A. (2004). The transcriptome profile of human embryonic stem cells as defined by SAGE. Stem Cells, 22, 51–64.PubMedGoogle Scholar
  37. 37.
    Sperger, J. M., Chen, X., Draper, J. S., et al. (2003). Gene expression patterns in human embryonic stem cells and human pluripotent germ cell tumors. Proceedings of the National Academy of Sciences of the United States of America, 100, 13350–5.PubMedCentralPubMedGoogle Scholar
  38. 38.
    Josephson, R., Ording, C. J., Liu, Y., et al. (2007). Qualification of embryonal carcinoma 2102Ep as a reference for human embryonic stem cell research. Stem Cells, 25, 437–46.PubMedGoogle Scholar
  39. 39.
    Houbaviy, H. B., Murray, M. F., & Sharp, P. A. (2003). Embryonic stem cell-specific MicroRNAs. Developmental Cell, 5, 351–8.PubMedGoogle Scholar
  40. 40.
    Lakshmipathy, U., Davila, J., & Hart, R. P. (2010). miRNA in pluripotent stem cells. Regenerative Medicine, 5, 545–55.PubMedCentralPubMedGoogle Scholar
  41. 41.
    Lakshmipathy, U., Love, B., Goff, L. A., et al. (2007). MicroRNA expression pattern of undifferentiated and differentiated human embryonic stem cells. Stem Cells and Development, 16, 1003–16.PubMedCentralPubMedGoogle Scholar
  42. 42.
    Suh, M. R., Lee, Y., Kim, J. Y., et al. (2004). Human embryonic stem cells express a unique set of microRNAs. Developmental Biology, 270, 488–98.PubMedGoogle Scholar
  43. 43.
    Chambers, I., Colby, D., Robertson, M., et al. (2003). Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell, 113, 643–55.PubMedGoogle Scholar
  44. 44.
    Mitsui, K., Tokuzawa, Y., Itoh, H., et al. (2003). The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell, 113, 631–42.PubMedGoogle Scholar
  45. 45.
    Baldassarre, G., Bianco, C., Tortora, G., et al. (1996). Transfection with a CRIPTO anti-sense plasmid suppresses endogenous CRIPTO expression and inhibits transformation in a human embryonal carcinoma cell line. International Journal of Cancer, 66, 538–43.Google Scholar
  46. 46.
    Nichols, J., Zevnik, B., Anastassiadis, K., et al. (1998). Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell, 95, 379–91.PubMedGoogle Scholar
  47. 47.
    Andrews, P. W. (2002). From teratocarcinomas to embryonic stem cells. Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences, 357, 405–17.PubMedCentralPubMedGoogle Scholar
  48. 48.
    Draper, J. S., Pigott, C., Thomson, J. A., & Andrews, P. W. (2002). Surface antigens of human embryonic stem cells: changes upon differentiation in culture. Journal of Anatomy, 200, 249–58.PubMedCentralPubMedGoogle Scholar
  49. 49.
    Henderson, J. K., Draper, J. S., Baillie, H. S., et al. (2002). Preimplantation human embryos and embryonic stem cells show comparable expression of stage-specific embryonic antigens. Stem Cells, 20, 329–37.PubMedGoogle Scholar
  50. 50.
    Pera, M. F., Reubinoff, B., & Trounson, A. (2000). Human embryonic stem cells. Journal of Cell Science, 113(Pt 1), 5–10.PubMedGoogle Scholar
  51. 51.
    Chan, E. M., Ratanasirintrawoot, S., Park, I. H., et al. (2009). Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells. Nature Biotechnology, 27, 1033–7.PubMedGoogle Scholar
  52. 52.
    Takahashi, K., & Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126, 663–76.PubMedGoogle Scholar
  53. 53.
    Ramirez, J. M., Gerbal-Chaloin, S., Milhavet, O., et al. (2011). Brief report: benchmarking human pluripotent stem cell markers during differentiation into the three germ layers unveils a striking heterogeneity: all markers are not equal. Stem Cells, 29, 1469–74.PubMedGoogle Scholar
  54. 54.
    Muthusamy, T., Mukherjee, O., Menon, R., Megha, P. B., & Panicker, M. M. (2014). A method to identify and isolate pluripotent human stem cells and mouse epiblast stem cells using lipid body-associated retinyl ester fluorescence. Stem Cell Reports, 3, 169–84.PubMedCentralPubMedGoogle Scholar
  55. 55.
    Santangelo, P., Nitin, N., & Bao, G. (2006). Nanostructured probes for RNA detection in living cells. Annals of Biomedical Engineering, 34, 39–50.PubMedGoogle Scholar
  56. 56.
    King, F. W., Liszewski, W., Ritner, C., & Bernstein, H. S. (2011). High-throughput tracking of pluripotent human embryonic stem cells with dual fluorescence resonance energy transfer molecular beacons. Stem Cells and Development, 20, 475–84.PubMedCentralPubMedGoogle Scholar
  57. 57.
    Graham, V., Khudyakov, J., Ellis, P., & Pevny, L. (2003). SOX2 functions to maintain neural progenitor identity. Neuron, 39, 749–65.PubMedGoogle Scholar
  58. 58.
    Kashyap, V., Rezende, N. C., Scotland, K. B., et al. (2009). Regulation of stem cell pluripotency and differentiation involves a mutual regulatory circuit of the NANOG, OCT4, and SOX2 pluripotency transcription factors with polycomb repressive complexes and stem cell microRNAs. Stem Cells and Development, 18, 1093–108.PubMedCentralPubMedGoogle Scholar
  59. 59.
    Kahler, D. J., Ahmad, F. S., Ritz, A., et al. (2013). Improved methods for reprogramming human dermal fibroblasts using fluorescence activated cell sorting. PLoS One, 8, e59867.PubMedCentralPubMedGoogle Scholar
  60. 60.
    Quintanilla, R. H., Jr., Asprer, J. S., Vaz, C., Tanavde, V., & Lakshmipathy, U. (2014). CD44 is a negative cell surface marker for pluripotent stem cell identification during human fibroblast reprogramming. PLoS One, 9, e85419.PubMedCentralPubMedGoogle Scholar
  61. 61.
    Cordie, T., Harkness, T., Jing, X., et al. (2014). Nanofibrous electrospun polymers for reprogramming human cells. Cellular and Molecular Bioengineering, 7, 379–93.Google Scholar
  62. 62.
    Kang, L., & Gao, S. (2012). Pluripotency of induced pluripotent stem cells. Journal of Animal Science and Biotechnology, 3, 5.PubMedCentralPubMedGoogle Scholar
  63. 63.
    Loring, J., Schwartz, P., & Wesselschmidt, R. (Eds.). (2007). Human stem cell manual. San Diego: Academic.Google Scholar
  64. 64.
    Kurosawa, H. (2007). Methods for inducing embryoid body formation: in vitro differentiation system of embryonic stem cells. Journal of Bioscience and Bioengineering, 103, 389–98.PubMedGoogle Scholar
  65. 65.
    Bauwens, C. L., Peerani, R., Niebruegge, S., et al. (2008). Control of human embryonic stem cell colony and aggregate size heterogeneity influences differentiation trajectories. Stem Cells, 26, 2300–10.PubMedGoogle Scholar
  66. 66.
    Watanabe, K., Ueno, M., Kamiya, D., et al. (2007). A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nature Biotechnology, 25, 681–6.PubMedGoogle Scholar
  67. 67.
    Nakazawa, K., Yoshiura, Y., Koga, H., & Sakai, Y. (2013). Characterization of mouse embryoid bodies cultured on microwell chips with different well sizes. Journal of Bioscience and Bioengineering, 116, 628–33.PubMedGoogle Scholar
  68. 68.
    Ng, E.S., Davis, R.P., Hatzistavrou, T., Stanley, E.G., Elefanty, A.G., (2008). Directed differentiation of human embryonic stem cells as spin embryoid bodies and a description of the hematopoietic blast colony forming assay. Curr Protoc Stem Cell Biol;Chapter 1:Unit 1D 3.Google Scholar
  69. 69.
    Kaur, J., & Tilkins, M. L. (2013). Methods for culturing human embryonic stem cells on feeders. Methods in Molecular Biology, 997, 93–113.PubMedGoogle Scholar
  70. 70.
    Pesl, M., Acimovic, I., Pribyl, J., et al. (2013). Forced aggregation and defined factors allow highly uniform-sized embryoid bodies and functional cardiomyocytes from human embryonic and induced pluripotent stem cells. Heart Vessels.Google Scholar
  71. 71.
    Phillips, B. W., Hentze, H., Rust, W. L., et al. (2007). Directed differentiation of human embryonic stem cells into the pancreatic endocrine lineage. Stem Cells and Development, 16, 561–78.PubMedGoogle Scholar
  72. 72.
    Grigoriadis, A. E., Kennedy, M., Bozec, A., et al. (2010). Directed differentiation of hematopoietic precursors and functional osteoclasts from human ES and iPS cells. Blood, 115, 2769–76.PubMedCentralPubMedGoogle Scholar
  73. 73.
    Hwang, Y., Suk, S., Lin, S., et al. (2013). Directed in vitro myogenesis of human embryonic stem cells and their in vivo engraftment. PLoS One, 8, e72023.PubMedCentralPubMedGoogle Scholar
  74. 74.
    Datta, I., Ganapathy, K., Tattikota, S. M., & Bhonde, R. (2013). Directed differentiation of human embryonic stem cell-line HUES9 to dopaminergic neurons in a serum-free defined culture niche. Cell Biology International, 37, 54–64.PubMedGoogle Scholar
  75. 75.
    Chambers, S. M., Fasano, C. A., Papapetrou, E. P., Tomishima, M., Sadelain, M., & Studer, L. (2009). Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nature Biotechnology, 27, 275–80.PubMedCentralPubMedGoogle Scholar
  76. 76.
    Chambers, S.M., Mica, Y., Lee, G., Studer, L., Tomishima, M.J., (2013). Dual-SMAD Inhibition/WNT Activation-Based Methods to Induce Neural Crest and Derivatives from Human Pluripotent Stem Cells. Methods Mol Biol.Google Scholar
  77. 77.
    Mica, Y., Lee, G., Chambers, S. M., Tomishima, M. J., & Studer, L. (2013). Modeling neural crest induction, melanocyte specification, and disease-related pigmentation defects in hESCs and patient-specific iPSCs. Cell Reports, 3, 1140–52.PubMedCentralPubMedGoogle Scholar
  78. 78.
    Borowiak, M., Maehr, R., Chen, S., et al. (2009). Small molecules efficiently direct endodermal differentiation of mouse and human embryonic stem cells. Cell Stem Cell, 4, 348–58.PubMedGoogle Scholar
  79. 79.
    Chen, S., Borowiak, M., Fox, J. L., et al. (2009). A small molecule that directs differentiation of human ESCs into the pancreatic lineage. Nature Chemical Biology, 5, 258–65.PubMedGoogle Scholar
  80. 80.
    Burridge, P. W., Keller, G., Gold, J. D., & Wu, J. C. (2012). Production of de novo cardiomyocytes: human pluripotent stem cell differentiation and direct reprogramming. Cell Stem Cell, 10, 16–28.PubMedCentralPubMedGoogle Scholar
  81. 81.
    Kattman, S. J., Witty, A. D., Gagliardi, M., et al. (2011). Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell Stem Cell, 8, 228–40.PubMedGoogle Scholar
  82. 82.
    Liu, Y., Liu, H., Sauvey, C., Yao, L., Zarnowska, E. D., & Zhang, S. C. (2013). Directed differentiation of forebrain GABA interneurons from human pluripotent stem cells. Nature Protocols, 8, 1670–9.PubMedCentralPubMedGoogle Scholar
  83. 83.
    Maroof, A. M., Keros, S., Tyson, J. A., et al. (2013). Directed differentiation and functional maturation of cortical interneurons from human embryonic stem cells. Cell Stem Cell, 12, 559–72.PubMedCentralPubMedGoogle Scholar
  84. 84.
    Menendez, L., Kulik, M. J., Page, A. T., et al. (2013). Directed differentiation of human pluripotent cells to neural crest stem cells. Nature Protocols, 8, 203–12.PubMedGoogle Scholar
  85. 85.
    Nicholas, C. R., Chen, J., Tang, Y., et al. (2013). Functional maturation of hPSC-derived forebrain interneurons requires an extended timeline and mimics human neural development. Cell Stem Cell, 12, 573–86.PubMedCentralPubMedGoogle Scholar
  86. 86.
    Shi, Y., Kirwan, P., & Livesey, F. J. (2012). Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks. Nature Protocols, 7, 1836–46.PubMedGoogle Scholar
  87. 87.
    Burridge, P. W., & Zambidis, E. T. (2013). Highly efficient directed differentiation of human induced pluripotent stem cells into cardiomyocytes. Methods in Molecular Biology, 997, 149–61.PubMedGoogle Scholar
  88. 88.
    Lian, X., Zhang, J., Azarin, S. M., et al. (2013). Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/beta-catenin signaling under fully defined conditions. Nature Protocols, 8, 162–75.PubMedCentralPubMedGoogle Scholar
  89. 89.
    Takasato, M., Er, P. X., Becroft, M., et al. (2014). Directing human embryonic stem cell differentiation towards a renal lineage generates a self-organizing kidney. Nature Cell Biology, 16, 118–26.PubMedGoogle Scholar
  90. 90.
    Xia, Y., Nivet, E., Sancho-Martinez, I., et al. (2013). Directed differentiation of human pluripotent cells to ureteric bud kidney progenitor-like cells. Nature Cell Biology, 15, 1507–15.PubMedGoogle Scholar
  91. 91.
    Roelandt, P., Pauwelyn, K. A., Sancho-Bru, P., et al. (2010). Human embryonic and rat adult stem cells with primitive endoderm-like phenotype can be fated to definitive endoderm, and finally hepatocyte-like cells. PLoS One, 5, e12101.PubMedCentralPubMedGoogle Scholar
  92. 92.
    Huang, S. X., Islam, M. N., O’Neill, J., et al. (2014). Efficient generation of lung and airway epithelial cells from human pluripotent stem cells. Nature Biotechnology, 32, 84–91.PubMedCentralPubMedGoogle Scholar
  93. 93.
    Kadzik, R. S., & Morrisey, E. E. (2012). Directing lung endoderm differentiation in pluripotent stem cells. Cell Stem Cell, 10, 355–61.PubMedCentralPubMedGoogle Scholar
  94. 94.
    Kearns, N. A., Genga, R. M., Ziller, M., et al. (2013). Generation of organized anterior foregut epithelia from pluripotent stem cells using small molecules. Stem Cell Research, 11, 1003–12.PubMedGoogle Scholar
  95. 95.
    Spence, J. R., Mayhew, C. N., Rankin, S. A., et al. (2011). Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature, 470, 105–9.PubMedCentralPubMedGoogle Scholar
  96. 96.
    Van Hoof, D., & Liku, M. E. (2013). Directed differentiation of human pluripotent stem cells along the pancreatic endocrine lineage. Methods in Molecular Biology, 997, 127–40.PubMedGoogle Scholar
  97. 97.
    Lancaster, M. A., Renner, M., Martin, C. A., et al. (2013). Cerebral organoids model human brain development and microcephaly. Nature, 501, 373–9.PubMedGoogle Scholar
  98. 98.
    McCracken, K. W., Howell, J. C., Wells, J. M., & Spence, J. R. (2011). Generating human intestinal tissue from pluripotent stem cells in vitro. Nature Protocols, 6, 1920–8.PubMedCentralPubMedGoogle Scholar
  99. 99.
    Tieng, V., Stoppini, L., Villy, S., Fathi, M., Dubois-Dauphin, M., & Krause, K. H. (2014). Engineering of midbrain organoids containing long-lived dopaminergic neurons. Stem Cells and Development, 23, 1535–47.PubMedGoogle Scholar
  100. 100.
    Gertow, K., Przyborski, S., Loring, J.F., et al. (2007). Isolation of human embryonic stem cell-derived teratomas for the assessment of pluripotency. Curr Protoc Stem Cell Biol; Chapter 1:Unit1B 4.Google Scholar
  101. 101.
    Przyborski, S. A. (2005). Differentiation of human embryonic stem cells after transplantation in immune-deficient mice. Stem Cells, 23, 1242–50.PubMedGoogle Scholar
  102. 102.
    Gertow, K., Cedervall, J., Jamil, S., et al. (2011). Early events in xenograft development from the human embryonic stem cell line HS181–resemblance with an initial multiple epiblast formation. PLoS One, 6, e27741.PubMedCentralPubMedGoogle Scholar
  103. 103.
    Gertow, K., Wolbank, S., Rozell, B., et al. (2004). Organized development from human embryonic stem cells after injection into immunodeficient mice. Stem Cells and Development, 13, 421–35.PubMedGoogle Scholar
  104. 104.
    Heins, N., Englund, M. C., Sjoblom, C., et al. (2004). Derivation, characterization, and differentiation of human embryonic stem cells. Stem Cells, 22, 367–76.PubMedGoogle Scholar
  105. 105.
    Vescovi, A. L., Parati, E. A., Gritti, A., et al. (1999). Isolation and cloning of multipotential stem cells from the embryonic human CNS and establishment of transplantable human neural stem cell lines by epigenetic stimulation. Experimental Neurology, 156, 71–83.PubMedGoogle Scholar
  106. 106.
    Tannenbaum, S. E., Turetsky, T. T., Singer, O., et al. (2012). Derivation of xeno-free and GMP-grade human embryonic stem cells–platforms for future clinical applications. PLoS One, 7, e35325.PubMedCentralPubMedGoogle Scholar
  107. 107.
    Yu, J., Vodyanik, M. A., Smuga-Otto, K., et al. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science, 318, 1917–20.PubMedGoogle Scholar
  108. 108.
    Peterson, S.E., Tran, H.T., Garitaonandia, I., et al. (2011). Teratoma generation in the testis capsule. J Vis Exp: e3177.Google Scholar
  109. 109.
    Gropp, M., Shilo, V., Vainer, G., et al. (2012). Standardization of the teratoma assay for analysis of pluripotency of human ES cells and biosafety of their differentiated progeny. PLoS One, 7, e45532.PubMedCentralPubMedGoogle Scholar
  110. 110.
    Miyazono, M., Lee, V. M., & Trojanowski, J. Q. (1995). Proliferation, cell death, and neuronal differentiation in transplanted human embryonal carcinoma (NTera2) cells depend on the graft site in nude and severe combined immunodeficient mice. Laboratory Investigation, 73, 273–83.PubMedGoogle Scholar
  111. 111.
    Wakitani, S., Takaoka, K., Hattori, T., et al. (2003). Embryonic stem cells injected into the mouse knee joint form teratomas and subsequently destroy the joint. Rheumatology (Oxford), 42, 162–5.Google Scholar
  112. 112.
    Buta, C., David, R., Dressel, R., et al. (2013). Reconsidering pluripotency tests: do we still need teratoma assays? Stem Cell Research, 11, 552–62.PubMedGoogle Scholar
  113. 113.
    Morris, S. A., Cahan, P., Li, H., et al. (2014). Dissecting engineered cell types and enhancing cell fate conversion via Cell Net. Cell, 158, 889–902.PubMedGoogle Scholar
  114. 114.
    Xu, X. Q., Soo, S. Y., Sun, W., & Zweigerdt, R. (2009). Global expression profile of highly enriched cardiomyocytes derived from human embryonic stem cells. Stem Cells, 27, 2163–74.PubMedGoogle Scholar
  115. 115.
    Chin, M. H., Pellegrini, M., Plath, K., & Lowry, W. E. (2010). Molecular analyses of human induced pluripotent stem cells and embryonic stem cells. Cell Stem Cell, 7, 263–9.PubMedCentralPubMedGoogle Scholar
  116. 116.
    Chung, H. C., Lin, R. C., Logan, G. J., Alexander, I. E., Sachdev, P. S., & Sidhu, K. S. (2012). Human induced pluripotent stem cells derived under feeder-free conditions display unique cell cycle and DNA replication gene profiles. Stem Cells and Development, 21, 206–16.PubMedCentralPubMedGoogle Scholar
  117. 117.
    Ghosh, Z., Wilson, K. D., Wu, Y., Hu, S., Quertermous, T., & Wu, J. C. (2010). Persistent donor cell gene expression among human induced pluripotent stem cells contributes to differences with human embryonic stem cells. PLoS One, 5, e8975.PubMedCentralPubMedGoogle Scholar
  118. 118.
    Guenther, M. G., Frampton, G. M., Soldner, F., et al. (2010). Chromatin structure and gene expression programs of human embryonic and induced pluripotent stem cells. Cell Stem Cell, 7, 249–57.PubMedCentralPubMedGoogle Scholar
  119. 119.
    Mallon, B. S., Hamilton, R. S., Kozhich, O. A., et al. (2013). Comparison of the molecular profiles of human embryonic and induced pluripotent stem cells of isogenic origin. Stem Cell Research, 12, 376–86.PubMedCentralPubMedGoogle Scholar
  120. 120.
    Newman, A. M., & Cooper, J. B. (2010). Lab-specific gene expression signatures in pluripotent stem cells. Cell Stem Cell, 7, 258–62.PubMedGoogle Scholar
  121. 121.
    Awe, J. P., Lee, P. C., Ramathal, C., et al. (2013). Generation and characterization of transgene-free human induced pluripotent stem cells and conversion to putative clinical-grade status. Stem Cell Research & Therapy, 4, 87.Google Scholar
  122. 122.
    Brandenberger, R., Khrebtukova, I., Thies, R. S., et al. (2004). MPSS profiling of human embryonic stem cells. BMC Developmental Biology, 4, 10.PubMedCentralPubMedGoogle Scholar
  123. 123.
    Wang, Z., Gerstein, M., & Snyder, M. (2009). RNA-Seq: a revolutionary tool for transcriptomics. Nature Reviews Genetics, 10, 57–63.PubMedCentralPubMedGoogle Scholar
  124. 124.
    Patel, S. N., Wu, Y., Bao, Y., Mancebo, R., Au-Young, J., & Grigorenko, E. (2013). TaqMan(R) OpenArray(R) high-throughput transcriptional analysis of human embryonic and induced pluripotent stem cells. Methods in Molecular Biology, 997, 191–201.PubMedGoogle Scholar
  125. 125.
    Muller, F. J., Schuldt, B. M., Williams, R., et al. (2011). A bioinformatic assay for pluripotency in human cells. Nature Methods, 8, 315–7.PubMedCentralPubMedGoogle Scholar
  126. 126.
    Fergus, J., Quintanilla, R.H., Lakshmipathy, U., (2014). Characterizing pluripotent stem cells using the TaqMan® hPSC Scorecard™ Panel. Springer Protocols.Google Scholar
  127. 127.
    Cahan, P., Li, H., Morris, S. A., da Lummertz, R. E., Daley, G. Q., & Collins, J. J. (2014). CellNet: network biology applied to stem cell engineering. Cell, 158, 903–15.PubMedGoogle Scholar
  128. 128.
    Williams, R., Schuldt, B., & Muller, F. J. (2011). A guide to stem cell identification: progress and challenges in system-wide predictive testing with complex biomarkers. Bioessays, 33, 880–90.PubMedGoogle Scholar
  129. 129.
    Xu, H., Baroukh, C., Dannenfelser, R., et al. (2013). ESCAPE: database for integrating high-content published data collected from human and mouse embryonic stem cells. Database (Oxford) 2013; bat045.Google Scholar
  130. 130.
    Hawkins, R. D., Hon, G. C., Lee, L. K., et al. (2010). Distinct epigenomic landscapes of pluripotent and lineage-committed human cells. Cell Stem Cell, 6, 479–91.PubMedCentralPubMedGoogle Scholar
  131. 131.
    Deng, J., Shoemaker, R., Xie, B., et al. (2009). Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming. Nature Biotechnology, 27, 353–60.PubMedCentralPubMedGoogle Scholar
  132. 132.
    Doi, A., Park, I. H., Wen, B., et al. (2009). Differential methylation of tissue- and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells, embryonic stem cells and fibroblasts. Nature Genetics, 41, 1350–3.PubMedCentralPubMedGoogle Scholar
  133. 133.
    Kim, H., Jang, M. J., Kang, M. J., & Han, Y. M. (2011). Epigenetic signatures and temporal expression of lineage-specific genes in hESCs during differentiation to hepatocytes in vitro. Human Molecular Genetics, 20, 401–12.PubMedGoogle Scholar
  134. 134.
    Lister, R., Pelizzola, M., Dowen, R. H., et al. (2009). Human DNA methylomes at base resolution show widespread epigenomic differences. Nature, 462, 315–22.PubMedCentralPubMedGoogle Scholar
  135. 135.
    Ricupero, C. L., Swerdel, M. R., & Hart, R. P. (2013). Epigenome analysis of pluripotent stem cells. Methods in Molecular Biology, 997, 203–16.PubMedGoogle Scholar
  136. 136.
    Grimm, C., & Adjaye, J. (2012). Analysis of the methylome of human embryonic stem cells employing methylated DNA immunoprecipitation coupled to next-generation sequencing. Methods in Molecular Biology, 873, 281–95.PubMedGoogle Scholar
  137. 137.
    Olkhov-Mitsel, E., & Bapat, B. (2012). Strategies for discovery and validation of methylated and hydroxymethylated DNA biomarkers. Cancer Medicine, 1, 237–60.PubMedCentralPubMedGoogle Scholar
  138. 138.
    Smith, Z. D., Gu, H., Bock, C., Gnirke, A., & Meissner, A. (2009). High-throughput bisulfite sequencing in mammalian genomes. Methods, 48, 226–32.PubMedCentralPubMedGoogle Scholar
  139. 139.
    Ball, M. P., Li, J. B., Gao, Y., et al. (2009). Targeted and genome-scale strategies reveal gene-body methylation signatures in human cells. Nature Biotechnology, 27, 361–8.PubMedCentralPubMedGoogle Scholar
  140. 140.
    Irizarry, R. A., Ladd-Acosta, C., Carvalho, B., et al. (2008). Comprehensive high-throughput arrays for relative methylation (CHARM). Genome Research, 18, 780–90.PubMedCentralPubMedGoogle Scholar
  141. 141.
    Buzzard, J. J., Gough, N. M., Crook, J. M., & Colman, A. (2004). Karyotype of human ES cells during extended culture. Nature Biotechnology, 22, 381–2. author reply 2.PubMedGoogle Scholar
  142. 142.
    Rosler, E. S., Fisk, G. J., Ares, X., et al. (2004). Long-term culture of human embryonic stem cells in feeder-free conditions. Developmental Dynamics, 229, 259–74.PubMedGoogle Scholar
  143. 143.
    Bickmore, W.A., (2001). Karyotype Analysis and Chromosome Banding. In: Encyclopedia of Life Sciences: Nature Publishing Group; 1–7.Google Scholar
  144. 144.
    Elliott, A. M., Elliott, K. A., & Kammesheidt, A. (2010). High resolution array-CGH characterization of human stem cells using a stem cell focused microarray. Molecular Biotechnology, 46, 234–42.PubMedGoogle Scholar
  145. 145.
    Maitra, A., Arking, D. E., Shivapurkar, N., et al. (2005). Genomic alterations in cultured human embryonic stem cells. Nature Genetics, 37, 1099–103.PubMedGoogle Scholar
  146. 146.
    Markovic, O., & Markovic, N. (1998). Cell cross-contamination in cell cultures: the silent and neglected danger. In Vitro Cellular and Developmental Biology - Animal, 34, 1–8.PubMedGoogle Scholar
  147. 147.
    Nelson-Rees, W. A., Daniels, D. W., & Flandermeyer, R. R. (1981). Cross-contamination of cells in culture. Science, 212, 446–52.PubMedGoogle Scholar
  148. 148.
    Stacey, G. N. (2000). Cell contamination leads to inaccurate data: we must take action now. Nature, 403, 356.PubMedGoogle Scholar
  149. 149.
    Thompson, R., Zoppis, S., & McCord, B. (2012). An overview of DNA typing methods for human identification: past, present, and future. Methods in Molecular Biology, 830, 3–16.PubMedGoogle Scholar
  150. 150.
    Butler, J. M. (2006). Genetics and genomics of core short tandem repeat loci used in human identity testing. Journal of Forensic Sciences, 51, 253–65.PubMedGoogle Scholar
  151. 151.
    Budowle, B., Moretti, T. R., Niezgoda, S. J., & Brown, B. L. (1998). CODIS and PCR-based short tandem repeat loci: law enforcement tools. In Second European Symposium on Human Identification; 1998; Innsbruck (pp. 73–88). Austria: Promega Corporation.Google Scholar
  152. 152.
    Josephson, R., Sykes, G., Liu, Y., et al. (2006). A molecular scheme for improved characterization of human embryonic stem cell lines. BMC Biology, 4, 28.PubMedCentralPubMedGoogle Scholar
  153. 153.
    Opelz, G., Susal, C., Ruhenstroth, A., & Dohler, B. (2010). Impact of HLA compatibility on lung transplant survival and evidence for an HLA restriction phenomenon: a collaborative transplant study report. Transplantation, 90, 912–7.PubMedGoogle Scholar
  154. 154.
    Petersdorf, E. W. (2008). Optimal HLA, matching in hematopoietic cell transplantation. Current Opinion in Immunology, 20, 588–93.PubMedCentralPubMedGoogle Scholar
  155. 155.
    Taylor C.J., Welsh, K.I., Gray, C.M., et al. (1993) Clinical and socioeconomic benefits of serological HLA-DR matching for renal transplantation over three eras of immunosuppression regimens at a single unit. Clin Transpl: 233–41.Google Scholar
  156. 156.
    IPD-IMGT/HLA Statistics. EMBL-EBI. (Accessed January, 2014, at
  157. 157.
    Gourraud, P. A., Gilson, L., Girard, M., & Peschanski, M. (2012). The role of human leukocyte antigen matching in the development of multiethnic “haplobank” of induced pluripotent stem cell lines. Stem Cells, 30, 180–6.PubMedGoogle Scholar
  158. 158.
    Nakatsuji, N., Nakajima, F., & Tokunaga, K. (2008). HLA-haplotype banking and iPS cells. Nature Biotechnology, 26, 739–40.PubMedGoogle Scholar
  159. 159.
    Taylor, C. J., Peacock, S., Chaudhry, A. N., Bradley, J. A., & Bolton, E. M. (2012). Generating an iPSC bank for HLA-matched tissue transplantation based on known donor and recipient HLA types. Cell Stem Cell, 11, 147–52.PubMedGoogle Scholar
  160. 160.
    Zimmermann, A., Preynat-Seauve, O., Tiercy, J. M., Krause, K. H., & Villard, J. (2012). Haplotype-based banking of human pluripotent stem cells for transplantation: potential and limitations. Stem Cells and Development, 21, 2364–73.PubMedGoogle Scholar
  161. 161.
    Okita, K., Matsumura, Y., Sato, Y., et al. (2011). A more efficient method to generate integration-free human iPS cells. Nature Methods, 8, 409–12.PubMedGoogle Scholar
  162. 162.
    Bontadini, A. (2012). HLA techniques: typing and antibody detection in the laboratory of immunogenetics. Methods, 56, 471–6.PubMedGoogle Scholar
  163. 163.
    Abbott, W. G., Tukuitonga, C. F., Ofanoa, M., Munn, S. R., & Gane, E. J. (2006). Low-cost, simultaneous, single-sequence genotyping of the HLA-A, HLA-B and HLA-C loci. Tissue Antigens, 68, 28–37.PubMedGoogle Scholar
  164. 164.
    Buyse, I., Decorte, R., Baens, M., et al. (1993). Rapid DNA typing of class II HLA antigens using the polymerase chain reaction and reverse dot blot hybridization. Tissue Antigens, 41, 1–14.PubMedGoogle Scholar
  165. 165.
    Cao, K., Chopek, M., & Fernandez-Vina, M. A. (1999). High and intermediate resolution DNA typing systems for class I HLA-A, B, C genes by hybridization with sequence-specific oligonucleotide probes (SSOP). Reviews in Immunogenetics, 1, 177–208.PubMedGoogle Scholar
  166. 166.
    Rottem, S., & Naot, Y. (1998). Subversion and exploitation of host cells by mycoplasmas. Trends in Microbiology, 6, 436–40.PubMedGoogle Scholar
  167. 167.
    Bolske, G. (1988). Survey of Mycoplasma infections in cell cultures and a comparison of detection methods. Zentralbl Bakteriol Mikrobiol Hyg A, 269, 331–40.PubMedGoogle Scholar
  168. 168.
    Timenetsky, J., Santos, L. M., Buzinhani, M., & Mettifogo, E. (2006). Detection of multiple mycoplasma infection in cell cultures by PCR. Brazilian Journal of Medical and Biological Research, 39, 907–14.PubMedGoogle Scholar
  169. 169.
    Weber, D. J. (2006). Manufacturing considerations for clinical uses of therapies derived from stem cells. Methods in Enzymology, 420, 410–30.PubMedGoogle Scholar
  170. 170.
    Uphoff, C. C., & Drexler, H. G. (2011). Detecting mycoplasma contamination in cell cultures by polymerase chain reaction. Methods in Molecular Biology, 731, 93–103.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Cell Biology, Life Sciences Solutions, Thermo Fisher ScientificCarlsbadUSA

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