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A simple and effective method for the isolation of inner cell mass samples from human blastocysts for gene expression analysis

  • Jian Xu
  • Yan Li
  • Yanwen Xu
  • Chenhui Ding
  • Tao Li
  • Canquan ZhouEmail author
Article

Abstract

The isolation of pure inner cell mass (ICM) and trophectoderm (TE) cells from a single human blastocyst is necessary to obtain accurate gene expression patterns of these cells, which will aid in the understanding of the primary steps of embryo differentiation. However, previously developed pure ICM isolation methods are either time-consuming or alter the normal gene expression patterns of these cells. Here, we demonstrate a simple and effective method of ICM samples isolation from human blastocysts. In total, 35 human blastocysts of all stages with expanded and good morphology were incubated in calcium/magnesium-free HEPES medium for 5 min before micromanipulation. With the aid of a laser, a biopsy pipette was inserted directly into the blastocoel for the suction-based removal of ICM samples. The ICM samples were obtained through simple mechanical pulling force or laser assistance, and each isolation process required 3–4 min. The isolated ICM and TE fractions were subjected to single-cell real-time quantitative RT-PCR to evaluate keratin 18 (KRT18) expression. Finally, 33 paired ICM and TE samples were verified using gene expression analysis. KRT18 was readily detectable in all TE cells but absent in 30 ICM counterparts, indicating a pure ICM isolation rate of 90.9% (30/33). The relative KRT18 expression of three TE samples compared with their three contaminated ICM counterparts was 19-fold (P < 0.001), indicating that the contamination was very weak. These results demonstrate that our ICM isolation method is simple and effective.

Keywords

Blastocyst Inner cell mass Isolation 

Notes

Acknowledgments

This study was supported by the following grants: Science and Technology Program of Guangzhou, China (201300000097); National Natural Science Foundation of China (31071272); National Basic Research Program of China (973 Program, grant No. 2012CB947604).

Supplementary material

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References

  1. Abruzzese R. V.; Fekete R. A. Single cell gene expression analysis of pluripotent stem cells. Methods Mol. Biol 997: 217–224; 2013.PubMedCrossRefGoogle Scholar
  2. Adjaye J.; Huntriss J.; Herwig R.; BenKahla A.; Brink T. C.; Wierling C.; Hultschig C.; Groth D.; Yaspo M. L.; Picton H. M.; Gosden R. G.; Lehrach H. Primary differentiation in the human blastocyst: comparative molecular portraits of inner cell mass and trophectoderm cells. Stem Cells 23: 1514–1525; 2005.PubMedCrossRefGoogle Scholar
  3. Buganim Y.; Faddah D. A.; Cheng A. W.; Itskovich E.; Markoulaki S.; Ganz K.; Klemm S. L.; van Oudenaarden A.; Jaenisch R. Single-cell expression analyses during cellular reprogramming reveal an early stochastic and a late hierarchic phase. Cell 150: 1209–1222; 2012.PubMedCentralPubMedCrossRefGoogle Scholar
  4. Cauffman G.; De Rycke M.; Sermon K.; Liebaers I.; Van de Velde H. Markers that define stemness in ESC are unable to identify the totipotent cells in human preimplantation embryos. Hum. Reprod 24: 63–70; 2009.PubMedCrossRefGoogle Scholar
  5. Cauffman G.; Van de Velde H.; Liebaers I.; Van Steirteghem A. DAZL expression in human oocytes, preimplantation embryos, and embryonic stem cells. Mol. Hum. Reprod 11: 405–411; 2005.PubMedCrossRefGoogle Scholar
  6. Cowan C. A.; Klimanskaya I.; McMahon J.; Atienza J.; Witmyer J.; Zucker J. P.; Wang S.; Morton C. C.; McMahon A. P.; Powers D.; Melton D. A. Derivation of embryonic stem-cell lines from human blastocysts. N. Engl. J. Med 350: 1353–1356; 2004.PubMedCrossRefGoogle Scholar
  7. De Spiegelaere W.; Erkens T.; De Craene J.; Burvenich C.; Peelman L.; Van den Broeck W. Elimination of amplification artifacts in real-time reverse transcription PCR using laser capture microdissected samples. Anal. Biochem 382: 72–74; 2008.PubMedCrossRefGoogle Scholar
  8. Emmert-Buck M. R.; Bonner R. F.; Smith P. D.; Chuaqui R. F.; Zhuang Z.; Goldstein S. R.; Weiss R. A.; Liotta L. A. Laser capture microdissection. Science 274: 998–1001; 1996.PubMedCrossRefGoogle Scholar
  9. Filliers M.; De Spiegelaere W.; Peelman L.; Goossens K.; Burvenich C.; Vandaele L.; Cornillie P.; Van Soom A. Laser capture microdissection for gene expression analysis of inner cell mass and trophectoderm from blastocysts. Anal Biochem 408: 169–171; 2011.PubMedCrossRefGoogle Scholar
  10. Gardner D. K.; Schoolcraft W. B. In vitro culture of human blastocysts. In: Jansen R.; Mortimer D. (eds) Towards Reproductive Certainty: Fertility and Genetics Beyond. Parthenon Publishing, Carnforth, UK, pp 378–388; 1999.Google Scholar
  11. Gardner R. L.; Johnson M. H. An investigation of inner cell mass and trophoblast tissues following their isolation from the mouse blastocyst. J Embryol Exp Morphol 28: 279–312; 1972.PubMedGoogle Scholar
  12. Goossens K.; De Spiegelaere W.; Stevens M.; Burvenich C.; De Spiegeleer B.; Cornillie P.; Van Zeveren A.; Van Soom A.; Peelman L. Differential microRNA expression analysis in blastocysts by whole mount in situ hybridization and reverse transcription quantitative polymerase chain reaction on laser capture microdissection samples. Anal. Biochem 423: 93–101; 2012.PubMedCrossRefGoogle Scholar
  13. Guo G.; Huss M.; Tong G. Q.; Wang C.; Li S. L.; Clarke N. D.; Robson P. Resolution of cell fate decisions revealed by single-cell gene expression analysis from zygote to blastocyst. Dev. Cell 18: 675–685; 2010.PubMedCrossRefGoogle Scholar
  14. Livak K. J.; Schmittgen T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods 25: 402–408; 2001.PubMedCrossRefGoogle Scholar
  15. Ozawa M.; Hansen P. J. A novel method for purification of inner cell mass and trophectoderm cells from blastocysts using magnetic activated cell sorting. Fertil Steri 95: 799–802; 2011.CrossRefGoogle Scholar
  16. Santalol J.; Grossmann M.; Egozcue J. Does Ca2+/Mg2+-free medium have an effect on the survival of the preimplantation mouse embryo after biopsy? Hum. Reprod. Update 2(3): 257–261; 1996.CrossRefGoogle Scholar
  17. Tanaka N.; Takeuchi T.; Neri Q. V.; Sills E. S.; Palermo G. D. Laser-assisted blastocyst dissection and subsequent cultivation of embryonic stem cells in a serum/cell free culture system: applications and preliminary results in a murine model. J. Transl. Med 4: 20; 2006.PubMedCentralPubMedCrossRefGoogle Scholar
  18. Wong C. C.; Loewke K. E.; Bossert N. L.; Behr B.; De Jonge C. J.; Baer T. M.; Reijo Pera R. A. Noninvasive imaging of human embryos before embryonic genome activation predicts development to the blastocyst stage. Nat. Biotechnol 28: 1115–1121; 2010.PubMedCrossRefGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2013

Authors and Affiliations

  • Jian Xu
    • 1
    • 2
  • Yan Li
    • 1
    • 2
  • Yanwen Xu
    • 1
    • 2
  • Chenhui Ding
    • 1
    • 2
  • Tao Li
    • 1
    • 2
  • Canquan Zhou
    • 1
    • 2
    Email author
  1. 1.Reproductive Medicine CenterFirst Affiliated Hospital of Sun Yat-Sen UniversityGuangzhouChina
  2. 2.The Key Laboratory of Reproductive Medicine of Guangdong ProvinceGuangzhouChina

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