Reproductive and Developmental Biology

The Korean Society of Animal Reproduction (한국동물번식학회)
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Effective Application of Multiplex RT-PCR for Characterization of Human Embryonic Stem Cells/ Induced Pluripotent Stem Cells

다중 역전사 중합효소 연쇄 반응(Multiplex RT-PCR)을 이용한 인간배아 줄기세포 및 유도만능 줄기세포의 효과적인 분화 양상 조사

  • Kim, Jung-Mo (Graduate School of Life Science, CHA Stem Cell Institute, CHA University, College of Medicine) ;
  • Cho, Youn-Jeong (Graduate School of Life Science, CHA Stem Cell Institute, CHA University, College of Medicine) ;
  • Son, On-Ju (Graduate School of Life Science, CHA Stem Cell Institute, CHA University, College of Medicine) ;
  • Hong, Ki-Sung (CHA Biotech & Diostech Co., Ltd.) ;
  • Chung, Hyung-Min (Graduate School of Life Science, CHA Stem Cell Institute, CHA University, College of Medicine)
  • 김정모 (차의과학대학 의생명과학대학원 줄기세포 연구실) ;
  • 조윤정 (차의과학대학 의생명과학대학원 줄기세포 연구실) ;
  • 손온주 (차의과학대학 의생명과학대학원 줄기세포 연구실) ;
  • 홍기성 (차바이오앤디오스텍) ;
  • 정형민 (차의과학대학 의생명과학대학원 줄기세포 연구실)
  • Received : 2010.11.04
  • Accepted : 2011.01.03
  • Published : 2011.03.31
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Abstract

Techniques to evaluate gene expression profiling, such as sufficiently sensitive cDNA microarrays or real-time quantitative PCR, are efficient methods for monitoring human pluripotent stem cell (hESC/iPSC) cultures. However, most of these high-throughput tests have a limited use due to high cost, extended turn-around time, and the involvement of highly specialized technical expertise. Hence, there is an urgency of rapid, cost-effective, robust, yet sensitive method development for routine screening of hESCs/hiPSCs. A critical requirement in hESC/hiPSC cultures is to maintain a uniform undifferentiated state and to determine their differentiation capacity by showing the expression of gene markers representing all three germ layers, including ectoderm, mesoderm, and endoderm. To quantify the modulation of gene expression in hESCs/hiPSC during their propagation, expansion, and differentiation via embryoid body (EB) formation, we developed a simple, rapid, inexpensive, and definitive multimarker, semiquantitative multiplex RT-PCR platform technology. Among the 9 gene primers tested, 5 were pluripotent markers comprising set 1, and 3 lineage-specific markers were combined as set 2, respectively. We found that these 2 sets were not only effective in determining the relative differentiation in hESCs/hiPSCs, but were easily reproducible. In this study, we used the hES/hiPS cell lines to standardize the technique. This multiplex RT-PCR assay is flexible and, by selecting appropriate reporter genes, can be designed for characterization of different hESC/hiPSC lines during routine maintenance and directed differentiation.

Keywords

References

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