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

서지정보

ㆍ발행기관 : 한국동물번식학회 ㆍ수록지정보 : Reproductive & developmental biology / 35권 / 1호
ㆍ저자명 : 김정모, 조윤정, 손온주, 홍기성, 정형민

목차

ABSTRACT 서론 재료 및 방법 인간 배아줄기세포(Human Embryonic Stem Cells: hESCs)와 유도만능 줄기세포(Induced Pluripotent Stem Cells:hiPSCs)의 배양 배상체(Embryoid Body) 형성 세포 배양(Cell Culture) 미분화 인간 배아줄기세포와 역분화줄기세포의 알칼라인포스파테이즈(AP) 염색 세포의 관찰 RNA 추출과 cDNA 합성 결과 실험에 시용된 세포들의 특성 확인 다중 역전사 중합연쇄 반응을 통해 단일 프라이머와 다중 프라이머의 제작 확인 다중 역전사 중합연쇄 반응을 통해 인간 배아줄기세포와 유도만능 줄기세포의 단일 콜로니로부터 미분화 유전자 발현확인 다중 역전사 중합연쇄 반응을 통해 인간배아 줄기세포로부터 분화된 세포로부터 삼배엽 분화 유전자의 발현량 확인 고찰 인용문헌

영어 초록

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.

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