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EXPERIMENTAL PROTOCOLS FOR EMBRYONIC STEM CELL RESEARCH

Methods for Derivation of Human Embryonic Stem Cells

^a Department of Obstetrics and Gynecology,
^b Institute of Reproductive Medicine and Population, Medical Research Center, College of Medicine, Seoul National University, Seoul, Korea;
^c Central Research Institute, Sam Jin Pharm. Co. Ltd., Hwasung, Korea;
^d Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, Seongnam, Korea;
^e Department of Physiology, Yonsei University College of Medicine, Seoul, Korea

Key Words. Embryonic stem cells • Derivation • Immunosurgery • Partial-embryo culture • Whole-embryo culture • Blastocyst quality • Inner cell mass

Correspondence: Shin Yong Moon, M.D., Ph.D., Department of Obstetrics and Gynecology, College of Medicine, Seoul National University, 28 Yongon-dong, Chonno-gu, Seoul 110-744, Korea. Telephone: 82-2-2072-2384; Fax: 82-2-3672-7601; e-mail: shmoon{at}plaza.snu.ac.kr; and Dong-Wook Kim, Ph.D., Department of Physiology, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-752, Korea. Telephone: 82-2-2228-1703; Fax: 82-2-393-0203; e-mail: dwkim2{at}yumc.yonsei.ac.kr

The expanded blastocysts, developed from 2PN-stage embryos, are generally divided into three categories: a good blastocyst containing a large and distinguishable inner cell mass (ICM), a blastocyst with a small and distinct ICM, and a blastocyst with a poorly defined ICM. In this study, we introduce methods for the derivation of human embryonic stem cells (hESCs) depending on the quality of the blastocysts. An immunosurgical method was used for the good expanded blastocysts. This method, however, raises the probability of ICM loss in cases of hESC derivation from blastocysts with smaller or indistinct ICMs. Furthermore, this method is also associated with a risk of the contamination of the hESCs with animal pathogens. To overcome these shortcomings, the partial- or whole-embryo culture method was used. For blastocysts with no visible ICM, the whole-embryo culture method was used to establish hESCs via the seeding of the entire blastocyst without its zona pellucida directly on a STO feeder layer. However, trophectodermal overgrowth tends to hinder the expansion of the ICM during the initial steps of hESC derivation. Therefore, the partial-embryo culture method was developed to establish hESCs from blastocysts with smaller ICMs. The surgical isolation of the region containing the ICM with an ultra-fine glass pipette alleviates trophectoderm overgrowth. This method is also applicable to blastocysts with large and distinct ICMs, and the efficiency of this method is comparable to that of the immunosurgical method.

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