Methods for derivation of human embryonic stem cells.
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.
Kim
Hee Sun
HS
Department of Obstetrics and Gynecology, College of Medicine, Seoul National University and Seoul National University Bundang Hospital, Seongnam, Korea.
Oh
Sun Kyung
SK
Park
Yong Bin
YB
Ahn
Hee Jin
HJ
Sung
Ki Cheong
KC
Kang
Moon Joo
MJ
Lee
Lim Andrew
LA
Suh
Chang Suk
CS
Kim
Seok Hyun
SH
Kim
Dong-Wook
DW
Moon
Shin Yong
SY
D001755:Blastocyst / Q000166:cytology* / Q000502:physiology; D002460:Cell Line; D003470:Culture Media; D046149:Embryo Culture Techniques / Q000379:methods*; D004622:Embryo, Mammalian / Q000166:cytology; D006801:Humans; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000502:physiology
D003470:Culture Media