Establishment of human embryonic stem cell lines from frozen-thawed blastocysts using STO cell feeder layers.
BACKGROUND
Recently, human embryonic stem (hES) cells have become very important resources for basic research on cell replacement therapy and other medical applications. The purpose of this study was to test whether pluripotent hES cell lines could be successfully derived from frozen-thawed embryos that were destined to be discarded after 5 years in a routine human IVF-embryo transfer programme and whether an STO cell feeder layer can be used for the culture of hES cells.


METHODS
Donated frozen embryos (blastocysts or pronuclear) were thawed, and recovered or in vitro developed blastocysts were immunosurgically treated. All inner cell masses were cultured continuously on an STO cell feeder layer and then presumed hES cell colonies were characterized.


RESULTS
Seven and two cell lines were established from frozen-thawed blastocysts (7/20, 35.0%) and pronuclear stage embryos (2/20, 10.0%), respectively. The doubling time of hES cells on the immortal STO cell feeder layer was approximately 36 h, similar to that of cells grown using fresh mouse embryonic fibroblast (MEF) feeder conditions. Subcultured hES cell colonies showed strong positive immunostaining for alkaline phosphatase, stage-specific embryonic antigen-4 (SSEA-4) and tumour rejection antigen 1-60 (TRA1-60) cell surface markers. Also, the hES colonies retained normal karyotypes and Oct-4 expression in prolonged subculture. When in vitro differentiation of hES cells was induced by retinoic acid, three embryonic germ layer cells were identified by RT-PCR or indirect immunocytochemistry.


CONCLUSIONS
This study indicates that establishment of hES cells from frozen-thawed blastocysts minimizes the ethical problem associated with the use of human embryos in research and that the STO cell feeder layer can be used for the culture of hES cells.
Maria Infertility Hospital Medical Institute/Maria Biotech, Dongdaemun-gu, Seoul 130-812, Korea. sppark@mariababy.com
Se-Pill
SP
Park
Young Jae
YJ
Lee
Keum Sil
KS
Lee
Hyun
H
Ah Shin
Hwang Yoon
HY
Cho
Kil Saeng
KS
Chung
Eun Young
EY
Kim
Jin Ho
JH
Lim
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000954:Antigens, Surface; D015415:Biomarkers / Q000032:analysis; D001755:Blastocyst / Q000166:cytology*; D002454:Cell Differentiation; D002460:Cell Line; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D015925:Cryopreservation; D004622:Embryo, Mammalian / Q000166:cytology; D005307:Fertilization in Vitro; D005347:Fibroblasts; D006023:Glycoproteins / Q000378:metabolism; D006028:Glycosphingolipids / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007621:Karyotyping; D051379:Mice; D011509:Proteoglycans; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055746:Stage-Specific Embryonic Antigens; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D014212:Tretinoin / Q000494:pharmacology
D000954:Antigens, Surface; D015415:Biomarkers; D006023:Glycoproteins; D006028:Glycosphingolipids; D011509:Proteoglycans; D055746:Stage-Specific Embryonic Antigens; C107064:TRA-1-60 antigen, human; C066584:stage-specific embryonic antigen-4; D014212:Tretinoin; D000469:Alkaline Phosphatase