Derivation and characterization of four new human embryonic stem cell lines: the Danish experience.
In September 2003, legislation approved in Denmark legalized work on surplus human embryos from IVF for clinical purposes to establish human embryonic stem (ES) cell cultures. The aim of this study was to establish such stem cell lines. Fresh surplus embryos were donated after informed consent from the donors. Embryos were cultured into blastocysts and using the immunosurgery procedure, inner cell masses were isolated and cultured on irradiated human foreskin fibroblasts in KnockOut D-MEM supplemented with KnockOut Serum Replacement, bFGF, and LIF. Within a period of 12 months, 198 embryos were donated. Four isolated inner cell masses developed into putative ES cell lines, CLS1, CLS2, CLS3, CLS4, which have now been continuously cultured for eight months, corresponding to 30 passages. These cells expressed markers for undifferentiated human ES cells: stage-specific embryonic antigen-4, tumour-related antigen (TRA)-1-60, TRA-1-81, OCT4, NANOG, SOX2, and FGF4. The cells expressed high levels of telomerase activity, had a normal karyotype, and have been successfully cryopreserved and thawed. Finally, the cells displayed the potential to differentiate in vitro into cell types originating from all three germ layers. It is thought that the cell lines described in this study are the first human ES cells established in Denmark.
Lysdahl
Helle
H
Laboratory for Stem Cell Research, Aalborg University, Fredrik Bajers Vej 3B, 9220 Aalborg Oest, Denmark. hellel@hst.aau.dk
Gabrielsen
Anette
A
Minger
Stephen L
SL
Patel
Minal J
MJ
Fink
Trine
T
Petersen
Karsten
K
Ebbesen
Peter
P
Zachar
Vladimir
V
D000954:Antigens, Surface / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D015925:Cryopreservation / Q000379:methods*; D003470:Culture Media; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism; D003718:Denmark; D004622:Embryo, Mammalian / Q000166:cytology*; D051519:Fibroblast Growth Factor 4 / Q000378:metabolism; D006028:Glycosphingolipids / Q000378:metabolism; D024002:HMGB Proteins / Q000378:metabolism; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007621:Karyotyping; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D011509:Proteoglycans / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055748:SOXB1 Transcription Factors; D055746:Stage-Specific Embryonic Antigens; D019098:Telomerase / Q000378:metabolism; D039901:Totipotent Stem Cells / Q000166:cytology* / Q000378:metabolism / Q000502:physiology; D014157:Transcription Factors / Q000378:metabolism
D000954:Antigens, Surface; D003470:Culture Media; D017931:DNA Primers; D004268:DNA-Binding Proteins; C496445:FGF4 protein, human; D051519:Fibroblast Growth Factor 4; D006028:Glycosphingolipids; D024002:HMGB Proteins; D018398:Homeodomain Proteins; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; D011509:Proteoglycans; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors; D055746:Stage-Specific Embryonic Antigens; C107064:TRA-1-60 antigen, human; D014157:Transcription Factors; C066584:stage-specific embryonic antigen-4; D019098:Telomerase