Embryonic Stem Cell Lines Derived from Human Blastocysts
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Thirty-six fresh or frozen-thawed donated human embryos produced by IVF were cultured to the blastocyst stage in G1.2 and G2.2 medium (25). Fourteen of the 20 blastocysts that developed were selected for ES cell isolation as described for rhesus monkey ES cells (5). The inner cell masses were isolated by immunosurgery (26) with a rabbit antiserum to BeWO cells and plated on irradiated (35 grays gamma irradiation) mouse embryonic fibroblasts. Culture medium consisted of 80% Dulbecco's modified Eagle's medium (no pyruvate high glucose formulation; Gibco-BRL) supplemented with 20% fetal bovine serum (Hyclone) 1 mM glutamine 0.1 mM β-mercaptoethanol (Sigma) and 1% nonessential amino acid stock (Gibco-BRL). After 9 to 15 days inner cell mass–derived outgrowths were dissociated into clumps either by exposure to Ca 2+ /Mg 2+ -free phosphate-buffered saline with 1 mM EDTA (cell line H1) by exposure to dispase (10 mg/ml; Sigma; cell line H7) or by mechanical dissociation with a micropipette (cell lines H9 H13 and H14) and replated on irradiated mouse embryonic fibroblasts in fresh medium. Individual colonies with a uniform undifferentiated morphology were individually selected by micropipette mechanically dissociated into clumps and replated. Once established and expanded cultures were passaged by exposure to type IV collagenase (1 mg/ml; Gibco-BRL) or by selection of individual colonies by micropipette. Clump sizes of about 50 to 100 cells were optimal. Cell lines were initially karyotyped at passages 2 to 7.
Counter C. M., Hirte H. W., Bacchetti S., Harley C. B., Proc. Natl. Acad. Sci. U.S.A. 91, 2900 (1994).
P. W. Andrews J. Oosterhuis I. Damjanov in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach E. Robertson Ed. (IRL Oxford 1987) pp. 207–248.
Alkaline phosphatase was detected with Vector Blue substrate (Vector Labs). SSEA-1 SSEA-3 SSEA-4 TRA-1-60 and TRA-1-81 were detected by immunocytochemistry with specific primary monoclonal antibodies and localized with a biotinylated secondary antibody and then an avidin or biotinylated horseradish peroxidase complex (Vectastain ABC system; Vector Laboratories) as previously described (5). The ES cell lines were at passages 8 to 12 at the time markers were analyzed.
hCG and α-fetoprotein were measured by specific radioimmunoassay (double AB hCG and AFP-TC kits; Diagnostic Products Los Angeles CA). hCG assays used the World Health Organization Third International Standard 75/537. H9 cells were allowed to grow to confluence (day 0) on plates of irradiated mouse embryonic fibroblasts. Medium was replaced daily. After 2 weeks of differentiation medium in triplicate wells conditioned for 24 hours was assayed for hCG and α-fetoprotein. No hCG or α-fetoprotein was detected in unconditioned medium.
R. O'Rahilly and F. Müller Developmental Stages in Human Embryos (Carnegie Institution of Washington Washington DC 1987).
We thank the personnel of the IVF clinics at the University of Wisconsin School of Medicine and at the Rambam Medical Center for the initial culture and cryopreservation of the embryos used in this study; D. Gardner and M. Lane for the G1.2 and G2.2 media; P. Andrews for the NTERA2 cl.D1 cells and the antibodies used to examine cell surface markers; C. Harris for karyotype analysis; and Geron Corporation for the 293 and MDA cell pellets and for assistance with the telomerase TRAP assay. Supported by the University of Wisconsin (UIR grant 2060) and Geron Corporation (grant 133-BU18).