ISOLATION AND CHARACTERIZATION OF CONTINUOUS HUMAN EMBRYONIC STEM CELL LINES
T. A. Krylova,1 V. V. Zenin,1 N. S. Musorina,1
V. S. Baranov,2 N. K. Bichevaya,2 V. S. Korsak,2 N. N. Nikolsky,1
G. P. Pinaev,1 G. G. Poljanskaya 1 *
1 Institute of Cytology RAS, St. Petersburg, and
2 Ott's Institute of Obstetrics and Gynaecology RAMS, St. Petersburg, Russia;
* e-mail: poljansk@mail.cytspb.rssi.ru
A long-term cultivation (5-8 months) of human blastocyst-derived embryonic cells (hES) was performed. Several
properties of hESs were examined to prove the state of continuous cell lines. These cells have passed through 100-175 population
doublings with the average population doubling time equal to 37.0 ± 1.5 h. Isolated hESs, referred to as HESC-1, HESC-2, HESC-3,
HESC-4, cultivated on mitotically inactivated mouse embryonic fibroblasts (STO continuous cell line), formed multilayer colonies of
various shape. The cells maintained stable proliferative activity, high activity of alkaline phosphatase and expression of transcription
factor Oct4, and all this characterizes embryonic stem cells of different origin. Expression of hES specific cell surface antigens (SSEA-3,
SSEA-4, TRA-1-81 and TRA-11-81 and TRA-1-60) was confirmed by immunofluorescence analysis with the corresponding monoclonal
antibodies. An additional prove for species specificity of HESC lines is the lack of expression of mouse specific surface antigen SSEA1.
The cell cycle of HESC-1 undifferentiated cells and embryoid bodies was analysed cytofluorimetrically.
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