THE CHARACTERS AND SPECIFIC FEATURES OF NEW HUMAN EMBRYONIC STEM CELLS LINES
T. A. Krylova,1 A. M. Koltzova,1 V. V. Zenin,1 O. F. Gordeeva,2
A. S. Musorina,1 T. S. Goryachaya,1 S. A. Shlykova,3 Ju. K. Kamenezkaya,3
G. P. Pinaev,1 G. G. Poljanskaya 1, *
1 Institute of Cytology RAS, St. Petersburg, 2 N. K. Koltzov Institute of Development Biology RAS and
3 AVA-PETER Company;
* e-mail: poljansk@mail.cytspb.rssi.ru
Four continuous human embryonic stem cell lines (SC1, SC2, SC3 and SC4), derived from the blastocysts has been described. The cell lines were cultivated on
mitotically inactivated human feeder cells. The cell lines SC1 and SC 2 have passed through 150 population doublings and the cell lines SC3 and SC4 - near 120
populations doublings, which exceeds Hayflick limit sufficiently. These cell lines maintain high activity of alkaline phosphatase, expression of transcription factor
OCT-4 and cell surface antigens (SSEA-4, TRA-1-60 è TRA-1-81), confirming their ESC status and human specificity. Immunofluorescent detection of antigens,
characteristic of ectoderm, endoderm and mesoderm confirms the ability of these cells to retain their pluripotency under in vitro condition. PCR analysis revealed
expression of six genes specific for pluripotent cells (OCT-4, NANOG, DPPA3/STELLA, TDGF/CRIPTO and LEFTYA). Correlation between the level of
proliferative activity and the character of DNA-bound fluorescent staining was found. Fluorescent dyes, Hoechst 33342 and PI, produced diffuse staining of the nuclei
in slowly proliferating cells of the SC1 and SC2 lines. In contrast, in actively proliferating cells of the SC3 and SC4 lines, the clear staining of the nuclei was observed.
Upon changing the cultivation condition, proliferative activity of SC3 and SC4 lines decreased and became similar to that of SC1 and SC2 lines. The character of the
fluorescent staining of all these lines was also shown to be similar. These results show that quality of the fluorescent staining with Hoechst 33342 and PI reflects the
level of proliferation. Possible causes and mechanisms of this feature of human ESC are discussed.
Key words: continuous human embryonic stem cell lines, immunofluorescent analysis, expression of genes, antibodies, fluorescent dyes
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