Characteristics and specific features of new human embryonic stem cell lines

Abstract

Four continuous human embryonic stem cell (ESC) lines (SC1, SC2, SC3, and SC4) isolated from preimplantational blastocysts obtained by artificial fertilization have been described. The cell lines were cultivated on mitotically inactivated human feeder cells. SC1 and SC2 cell lines passed through 150 population doublings, while the SC3 and SC4 cell lines passed through about 120 population doublings, which significantly exceeded Hayflick’s limit. All of these cell lines maintain the high activity of alkaline phosphatase and the expression of transcription factor OCT-4 and cell surface antigens (SSEA-4, TRA-1-60, TRA-1-81), which confirms the status of ESC and human specificity. An immunofluorescent analysis of the expression of antigens that characterize the ectoderm, endoderm, and mesoderm has confirmed the capacity of these cells for pluripotency in all four lines under in vitro conditions. Using PCR analysis, the expression of six genes specific to pluripotent cells, i.e., OCT-4, NANOG, DPPA3/STELLA, TDGF/CRIPTO, and LEFTYA, has been revealed. The correlation between the level of proliferative activity and the character of staining with DNA-bound fluorescent dyes has been found. Hoechst 33342 and PI dyes produced a diffuse staining of nuclei in slowly proliferating cells of the SC1 and SC2 lines. On the contrary, in actively proliferating cells of the SC3 and SC4 lines, a distinct staining of nuclei was observed. Upon changing the cultivation conditions, proliferative activity of the SC3 and SC4 lines decreased and the character of the fluorescent staining became similar to that of the SC1 and SC2 lines. The quality of the fluorescent staining with Hoechst 33342 and propidium iodide reflects the level of proliferation. Possible causes and mechanisms of this peculiarity of human ESCs are discussed.