engleski

Rapid loss of pluripotency regulator Oct4 expression in cultured embryonic stem cells of non-obese diabetic mice

Background and purpose. Non-obese diabetic (NOD) mouse strain is highly nonpermissive for in vitro derivation of embryonic stem (ES) cell line. Namely, NOD ES cells display strong tendency to differentiate in primary culture. We presumed that this feature might be due to disrupted Oct4 expression, because this transcription factor is the master regulator of stem cell pluripotency. Therefore, we analyzed the expression of Oct4 in NOD ES cells and compared it with ES cells of mouse strains confirmed as permissive for ES cell line derivation (BALB/c, C57BL/6 and C3H). Materials and Methods. ES cells of all tested mouse strains were obtained from 3.5 days old blastocysts that were cultured on mouse primary fibroblast layer in culture medium supplemented with foetal calf serum and leukemia inhibitory factor (LIF). Blastocyst-derived primary outgrowths were removed from the culture on the third, fourth and fifth day successively, and Oct4 expression was detected by RT-PCR. Results. NOD ES cell outgrowths were Oct4 positive after three days of culture. In the next 2 days Oct4 was undetectable. Parallel with Oct4 loss, after 4 days of culture, the colonies acquired differentiated phenotype. Outgrowths of other tested strains (C57BL/6, BALB/c, C3H) displayed detectable levels of Oct4 expression after 3, 4 and 5 days of culture, and retained undifferentiated phenotype throughout whole five-day culture period. Conclusions. These observations suggest that rapid loss of Oct4 expression, accompanied with morphological differentiation and consequent loss of pluripotency in cultured NOD ES cells, could represent the major obstacle in obtaining ES cell line from NOD mice source.

Embryonic stem cells ; Non-obese diabetic mice

nije evidentirano