Abstract:

Objective To investigate the pluripotency of residual undifferentiated embryonic stem (ES) cells from differentiated embryoid bodies (EBs), and explore the relationship between pluripotency maintenance and exogenous leukemia inhibitory factor (LIF). Methods Mouse R1 ES cells were differentiated for 20 days to form EBs. EBs were then trypsinized and re-plated on tissue culture plate in DMEM without LIF. The expression of phenotypes (CD9, SSEA1 and Flk-1）of expanded cells was analysed by flow cytometry. The expression of Oct-4, Nanog, Rex1, FGF5, Nestin, Brachyury, Flk-1 and GATA6 was detected by RT-PCR before and after secondary EB differentiation. The residual cells after expansion were subcutaneously injected into nude mice, and the ability to form teratoma was examined. Single Oct-4/GFP transgenic mouse ES cells were differentiated in semi-solid media to determine the residual rates after 20 days. Results The residual cells grew out to form ES-like colonies in DMEM without LIF, and expressed undifferentiated ES markers such as CD9, SSEA1, Oct-4, Nanog and Rex1. Meanwhile, the residual cells could be redifferentiated to form EBs and express Nestin, Brachyury, Flk-1 and GATA6. Teratoma was formed 6 weeks after subcutaneous injection of residual cells into nude mice. Single cell differentiation of Oct-4-GFP cells showed that about 14% ES cells in primary culture possessed the potential to generate residual undifferentiated cells after long-term differentiation. Conclusion The pluripotency of residual cells from differentiated EBs can be maintained without exogenous LIF. Only a part of the primary cultured ES cells possess the potential to generate residual undifferentiated cells after long-term differentiation.

Key words: embryonic stem cell, differentiation, residue, pluripotency, leukemia inhibitory factor