Evidence for self-maintaining pluripotent murine stem cells in embryoid bodies
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Pluripotent stem cells have great potential for regenerative medicine; however, their clinical use is associated with a risk of tumor formation. We utilized pluripotent cells expressing green fluorescent protein and puromycin resistance under control of the Oct4 promoter to study the persistence of potential pluripotent cells under embryoid body (EB) culture conditions, which are commonly used to obtain organotypic cells. We found that i.) OCT4-expressing cells dramatically decrease during the first week of differentiation, ii.) the number of OCT4-expressing cells recovers from day 7 on, iii.) the OCT4-expressing cells are similar to embryonic stem cells grown in the presence of leukemia inhibitory factor LIF but express several markers associated with germ cell formation, such as DAZL and STRA-8 and iv.) the persistence of potentially pluripotent cells is independent of supportive cells in EBs. Finally, OCT4-expressing cells, isolated from EBs after 2-month of culture, were further maintained under feeder-free conditions in absence of LIF and continued to express OCT4 in 95 % of the population for at least 36 days. These findings point to an alternative state of stable OCT4 expression. In the frame of the landscape model of differentiation two attractors of pluripotency might be defined based on their different characteristics.
KeywordsOCT4 Pluripotency Self-renewal Stem cells Embryoid bodies Landscape model
We thank Professor Austin Smith (Institute for Stem Cell Biology University of Cambridge, UK) for providing the Oct4-GFP-ires-PAC ES cell line and Christoph Göttlinger for help with the FACS sorter. Thanks to Manoj Gupta and Daniel Derichsweiler for support with flow cytometry and Moritz Haustein for statistical analysis. This project was supported by grants from the German Academic Exchange Service (DAAD) to Wael A Attia and Osama Abd El Aziz and the European Union funded RAMSES project to KB (7th framework program), by grants of the Imhoff Foundation (Imhoff Stiftung) and the Maria Pesch Foundation (Maria Pesch Stiftung) to KP and by grants of the National Medical Research Council and the Ministry of Education of Singapore (PD).
Author Disclosure Statement
No conflicts of interests were declared.
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