Abstract
The use of embryonic stem (ES) cells in regenerative medicine is one of the most important promises of this decade. The knowledge about culture medium, isolation protocols and pluripotency related genes is growing but their therapeutic use is still restricted by a risk of teratoma formation. To test the hypothesis that some cells remain undifferentiated after a differentiation culture process, we have electroporated mouse ES cells with a construct comprising the mTert promoter coupled to green fluorescent protein (GFP). When ES cells colonies are in culture, they express the green fluorescent protein as a consequence of their telomerase activity. After LIF removal a natural differentiation process into embryoid bodies (EB) occurs, such that the GFP signal gradually diminishes due to this differentiation. However, after 2 weeks of incubation, we could observe small groups of fluorescent cells remaining in the differentiated monolayer. After isolation and expansion of these groups under ES cell culture conditions, ES cell morphology was recovered. Molecular analysis showed that after several passages in ES medium, pluripotency quality characteristics of these cells are completely recovered. According to our results, a specific screening of differentiated cells should be performed before their therapeutic use to avoid the risk of teratoma formation stimulated by an adequate environment. Moreover, to maintain undifferentiated stem cells in defined conditions is of critical importance to improve their in vitro culture. We evaluated the effects of the replacement of fibroblast feeder layer (mEF) by gelatin or the glycosaminoglycan hyaluronan (HA). Long-term culture onto HA was more effective in maintaining the pluripotent state of mES cells when compared to gelatin. The rate of terminal differentiation was the highest onto mEF, intermediate onto HA and reduced onto gelatin. In addition, our findings strongly suggest that the use of hyaluronan, without feeder cells, may offer a valuable alternative for unifying and standardizing the conditions for long-term defined culture conditions of undifferentiated mES cells.
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Ramos-Ibeas, P., Pericuesta, E., Miranda, A., Fernández-González, R., Gutiérrez-Adán, A., Ramírez, M.Á. (2012). Maintenance of Pluripotency in Mouse Stem Cells: Use of Hyaluronan in the Long-Term Culture. In: Hayat, M. (eds) Stem Cells and Cancer Stem Cells, Volume 7. Stem Cells and Cancer Stem Cells, vol 7. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-4285-7_11
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DOI: https://doi.org/10.1007/978-94-007-4285-7_11
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