Abstract
Embryonic stem (ES) cells have been in the fore front of scientific literature lately as having the potential for regeneration of many tissue types. Two important issues that need to be addressed are the culture conditions for maintaining ES cells and the accuracy of ES cell markers in monitoring the undifferentiated state. Leukaemia inhibitory factor (LIF) is routinely used to sustain mouse ES cells (mES) in a pluripotent fashion. In this paper, we assessed three markers during long-term maintenance of ES cells with various concentrations of LIF to see if decreasing concentration would lead to changes in marker expressions and growth behavior. Common markers of pluripotency such as alkaline phosphatase enzyme activity (ALP), surface staining for stage specific embryonic antigen 1 (SSEA-1), Oct-4 transcription factor, cell doubling time, as well as visual observations of cell morphology were analyzed during long-term maintenance of mES cells with LIF concentrations ranging from 0 to 500 pM. The morphology of the cells at LIF concentrations of 0 25 pM changed from being tight clusters to more flattened shapes while cells in 50–500 pM retained the clustered shape but growth rates remained essentially identical at between 10 and 16 h. ES cells at all concentrations of LIF continued expressing ALP, SSEA-1 and Oct-4 markers over a period of 6 weeks, which indicate that mES cells are capable of either producing autocrine LIF or are able to proliferate at very low levels of LIF. Pluripotency markers such as Oct-4 and SSEA-1 are only moderately reduced after 5–6 weeks. Oct-4 mRNA expression levels were partially diminished in LIF free conditions only at weeks 5 and 6 compared to controls with LIF at 500 pM. Changes in morphology of cells by visual observation seemed to be a faster indication of the onset of differentiation in mES cells, although other reliable means also include decreased levels of Oct-4, SSEA-1 and ALP markers. It is preferable to maintain long-term cultures of mES cells above 50 pM of LIF to have a more homogenous, stable population of pluripotent cells.
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References
Burdon T., Smith A. and Savatier P. 2002. Signaling, cell cycle and pluripotency in embryonic stem cells. Trends Cell Biol. 12: 432–438.
Evans M.J. and Kaufman M.H. 1981. Establishment in culture of pluripotent cells from mouse embryos. Nature 292: 154–156.
Hahnel A.C., Rappolee D.A., Millan J.L., Manes T., Ziome C.A., Theodosiou N.G., Werb Z., Pedersen R. and Schultz G.A. 1990. Two alkaline phosphatase genes are expressed during early development in the mouse embryo. Development 110: 555–564.
Heinrich P.C., Behrmann I., Mü ller-Newen G., Schaper F. and Graeve L. 1998. Interleukin-6-type cytokine signaling through the gp130/Jak/STAT pathway. Biochem. J. 334: 297–314.
Ling V. and Neben S. 1997. In vitro differentiation of embryonic stem cells: immunophenotypic analysis of cultured embryoid bodies. J. Cell Physiol. 171: 104–115.
Livak K.J. and Schmittgen T.D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-Delta Delta C(T)) method. Methods 25: 402–408.
Martin G.R. 1981. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinomas stem cells. Proc. Natl. Acad. Sci. USA 78: 7634–7638.
Murray P. and Edgar D. 2001. The regulation of embryonic stem cell differentiation by leukaemia inhibitory factor. Differentiation 68: 227–234.
Nichols J., Zevnik B., Anastassiadis K., Niwa H., Klewe-Nebenius D., Chambers I., Schö ler H. and Smith A. 1998. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct 4. Cell 95: 379–391.
Niwa H., Miyazaki J. and Smith A.G. 2000. Quantitative expression of Oct3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat. Genet 24: 372–376.
Oka M., Tagoku K., Russell T.L., Nakano Y., Hamazaki T., Meyer E.M., Yokota T. and Terada N. 2002. CD9 is associated with leukemia inhibitory factor-mediated maintenance of embryonic stem cells. Mol. Biol. Cell 4: 1274–1281.
Pesce M. and Schö ler H.R. 2001. Oct-4: Gatekeeper in the beginnings of mammalian development. Stem Cells 19: 271–278.
Rathjen P.D., Toth S., Willis A., Heath J.K. and Smith A.G. 1990. Differentiation inhibiting activity is produced in matrix associated and diffusible forms that are generated by alternate promoter usage. Cell 62: 1105–1114.
Schö ler H.R. 1991. Octamania: the POU factors in murine development. Trends Genet. 7: 323–329.
Smith A.G., Heath J.K., Donaldson D.D., Wong G.G., Moreau J., Stall M. and Rogers D. 1988. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 336: 688–690.
Solter D. and Knowles B.B. 1978. Monoclonal antibody defining a stage-specific mouse embryonic antigen (SSEA-1). Proc. Natl. Acad. Sci. USA 75: 5565–5569.
Viswanathan S., Benatar T., Rose-John S., Lauffenburger D.A. and Zandstra P.W. 2002. Ligand/receptor signaling threshold (LIST) model accounts for gp130-mediated embryonic stem cell self-renewal responses to LIF and HIL-6. Stem Cells 20: 119–138.
Williams M.A., Gross S.K., Evans J.E. and McCluer R.H. 1988a. Glycolipid stage-specific embryonic antigens (SSEA-1) in kidneys of male and female C57BL/6J and beige adult mice. J. Lipid Res. 29: 1613–1619.
Williams R.L., Hilton D.J., Pease S., Willson T.A., Stewart C.L., Gearing D.P., Wagner E.F., Metcalf D., Nicola N.A. and Gough N.M. 1988b. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 336: 684–687.
Yin Y., Lim Y.K., Salto-Tellez M., Ng S.C., Lin C.-S. and Lim S.K. 2002. AFP+, ESC derived cells engraft and differentiate into hepatocytes in vivo. Stem Cells 20: 338–346.
Yoshida K., Chambers I., Nichols J., Smith A., Saito M., Yasukawa K., Shoyab M., Taga T. and Kishimoto T. 1994. Maintenance of the pluripotent phenotype of embryonic stem cells through direct activation of gp130 signalling pathways. Mech. Dev. 45: 163–171.
Zandstra P.W., Le H.V., Daley G.Q., Griffith L.G. and Lauffenburger D.A. 2000. Leukaemia inhibitory factor (LIF) concentration modulates embryonic stem cell self-renewal and differentiation independently of proliferation. Biotechnol. Bioeng. 69: 607–617.
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Berrill, A., Tan, H., Wuang, S. et al. Assessment of Stem Cell Markers During Long-Term Culture of Mouse Embryonic Stem Cells. Cytotechnology 44, 77–91 (2004). https://doi.org/10.1023/B:CYTO.0000043414.90681.c2
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DOI: https://doi.org/10.1023/B:CYTO.0000043414.90681.c2