Self-renewal and Proliferation of Murine Embryonic Stem Cells: A Study of Glycosaminoglycans Effect on Feeder-Free Cultures

Abstract

The self-renewal and proliferation of murine embryonic stem (ES) cells can be preserved indefinitely in the presence of the mouse embryonic fibroblast (MEF) feeder layer. Since the feeder layer has several drawbacks, including viral contamination and large-scale production, gelatin solutions are the most prominent alternative to replacing it. In this investigation, self-renewal and proliferation of ES cells was carried out by supplementing the gelatin solution with glycosaminoglycan components, such as chondroitin sulfate and heparin. The methylcellulose-based embryoid body (EB) assay was used to evaluate the self-renewal ability of the ES cells. Chondroitin sulfate and heparin were mixed with 0.1% gelatin solution in 0:100, 50:50 and 100:0 (heparin/chondroitin sulfate) ratios and compared with 0.1% gelatin (positive control) and MEF (negative control) in three independent parallel experiments. Chondroitin sulfate addition to 0.1% gelatin enhanced the cell population and the number of EBs by 57 and 32%, respectively.

Keywords

embryonic stem cells glycosaminoglycans embryoid body chondroitin sulfate heparin methylcellulose.

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References

Thomson, J.A. , Itskovitz-Eldor, J. , Shapiro, S.S. , Waknitz, M.A. , Swiergiel, J.J. , Marshall, V.S. , and Jones, J.M. (1998). Embryonic stem cell lines derived from human blastocysts, Science, 282: 1145—1147.

Reubinoff, B.E. , Pera, M.F. , Fong, C.Y. , Trounson, A. , and Bongso, A. (2000). Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro, Nat. Biotech., 18: 399—404.

Xu, C.H. , Inokuma, M.S. , Denham, J. , Golds, K. , Kundu, P. , Gold, J.D. , and Carpenter , M.K. (2001). Feeder-free growth of undifferentiated human embryonic stem cells, Nat. Biotech., 19: 971—974.

Smith, A.G. , Heat, J.K. , Donaldson, D.D. , Wong, G.G. , Moreau, J. , Stahl, J.M. , and Rojers, D. (1988). Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides, Nature, 336: 688—690.

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. (1988). Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells, Nature, 336: 684—687.

Murray, P. and Edgar, D. (2001). The regulation of embryonic stem cell differentiation by leukaemia inhibitory factor (LIF), Differentiation, 68: 227—234.

Amit, M. , Shariki, C. , Margulets, V. , and Itskovitz-Eldor, J. (2004). Feeder layer- and serum-free culture of human embryonic stem cells, Biol. Reprod., 70: 837—845.

Prudhomme, W. , Daley, G.Q. , Zandstra, P. , and Lauffenburger , D.A. (2004). Multivariate proteomic analysis of murine embryonic stem cell self-renewal versus differentiation signaling, Proc. Natl. Acad. Sci. USA , 101: 2900—2905.

Sato, N. , Meijer, N.L. , Skaltsounis, L. , Greengard, P. , and Brivanlou, A.H. (2004). Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor, Nat. Med., 10: 55—63.

10.

Vallier, L. , Alexander, M. , and Pedersen, R.A. (2004). Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway, Dev. Biol., 275: 403—421.

11.

James, D. , Levine, A.J. , Besser, D. , and Hemmati-Brivanlou, A. (2005). TGF-b/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells , Development, 132: 1273—1282.

12.

Beattie, G.M. , Lopez, A.D. , Bucay, N. , Hinton, A. , Firpo, M.T. , King, C.C. , and Hayek, A. (2005). Activin A maintains pluripotency of human embryonic stem cells in the absence of feeder layers, Stem Cells, 23: 489—495.

13.

Xu, R.H. , Peck, R.M. , Li, D.S. , Feng, X. , Ludwig, T. , and Thomson, J.A. (2005). Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells, Nat. Methods , 2: 185—190.

14.

Indiana State University [Internet] (2006). Terre Haute (Indiana): Glycosaminoglycans, The Medical Biochemistry Page [cited October 6, 2006]. Available from: http://web.indstate.edu/thcme/mwking/glycans.html .

15.

Atala, A. and Lanza, R.P. (2001). Methods of Tissue Engineering, Academic Press, San Diego, CA.