ABSTRACT Mouse embryonic stem (ES) cells are continuous cell lines derived directly from the fetal founder tissue of the pre-im- plantation embryo and can be expanded in vitro and give rise to cells from ectodermal, mesodermal and endodermal layers. Mouse ES cells can be maintained and their numbers expanded by culture on feeder layer cells with LIF present in the culture medium. This study shows that changes in seeding density can significantly influence cell number expansion rates. Culturing ES cells in the absence of feeder layer cells and LIF stimulates EB formation when cultured in non-adherent culture plates. Formation of EBs particularly numbers, size of EBs formed, rates of cell proliferation within EBs and viability of cells can also be controlled based on seeding density. All these factors are important for optimizing approaches to co-ordinate differentiation towards a specific cell type. A key goal of ES cell research is to develop specific functional cell types which can be potentially used to study mechanisms of tissue development and as a therapy to repair or replace damaged or diseased tissues.
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nullG. Adams, L. Buttery, S. Stolnik, S. Harding and N. Wang, "Assessment of Biological Properties of Mouse Embryonic Stem Cells Characteristics Prior To Differentiation," Journal of Biomaterials and Nanobiotechnology, Vol. 2 No. 4, 2011, pp. 378-389. doi: 10.4236/jbnb.2011.24047.
 A. G. Smith, “Embryo-Derived Stem Cells: Of Mice and Men,” Annual Review of Cell and Developmental Biology, Vol. 17, 2001, pp. 435-462.
 P. W Andrews, et al., “Pluripotent Embryonal Carcinoma Clones Derived from the Human Teratocarcinoma Cell Line Tera-2,” Differentiation in Vivo and in Vitro. Labo- ratory Investigation, Vol. 50, No. 2, 1984, pp. 147-162.
 G. Hopfl, M. Gassmann, and I. Desbaillets, “Differenti- ating Embryonic Stem Cells into Embryoid Bodies,” Me-thods in Molecular Biology, Vol. 254, 2004, pp. 79- 98.
 T. C. Doetschman, et al., “The in Vitro Development of Blastocyst-Derived Embryonic Stem Cell lines: Formation of Visceral Yolk Sac, Blood Islands and Myocar- dium,” Journal of Embryology & Experimental Morpho- logy, Vol. 87, 1985, pp. 27-45.
 Itskovitz-Eldor, J., et al., Differentiation of human em- bryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Molecular Medicine Vol. 6, No. 2, 2000, pp. 88-95.
 S. M .Dang, et al., “Efficiency of Embryoid Body For- mation and Hematopoietic Development from Embryonic Stem Cells in Different Culture Systems,” Biotechnology and Bioengineering, Vol. 78, No. 4, 2002, pp. 442-453. doi:10.1002/bit.10220
 C. M. Cameron, W. S. Hu and D. S. Kaufman, “Improved Development of Human Embryonic Stem Cell-Derived Embryoid Bodies by Stirred Vessel Cultivation,” Bio- technology and Bioengineering, Vol. 94, No. 5, 2006, pp. 938-948. doi:10.1002/bit.20919
 S. M. Dang, and P. W. Zandstra, “Scalable Production of Embryonic Stem Cell-Derived Cells,” Methods in Mole-cular Biology, Vol. 290, No. 1, 2005, pp. 353-364.
 S. Gerecht-Nir, S. Cohen, and J. Itskovitz-Eldor, “Bio- reactor Cultivation Enhances the Efficiency of Human Embryoid Body (HEB) Formation and Differentiation,” Biotechnology and Bioengineering, Vol. 86, No. 5, 2004, pp. 493-502. doi:10.1002/bit.20045
 Y. Suda, et al., “Mouse Embryonic Stem Cells Exhibit Indefinite Proliferative Potential,” Journal of Cellular Physiology, Vol. 133, No. 1, 1987, pp. 197-201.
 T. Burdon, et al., “Signaling Mechanisms Regulating Selfrenewal and Differentiation of Pluripotent Embryonic Stem Cells,” Cells Tissues Organs, Vol. 165, No. 3-4, 1999, pp. 131-143.
 H. Niwa, J. Miyazaki, and A. G. Smith, “Quantitative Expression of Oct-3/4 Defines Differentiation, Dediffe- rentiation or Self-Renewal of ES Cells,” Nature Genetics, Vol. 24, No. 4, 2000, pp. 372-376. doi:10.1038/74199
 M. J Martin, et al., “Human Embryonic Stem Cells Ex-press an Immunogenic Nonhuman Sialic Acid,” Nature Medicine, Vol. 11, No. 2, 2005, pp. 228-232.
 J. L. Spees, et al., “Internalized Antigens Must Be Re- moved to Prepare Hypoimmunogenic Mesenchymal Stem Cells for Cell and Gene Therapy,” Molecular Therapy, Vol. 9, No. 5, 2004, pp. 747-756.
 A. Heiskanen, et al., “N-Glycolylneuraminic Acid Xe- noantigen Contamination of Human Embryonic and Me-senchymal Stem Cells is Substantially Reversible,” Stem Cells, Vol. 25, No. 1, 2007, pp. 197-202.
 N. Plachta, et al., “Developmental Potential of Defined Neural Progenitors Derived from Mouse Embryonic Stem Cells,” Development, Vol. 131, No. 21, 2004, pp. 5449- 5456. doi:10.1242/dev.01420
 W. Risau, et al., “Vasculogenesis and Angiogenesis in Embryonic-Stem-Cell-Derived Embryoid Bodies,” De- velopment, Vol. 102, No. 3, 1988, pp. 471-478.
 O. Feraud, Y. Cao, and D. Vittet, “Embryonic Stem Cell- Derived Embryoid Bodies Development in Collagen Gels Recapitulates Sprouting Angiogenesis,” Laboratory In-vestigation, Vol. 81, No. 12, 2001, pp. 1669-1681.
 M. G Klug, et al., “Genetically Selected Cardiomyocytes from Differentiating Embronic Stem Cells form Stable Intracardiac Grafts,” Journal of Clinical Investigation, Vol. 98, No. 1, 1996, pp. 216-224.
 N. S. Hwang, et al., “Enhanced Chondrogenic Differen- tiation of Murine Embryonic Stem Cells in Hydrogels with Glucosamine,” Biomaterials, Vol. 27, No. 36, 2006, pp. 6015-6023. doi:10.1016/j.biomaterials.2006.06.033
 E. A Jones, et al., “Hepatic Differentiation of Murine Embryonic Stem Cells,” Experimental Cell Research, Vol. 272, No. 1, 2002, pp. 15-22.
 Y. Moritoh, et al., “Analysis of Insulin-Producing Cells during in Vitro Differentiation from Feeder-Free Em- bryonic Stem Cells,” Diabetes, Vol. 52, No. 5, 2004, pp. 1163-1168. doi:10.2337/diabetes.52.5.1163
 N. Lumelsky, et al., “Differentiation of Embryonic Stem Cells to Insulin-Secreting Structures Similar to Pancreatic Islets,” Science, Vol. 292, No. 5520, 2001, pp. 1389- 1394. doi:10.1126/science.1058866
 Y. Toyooka, et al., “Embryonic Stem Cells can Form Germ Cells in Vitro,” Proceedings of the National Aca- demy of Sciences, USA, Vol. 100, No. 20, 2003, pp. 11457-11462. doi:10.1073/pnas.1932826100
 I. Kehat, et al., “Human Embryonic Stem Cells can Dif-ferentiate into Myocytes with Structural and Fun- ctional Properties of Cardiomyocytes,” Journal of Clinical In-vestigation, Vol. 108, No. 3, 2001, pp. 407- 414.
 S. Assady, et al., “Insulin Production by Human Em- bryonic Stem Cells,” Diabetes, Vol. 50, No. 8, 2001, pp. 1691-1697. doi:10.2337/diabetes.50.8.1691
 S. Levenberg, et al., “Endothelial Cells Derived from Human Embryonic Stem Cells,” Proceedings of the Na-tional Academy of Sciences, U S A, Vol. 99, No. 7, 2002, pp. 4391-4396. doi:10.1073/pnas.032074999
 R. Zweigerdt, et al., “Generation of Confluent Cardio- myocyte Monolayers Derived from Embryonic Stem Cells in Suspension: a Cell source for New Therapies and Screening Strategies,” Cytotherapy, Vol. 5, No. 5, 2003, pp. 399-413. doi:10.1080/14653240310003062
 M. A. Rundnick and M. W, “Cell Culture Methods and Induction of Differentiation of Embryonal Carcinoma Cell lines,” In: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach., Ed., IRL Press, Washington, D.C., 1987, pp. 19-49.
 A. Goedecke, M. Wobus, M. Krech, N. Münch, K. Rich-ter, K. H?lig and M. Bornhauser, “Differential Effect of Platelet-Rich Plasma and Fetal Calf Serum on Bone Marrow-Derived Human Mesenchymal Stromal Cells eExpanded in Vitro,” Journal of Tissue Engineering and Regenerative Medicine, Vol. 5, No. 8, 2011, p. 648.
 T. M. Maul, D. W. Chew, A. Nieponice and D. A. Vorp, “Mechanical Stimuli Differentially Control Stem Cell Behavior: Morphology, Proliferation, and Differentia- tion,” Biomechanics and Modeling in Mechanobiology, Vol. 10, No. 1, 2011, pp. 145-160.
 Z. He, et al., “Effect of Leukemia Inhibitory Factor on Embryonic Stem Cell Differentiation: Implications for Supporting Neuronal Differentiation,” Acta Pharmacolo-gica Sinica, Vol. 27, No. 1, 2006, pp. 80-90.
 A. J. Potocnik, H. Kohler and K. Eichmann, “Hemato- lymphoid in Vivo Reconstitution Potential of Sub-populations Derived from in Vitro Differentiated Embryonic Stem Cells,” Proceedings of the National Academy of Sciences, USA, Vol. 94, No. 19, 1997. pp. 10295-10300. doi:10.1073/pnas.94.19.10295
 J. M Metzger, W. I. Lin and L. C. Samuelson, “Transition in Cardiac Contractile Sensitivity to Calcium during the in Vitro Differentiation of Mouse Embryonic Stem Cells,” Journal of Cell Biology, Vol. 126, No. 3, 1994, pp. 701-711. doi:10.1083/jcb.126.3.701
 T. Yamada, et al., “In Vitro Functional Gut-Like Organ Formation from Mouse Embryonic Stem Cells,” Stem Cells, Vol. 20, No. 1, 2002, pp. 41-49.
 C. Hegert, et al., “Differentiation Plasticity of Chon-m drocytes Derived from Mouse Embryonic Stem Cells,” Journal of Cell Science, Vol. 115, No. 23, 2002, pp. 4617-4628. doi:10.1242/jcs.00171
 J. Kramer, et al., “Cells Differentiated from Mouse Em-bryonic Stem Cells via EmbryoidBodies Express Renal Marker Molecules,” Differentiation, Vol. 74, No. 2-3, 2006, pp. 91- 104.
 C. Dani, et al., “Differentiation of Embryonic Stem Cells into Adipocytes in vitro,” Journal of Cell Science, Vol. 110, No. 11, 1997, pp. 1279-1285.
 T. Yamada, et al., “In Vitro Differentiation of Embryonic Stem Cells into Hepatocyte-Like Cells Identified by Cel-lular Uptake of Indocyanine Green,” Stem Cells, Vol. 20, No. 2, 2002, pp. 146-154.
 A. Shiroi, et al., “Identification of Insulin-Producing Cells Derived from Embryonic Stem Cells by Zinc-Chelating Dithizone,” Stem Cells, Vol. 20, No. 4, 2002, pp. 284-292. doi:10.1634/stemcells.20-4-284
 N. Geijsen, et al., “Derivation of Embryonic Germ Cells and Male Gametes from Embryonic Stem Cells,” Nature, Vol. 427, No. 6970, 2004, pp. 148-154.
 E. Y. Fok and P. W. Zandstra, “Shear-Controlled Single- Step Mouse Embryonic Stem Cell Expansion and Em- bryoid Body-Based Differentiation,” Stem Cells, Vol. 23, No. 9, 2005. pp. 1333-1342.
 K. Sasaki, I. H., S. Takei, H. S. No, D. Tomotsune, Y. Kano, T. Yokoyama, S. Sirasawa, A. Mogi, S. Yoshie, S. Sasaki, S. Yamada, K. Matsumoto, M. Mizuguchi, F. Yue and Y. Tanaka, “Hepatocyte Differentiation from Human ES Cells Using the Simple Embryoid Body Formation Method and the Staged-Additional Cocktail,” Scientific World Journal, Vol. 1, No. 9, 2009, pp. 884-890.
 A. Sen, M. S. Kallos, and L. A. Behie, “Expansion of Mammalian Neural Stem Cells in Bioreactors: Effect of Power Input and Medium Viscosity,” Brain Research, Vol. 134, No. 1-2, 2002, pp. 103-113.