Defined culture conditions of human embryonic stem cells

doi:10.1073/pnas.0601383103

Defined culture conditions of human embryonic stem cells

Departments of *Molecular, Cellular, and Developmental Biology and
**Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511;
^†Department of Medicine, Hospital of Saint Raphael, New Haven, CT 06511;
^§Section of Comparative Medicine and
^¶Department of Neurology, Yale University School of Medicine, New Haven, CT 06510; and
^‖Division of Neurosurgery, Department of Surgery, National Taiwan University Hospital, Taipei 100, Taiwan

Communicated by Sherman M. Weissman, Yale University School of Medicine, New Haven, CT, February 21, 2006
↵ ^‡R.H. and C.J.B. contributed equally to this work. (received for review January 19, 2006)

Abstract

Human embryonic stem cells (hESCs) are pluripotent cells that have the potential to differentiate into any tissue in the human body; therefore, they are a valuable resource for regenerative medicine, drug screening, and developmental studies. However, the clinical application of hESCs is hampered by the difficulties of eliminating animal products in the culture medium and/or the complexity of conditions required to support hESC growth. We have developed a simple medium [termed hESC Cocktail (HESCO)] containing basic fibroblast growth factor, Wnt3a, April (a proliferation-inducing ligand)/BAFF (B cell-activating factor belonging to TNF), albumin, cholesterol, insulin, and transferrin, which is sufficient for hESC self-renewal and proliferation. Cells grown in HESCO were maintained in an undifferentiated state as determined by using six different stem cell markers, and their genomic integrity was confirmed by karyotyping. Cells cultured in HESCO readily form embryoid bodies in tissue culture and teratomas in mice. In both cases, the cells differentiated into each of the three cell lineages, ectoderm, endoderm, and mesoderm, indicating that they maintained their pluripotency. The use of a minimal medium sufficient for hESC growth is expected to greatly facilitate clinical application and developmental studies of hESCs.

Footnotes

^††To whom correspondence should be addressed at:
Department of Molecular, Cellular, and Developmental Biology, P.O. Box 208103, Yale University, New Haven, CT 06520-8103.
E-mail: michael.snyder{at}yale.edu
Author contributions: J.L. designed research; J.L., R.H., C.J.B., and S.-H.Y. performed research; J.L. contributed new reagents/analytic tools; J.L. and C.J.B. analyzed data; and J.L., R.H., and M.S. wrote the paper.
Conflict of interest statement: M.S. has financial interests with Invitrogen.
Abbreviations:

Abbreviations:

April,

a proliferation-inducing ligand;

BAFF,

B cell-activating factor belonging to TNF;

bFGF,

basic fibroblast growth factor;

hESC,

human embryonic stem cell;

HESCO,

hESC Cocktail;

MEF,

mouse embryonic fibroblast;

SSEA,

stage-specific embryonic antigen.