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Stem Cells 2005;23:544-549 www.StemCells.com
© 2005 AlphaMed Press

Derivation of Human Embryonic Stem Cell Lines in Serum Replacement Medium Using Postnatal Human Fibroblasts as Feeder Cells

José Inzunzaa,b, Karin Gertowc, Marie A. Strömberga, Eija Matilainena, Elisabeth Blennowd, Heli Skottmana, Susanne Wolbankc, Lars Ährlund-Richterc, Outi Hovattaa

a Department of Clinical Sciences, Division of Obstetrics and Gynecology,
b Department of Medical Nutrition at Novum,
c Department of Laboratory Medicine, and
d Department of Molecular Medicine, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden

Key Words. Human embryonic stem cells • Serum replacement medium • Foreskin fibroblasts • Characterization • Pluripotency

Correspondence: Outi Hovatta, M.D., Ph.D, Department of Clinical Sciences, Division of Obstetrics and Gynecology, Karolinska Institutet, Karolinska University Hospital, Huddinge, S-141 86 Stockholm, Sweden; Telephone: 46-8-58580000; Fax: 46-8-58587575; e-mail: Outi.Hovatta{at}klinvet.ki.se

Derivation and culture of human embryonic stem cells (hESCs) without animal-derived material would be optimal for cell transplantation. We derived two new hES (HS293 and HS306) and 10 early cell lines using serum replacement (SR) medium instead of conventional fetal calf serum and human foreskin fibroblasts as feeder cells. Line HS293 has been in continuous culture, with a passage time of 5–8 days, since October 2003 and is at passage level 56. Line HS306 has been cultured since February 2004, now at passage 41. The lines express markers of pluripotent hESCs (Oct-4, SSEA-4, TRA-1-60, TRA-1-81, GCTM-2, and alkaline phosphatase). The pluripotency has been shown in embryoid bodies in vitro, and the pluripotency of line 293 has also been shown in vivo by teratoma formation in severe combined immunodeficiency/beige mice. The karyotype of HS293 is 46,XY, and that of HS306 is 46,XX. Ten more early lines have been derived under similar conditions since September 2004. We conclude that hESC lines can be successfully derived using SR medium and postnatal human fibroblasts as feeder cells. This is a step toward xeno-free conditions and facilitates the use of these cells in transplantation.




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