Feeder layer- and serum-free culture of human embryonic stem cells.
In addition to their contribution to the research on early human development, human embryonic stem (hES) cells may also be used for cell-based therapies. Traditionally, these cells have been cultured on mouse embryonic fibroblast feeder layers, which allow their continuous growth in an undifferentiated state. However, the use of hES cells in human therapy requires an animal-free culture system, in which exposure to mouse retroviruses is avoided. In this study we present a novel feeder layer-free culture system for hES cells, based on medium supplemented with 15% serum replacement, a combination of growth factors including transforming growth factor beta1 (TGFbeta1), leukemia inhibitory factor, basic fibroblast growth factor, and fibronectin matrix. Human ES cells grown in these conditions maintain all ES cell features after prolonged culture, including the developmental potential to differentiate into representative tissues of the three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype. The culture system presented here has two major advantages: 1) application of a well-defined culture system for hES cells and 2) reduced exposure of hES cells to animal pathogens. The feeder layer-free culture system reported here aims at facilitating research practices and providing a safer alternative for future clinical applications of hES cells.
Amit
M
M
Department of Obstetrics and Gynecology, Rambam Medical Center, Haifa, and The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, 31096 Haifa, Israel.
Shariki
C
C
Margulets
V
V
Itskovitz-Eldor
J
J
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D016895:Culture Media, Serum-Free / Q000494:pharmacology*; D005333:Fetus; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D006801:Humans; D015850:Interleukin-6; D053605:Leukemia Inhibitory Factor; D008297:Male; D051379:Mice; D016513:Mice, SCID; D011506:Proteins / Q000494:pharmacology; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology*; D013724:Teratoma; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D053773:Transforming Growth Factor beta1
D016895:Culture Media, Serum-Free; D015850:Interleukin-6; C507837:LIF protein, human; D053605:Leukemia Inhibitory Factor; C507838:Lif protein, mouse; D011506:Proteins; C508960:TGFB1 protein, human; C508961:Tgfb1 protein, mouse; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D016222:Fibroblast Growth Factor 2