title: Activin A is essential for Feeder-free culture of human induced pluripotent stem cells.
abstract: Feeder-free culture of human induced pluripotent stem (hiPS) cells is necessary for their clinical application to avoid adverse effects of foreign proteins. hiPS cells were cultured with combinations of activin (A), CHIR99021 (C), basic fibroblast growth factor (F), and leukemia inhibitory factor (L) under feeder-free conditions. Culture was terminated after 12 passages or when the cell morphology changed from pluripotency. Pluripotency was analyzed by alkaline phosphatase (ALP) staining and immunostaining with antibodies to Oct3/4, Nanog, SSEA4, and TRA-1-60. SB431542 (SB), an activin inhibitor, was added to the culture, and the morphology of the cells was observed. hiPS cells cultured with A, AC, and ACL after 12 passages were positive for ALP staining. Oct3/4 was positive in hiPS cells cultured with A, AC, and ACL. hiPS cells were positive for Nanog when cultured with A and AC; however, Nanog signal was weaker in cells cultured with ACL. SSEA4 was positive in hiPS cells cultured with A and AC but almost negative in those cultured with ACL. hiPS cells were positive for TRA-1-60 when cultured with A, AC, and ACL. hiPS cells lose their undifferentiated morphology at six passages when cultured with A + SB, five passages with AC + SB, and nine passages with ACL. We conclude that feeder-free culture of hiPS cells requires A or AC to maintain pluripotency.
authors.affiliation: Department of Gastroenterology, National Hospital Organization Shimoshizu Hospital, 934-5 Shikawatashi, Yotsukaido City, Chiba 284-0003, Japan. nihminor-cib@umin.ac.jp
authors.forename: Minoru
authors.initials: M
authors.lastname: Tomizawa
authors.forename: Fuminobu
authors.initials: F
authors.lastname: Shinozaki
authors.forename: Takao
authors.initials: T
authors.lastname: Sugiyama
authors.forename: Shigenori
authors.initials: S
authors.lastname: Yamamoto
authors.forename: Makoto
authors.initials: M
authors.lastname: Sueishi
authors.forename: Takanobu
authors.initials: T
authors.lastname: Yoshida
mesh_terms: D028341:Activins / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D000469:Alkaline Phosphatase / Q000032:analysis; D000818:Animals; D000954:Antigens, Surface / Q000032:analysis; D001549:Benzamides / Q000494:pharmacology; D018929:Cell Culture Techniques; D003094:Collagen; D004149:Dioxoles / Q000494:pharmacology; D004338:Drug Combinations; D061252:Feeder Cells; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D018398:Homeodomain Proteins / Q000032:analysis; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism*; D007797:Laminin; D053605:Leukemia Inhibitory Factor / Q000494:pharmacology; D051379:Mice; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000032:analysis; D011509:Proteoglycans / Q000032:analysis; D011725:Pyridines / Q000494:pharmacology*; D011743:Pyrimidines / Q000494:pharmacology*; D055746:Stage-Specific Embryonic Antigens / Q000032:analysis
chemical_list: C459179:4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide; D000954:Antigens, Surface; D001549:Benzamides; C473711:Chir 99021; D004149:Dioxoles; D004338:Drug Combinations; D018398:Homeodomain Proteins; D007797:Laminin; D053605:Leukemia Inhibitory Factor; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D011509:Proteoglycans; D011725:Pyridines; D011743:Pyrimidines; D055746:Stage-Specific Embryonic Antigens; C107064:TRA-1-60 antigen, human; C422405:activin A; C066584:stage-specific embryonic antigen-4; D016222:Fibroblast Growth Factor 2; D028341:Activins; C059485:matrigel; D003094:Collagen; D000469:Alkaline Phosphatase