Hum. Reprod. Advance Access published online on February 6, 2009
Human Reproduction, doi:10.1093/humrep/dep003
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Novel autogenic feeders derived from human embryonic stem cells (hESCs) support an undifferentiated status of hESCs in xeno-free culture conditions
1 Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, College of Medicine and the Hospital, National Taiwan University, Taipei, Taiwan, Republic of China 2 Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China 3 Institute of Biotechnology, College of Bio-Resources and Agriculture, National Taiwan University, Taipei, Taiwan, Republic of China 4 Stem Cell Program, Institute of Cellular and Organismic Biology and Genomics Research Center, Academia Sinica, No. 128, Sec. 2, Academia Road, Nankang, Taipei, Taiwan, Republic of China 5 Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan, Republic of China 6 Graduate Institute of Immunology, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China
7 Correspondence address. Tel: +886-2-278-99580 x201, E-mail: kuohuch{at}gate.sinica.edu.tw
BACKGROUND: Clinical-grade human embryonic stem cells (hESCs) ideally should be derived and maintained in xeno-free culture conditions using defined chemicals or materials of human origin. This will reduce the possibility of xeno-derived pathogenic infection and/or unfavorable immune reaction in clinical application. The present study therefore aimed to derive autogenic feeders from hESCs and evaluate their capability to support the pluripotency of hESCs in xeno-free culture conditions.
METHODS AND RESULTS: H9 hESCs were cultured in media containing human serum (HS), serum replacement (SR) or KFM combination, to generate autogenic feeders (named HSdF, SRdF and KFMdF, respectively). Reverse transcription polymerase chain reaction, flow cytometry and immunofluorescence analysis using pluripotent stem cell markers, markers of early cell lineages and surface markers revealed that HSdF, SRdF and KFMdF likely belonged to different cellular subpopulations. The efficiency of the autogenic feeders in maintaining pluripotency of H9 hESCs using media containing SR, fetal bovine serum, HS or 1% HS plus various combinations of growth factors was evaluated by flow cytometric analysis of Oct4 expression. All three autogenic feeders were shown to be capable of maintaining the undifferentiated status of H9 hESCs in SR-containing media in long-term culture. When supplemented with bFGF, activin A and noggin, hESCs could also be maintained favorably on KFMdF in a medium containing 1% HS without losing their pluripotent potentials both in vitro and in vivo.
CONCLUSIONS: Novel autogenic feeders can be derived from hESCs under xeno-free conditions and they can robustly maintain the pluripotent identity of hESCs in xeno-free media containing a low concentration of HS.
Key words: autogenic feeder/embryonic stem cells/pluripotency/human serum/xeno-free culture
Submitted on October 23, 2008; resubmitted on December 19, 2008; accepted on January 5, 2009.