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Hum. Reprod. Advance Access originally published online on October 27, 2006
Human Reproduction 2007 22(2):567-577; doi:10.1093/humrep/del412
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© The Author 2006. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Derivation, characterization and differentiation of human embryonic stem cells: comparing serum-containing versus serum-free media and evidence of germ cell differentiation

H.-F. Chen1, H.-C. Kuo2,3, C.-L. Chien4, C.-T. Shun5, Y.-L. Yao1, P.-L. Ip1,6, C.-Y. Chuang3, C.-C. Wang7, Y.-S. Yang1 and H-N. Ho1,8,9

1 Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, College of Medicine and the Hospital, National Taiwan University, Taipei, Taiwan 2 Division of Life Sciences, Institute of Cellular and Organismic Biology 3 Stem Cell Program, Genomics Research Center, Academia Sinica 4 Department of Anatomy and Cell Biology, College of Medicine 5 Department of Pathology, College of Medicine and Hospital, National Taiwan University, Taipei, Taiwan 6 Department of Biology, New York University, New York, NY, USA 7 Graduate Institute of Clinical Medicine, College of Medicine and 8 Graduate Institute of Immunology, College of Medicine, National Taiwan University, Taipei, Taiwan

9 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, National Taiwan University Hospital, No. 7 Chung-Shan South Road, Taipei 100, Taiwan. E-mail: hnho{at}ha.mc.ntu.edu.tw

BACKGROUND: This study was designed to establish human embryonic stem cell (hESC) lines, to identify the differences when maintained in serum-containing versus serum-free medium and to test their potential of in vitro differentiation. METHODS: Procedures including immunosurgery were performed on 11 donated human blastocysts to establish hESC lines. The cell lines were characterized and maintained using either serum-free or serum-containing media to compare their morphology, Oct-4 expression, apoptosis and growth speed. Differentiation of these lines was evaluated by the morphology and the expression of genes belonging to the three embryonic germ layers and the germ cell lineage. RESULTS: Three hESC lines were established, and they grew at similar speed in both media (serum-containing or serum-free), but hESC cultured in serum-containing medium yielded significantly higher percentages of morphologically good colonies and cells expressing Oct-4. These cell lines differentiated spontaneously in vitro into cells expressing markers belonging to all three embryonic germ layers and germ cell markers, including c-Kit, STELLA, VASA and growth differentiation factor 9 (GDF9), in directly adherent culture. CONCLUSIONS: Three hESC lines with Taiwanese ancestry have been established, and they retain the in vitro differentiation potential with or without embryoid body (EB) formation. The data support that hESC may be capable of differentiation into germ cells although further confirmation is needed. It is also suggested that strategies such as stepwise adaptation will be needed before implementing a serum-free culture condition for hESC lines that have previously been derived in a medium containing serum.

Key words: differentiation/embryoid body/embryonic stem cells/germ cell/serum-free medium


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