Derivation, characterization and differentiation of human embryonic stem cells: comparing serum-containing versus serum-free media and evidence of germ cell differentiation.
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.
Chen
H-F
HF
Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, College of Medicine and Hospital, National Taiwan University, Taipei, Taiwan.
Kuo
H-C
HC
Chien
C-L
CL
Shun
C-T
CT
Yao
Y-L
YL
Ip
P-L
PL
Chuang
C-Y
CY
Wang
C-C
CC
Yang
Y-S
YS
Ho
H-N
HN
D017209:Apoptosis; D055430:Bone Morphogenetic Protein 15; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002868:Chromosomal Proteins, Non-Histone; D003470:Culture Media; D016895:Culture Media, Serum-Free; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D005260:Female; D005854:Germ Cells / Q000166:cytology*; D055429:Growth Differentiation Factor 9; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis; D050814:Octamer Transcription Factor-3 / Q000096:biosynthesis; D039904:Pluripotent Stem Cells / Q000166:cytology; D011247:Pregnancy; D011506:Proteins / Q000378:metabolism; D019009:Proto-Oncogene Proteins c-kit / Q000096:biosynthesis
C525901:BMP15 protein, human; D055430:Bone Morphogenetic Protein 15; D002868:Chromosomal Proteins, Non-Histone; D003470:Culture Media; D016895:Culture Media, Serum-Free; C466105:DPPA3 protein, human; C525897:GDF9 protein, human; D055429:Growth Differentiation Factor 9; D036341:Intercellular Signaling Peptides and Proteins; D050814:Octamer Transcription Factor-3; D011506:Proteins; D019009:Proto-Oncogene Proteins c-kit