Generation of embryonic stem cell lines from mouse blastocysts developed in vivo and in vitro: relation to Oct-4 expression.
Embryonic stem (ES) cells are the source of all embryonic germ layer tissues. Oct-4 is essential for their pluripotency. Since in vitro culture may influence Oct-4 expression, we investigated to what extent blastocysts cultured in vitro from the zygote stage are capable of expressing Oct-4 and generating ES cell lines. We compared in vivo with in vitro derived blastocysts from B6D2 mice with regard to Oct-4 expression in inner cell mass (ICM) outgrowths and blastocysts. ES cells were characterized by immunostaining for alkaline phosphatase (ALP), stage-specific embryonic antigen-1 (SSEA-1) and Oct-4. Embryoid bodies were made to evaluate the ES cells' differentiation potential. ICM outgrowths were immunostained for Oct-4 after 6 days in culture. A quantitative real-time PCR assay was performed on individual blastocysts. Of the in vitro derived blastocysts, 17% gave rise to ES cells vs 38% of the in vivo blastocysts. Six-day old outgrowths from in vivo developed blastocysts expressed Oct-4 in 55% of the cases vs 31% of the in vitro derived blastocysts. The amount of Oct-4 mRNA was significantly higher for freshly collected in vivo blastocysts compared to in vitro cultured blastocysts. In vitro cultured mouse blastocysts retain the capacity to express Oct-4 and to generate ES cells, be it to a lower level than in vivo blastocysts.
Tielens
S
S
Department of Anatomy, Embryology, Histology and Medical Physics, Ghent University, L. Pasteurlaan 2, B-9000 Ghent, Belgium.
Verhasselt
B
B
Liu
J
J
Dhont
M
M
Van Der Elst
J
J
Cornelissen
M
M
D000199:Actins / Q000032:analysis; D000469:Alkaline Phosphatase / Q000032:analysis; D000818:Animals; D015415:Biomarkers / Q000032:analysis; D001755:Blastocyst / Q000166:cytology*; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002460:Cell Line; D047108:Embryonic Development; D015870:Gene Expression; D006651:Histocytochemistry; D016256:Lewis X Antigen / Q000032:analysis; D051379:Mice; D016900:Neurofilament Proteins / Q000032:analysis; D050814:Octamer Transcription Factor-3 / Q000032:analysis / Q000235:genetics*; D039904:Pluripotent Stem Cells / Q000166:cytology*; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018709:Statistics, Nonparametric; D015053:Zygote / Q000166:cytology; D000509:alpha-Fetoproteins / Q000032:analysis
D000199:Actins; D015415:Biomarkers; D016256:Lewis X Antigen; D016900:Neurofilament Proteins; D050814:Octamer Transcription Factor-3; D012333:RNA, Messenger; D000509:alpha-Fetoproteins; D000469:Alkaline Phosphatase