The use of parthenotegenetic and IVF bovine blastocysts as a model for the creation of human embryonic stem cells under defined conditions.
OBJECTIVE
Clinical application of human embryonic stem cells will be possible, when cell lines are created under xeno-free and defined conditions. We aimed to establish methodologies for parthenogenetic activation, culture to blastocyst and mechanical isolation of the inner cell mass (ICM) using bovine oocytes, as a model for derivation and proliferation of human embryonic stem cells under defined xeno-free culture conditions.


METHODS
Cumulus-oocyte-complexes were in vitro matured and activated using Ca(2+)Ionophore and 6-DMAP or in vitro fertilized (IVF). Parthenotes and biparental embryos were cultured to blastocysts, when their ICM was mechanically isolated and placed onto a substrate of fibronectin in StemPro medium. After attachment, primary colonies were left to proliferate and stained for pluripotency markers, alkaline phosphatase and Oct-4.


RESULTS
Parthenogenesis and fertilization presented significantly different success rates (91 and 79 %, respectively) and blastocyst formation (40 and 43 %, respectively). ICMs from parthenogenetic and IVF embryos formed primary and expanded colonies at similar rates (39 % and 33 %, respectively). Six out of eight parthenogenetic colonies tested positive for alkaline phosphatase. Three colonies were analyzed for Oct-4 and they all tested positive for this pluripotency marker.


CONCLUSIONS
Our data show that Ca(2+) Ionophore, and 6-DMAP are efficient in creating large numbers of blastocysts to be employed as a model for human oocyte activation and embryo development. After mechanical isolation, parthenogetic derived ICMs showed a good rate of derivation in fibronectin and Stem-Pro forming primary and expanded colonies of putative embryonic stem cells. This methodology may be a good strategy for parthenogenetic activation of discarded human oocytes and derivation in defined conditions for future therapeutic interventions.
Department of Morphological Sciences, ICBS, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil 90050-170.
R R
RR
Ruggeri
Y
Y
Watanabe
F
F
Meirelles
F F
FF
Bressan
N
N
Frantz
A
A
Bos-Mikich
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D001755:Blastocyst / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D053624:Blastocyst Inner Cell Mass / Q000166:cytology; D002417:Cattle; D046149:Embryo Culture Techniques; D053595:Embryonic Stem Cells / Q000166:cytology*; D005260:Female; D005307:Fertilization in Vitro; D005353:Fibronectins / Q000737:chemistry; D008297:Male; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D010312:Parthenogenesis; D039904:Pluripotent Stem Cells / Q000378:metabolism
D015415:Biomarkers; D005353:Fibronectins; D050814:Octamer Transcription Factor-3; C001020:N(6),N(6)-dimethyladenine; D000469:Alkaline Phosphatase; D000225:Adenine