Development of culture conditions for the isolation of pluripotent porcine embryonal outgrowths from in vitro produced and in vivo derived embryos.
In the present study we examined the effect of culture media and protein source on the formation of pluripotent primary outgrowths from in vitro produced and in vivo derived porcine embryos as the first step towards the isolation of embryonic stem cells (ESCs). To do this we compared high glucose Dulbeccos Modified Eagles Medium (DMEM) with Minimal Essential Alpha Medium (αMEM) both supplemented with fetal bovine serum (FBS) or serum replacement (SR) in a 2 × 2 factorial design. Culture in DMEM or αMEM supplemented with 10% SR resulted in the establishment of homogenous populations of cells which expressed Oct 4 and Nanog. In contrast culture in either media with FBS resulted in the formation of embryonal outgrowths composed entirely of differentiated cells or a mixture of differentiated cells and putative ESCs which grew poorly and could not be passaged. Using αMEM medium containing 10% SR and culturing in 5% oxygen, putative ESC lines were isolated from in vitro and in vivo derived embryos at efficiencies of 2 and 10% respectively.
Reproductive Biotechnology Group, Discipline of Obstetrics and Gynaecology, School of Paediatrics and Reproductive Health and Centre for Stem Cell Research, Robinson Institute, University of Adelaide, Australia.
Ivan
I
VASSILIEV
Svetlana
S
VASSILIEVA
Luke F S
LF
BEEBE
Stephen M
SM
MCILFATRICK
Sharon J
SJ
HARRISON
Mark B
MB
NOTTLE
D000818:Animals; D001755:Blastocyst / Q000166:cytology* / Q000187:drug effects; D001798:Blood Proteins / Q000494:pharmacology; D003470:Culture Media / Q000494:pharmacology*; D046149:Embryo Culture Techniques / Q000379:methods* / Q000662:veterinary; D047108:Embryonic Development / Q000187:drug effects / Q000502:physiology; D053595:Embryonic Stem Cells / Q000166:cytology*; D005260:Female; D005947:Glucose / Q000494:pharmacology; D039904:Pluripotent Stem Cells / Q000166:cytology*; D011247:Pregnancy; D013552:Swine
D001798:Blood Proteins; D003470:Culture Media; D005947:Glucose